iGEM 2024 — Team ju-krakow

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Lab Notebooks

# Wet Lab

---

During our iGEM year, we organized our project by forming multiple subgroups, each dealing with different
parts of our project.

## Sherlock Lab

---

### 08.04. - 14.04

### Pre-amplification of Synthetic DNA 1

Since the RPA kit was not yet available, the
pre-amplification was completed using a PCR reaction. Moving
forward, **Synthetic DNA
1** will be referred to
as **SynDNA**.

1. Primers were suspended in water according to the
   manual to create stocks (100 μM).

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/1-sher.png)

Figure 1: Manual detailing the
parameters of the oligonucleotides used

**SynF:**AATTCTAATACGACTCACTATAGGGATCCTCTAGAAATATGGATTACTTGGTAGAACAG

**SynR:** GATAAACACAGGAAACAGCTATGACCATGATTACG

**T7-3GIVT
primer:**GAAATTAATACGACTCACTATAAGGG

2. 10-fold dilutions of the primers were prepared to
   create **10
   μM** samples.
3. SynDNA was diluted
   according to the manual. The concentration of SynDNA was calculated from the
   data in the said manual.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/2-sher.png)

Figure 2: Manual detailing the
parameters of the oligonucleotides used

**SynDNA:**GGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAAATATGGATTACTTGGTAGAACAGCAATCTACTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAG

**SyncrRNA:**CTCGACCTGCAGGCATGCAAGCTTGGCGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC

(53386 g \* 100 μmol) / 1000
000 μmol = 5.34 g

(5.34 g \* 1 ml) / 1000 ml =
5.34 mg (of Synthetic DNA 1 in 1 ml of stock) =

**5.34 μg /μl
(concentration of the stock)**

4. 8,4 μl was taken from the stock and added to
   491.6 μl of water creating a diluted stock sample
   with **89.7
   ng/μl**concentration.
5. This stock solution was further diluted 10-fold to
   create a workable sample with **8.97 ng/μl
   concentration.**
6. Six samples were prepared (including negative
   control), as follows:

Table 1: PCR Reaction Master
Mix components (for a single reaction):

|  |  |
| --- | --- |
| **Component** | **Volume [µl]** |
| Q5 mix | 10 |
| Template DNA (water in negative control) | 2 |
| SynF primer (10 µM) | 1 |
| SynR primer (10 µM) | 1 |
| H₂O | 6 |
| **Total** | **20** |

**Reaction conditions** (steps in *italics* indicate the cyclically repeated ones):

* pre-denaturation – 98°C, 30 sec

* *denaturation – 98°C, 10 sec*

* **annealing – temperature gradient (58°C, 60°C, 62°C, 64°C, 66°C) 30
  sec**

* elongation – 72°C, 10 sec

* final elongation – 72°C, 2 min

* **number of cycles – 35**

**Electrophoresis**

1. Pre-prepared**TAE-Agarose gel (1%)** was melted.
2. Once fully melted and cooled, 35 ml of the solution
   was transferred to a falcon tube, and 1.75 μl of Syngen GreenDNA Gel
   Stain (20,000x) was added.
3. 5 μl of the PCR product was mixed with 1 μl
   of a loading buffer. The resulting 5 μl samples were then loaded onto the
   gel alongside a DNA ladder (Thermo Scientific GeneRuler 100 bp, 0.1
   μg/μl).
4. Electrophoresis was carried out at 90 V for 50
   minutes in a TAE buffer.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/3-sher.png)

Figure 3: Gel electrophoresis
of the obtained SynDNA amplification products.

**Conclusions:** The expected product length was 127 bp. All tested
temperatures yielded satisfactory results, except for the reaction conducted at an
annealing temperature of 66°C. **The product obtained at 62°C was selected for
further testing**.

### Annealing of syncrRNA template and T7-polymerase primer; IVT

1. The annealing reaction was conducted according to
   the Kellner et al. [1]

Table 2: Annealing Reaction Mix
components (for a single reaction):

|  |  |
| --- | --- |
| **Component** | **Volume [µl]** |
| crRNA template, 100 µM | 1 |
| T7-3G oligonucleotide, 100 µM | 2 |
| Standard Taq buffer, 10x | 1 |
| H₂O | 7 |
| **Total** | **11** |

2. A 5-minute denaturation was conducted, after which
   the reaction was slowly cooled in a thermocycler to 4°C, at a rate of
   0.1°C/s.

### IVT reaction

IVT (*in
vitro* transcription) reaction was
performed. The reaction mix content was as follows:

Table 3: IVT Reaction Mix
components (for a single reaction):

|  |  |
| --- | --- |
| **Component** | **Volume [µl]** |
| H₂O | 15 |
| Annealing reaction mix | 10 |
| 5x Buffer (from TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific) | 4 |
| rNTP mix | 8 |
| Enzyme mix | 2 |
| **Total** | **39** |

The reaction was incubated for 4 hours at
37°C.

Note: DNA sequences of SynDNA and SynCrRNA were introduced by
Kellner et al. [1]

### SyncrRNA purification after IVT

1. 2 U of DNase I, RNase-free was added per 1 μg of
   template DNA directly to a transcription reaction mixture.
2. The reaction was incubated at 37°C
   for **15
   minutes**.
3. **Dilute the sample to 200 μl with RNase-free
   water.**
4. DNase was inactivated by phenol/chloroform
   extraction.
5. **100 μl of phenol, 100 μl of
   chloroform and 20 μl of sodium acetate** were added to the sample, then the sample was
   mixed thoroughly.
6. The mixture was centrifuged
   for **10
   minutes** (max speed,
   4°C).
7. The upper phase was taken to continue the
   experiment.
8. **100 μl of
   chloroform** were added
   and the sample was thoroughly mixed.
9. Centrifugation for**5 minutes** (max speed, 4°C)
10. The upper phase was taken to continue the
    experiment.
11. 1:1 volume of isopropanol was added to the sample,
    the sample was mixed.
12. The sample was incubated at –20°C
    for **5
    minutes**.
13. The sample was centrifuged
    for **10
    minutes** (max speed,
    4°C).
14. The pellet was washed
    with **70% EtOH
    twice** (centrifuged in
    between for 5 min, max speed, 4°C) and then left to dry.
15. **20 μl of DEPC-treated
    water** was added to
    the pellet without pipetting and the sample was left to incubate on ice
    for **5
    minutes**.
16. The pellet was then resuspended and the
    concentration of RNA was measured. The RNA purification protocol post IVT
    can be accessed [here](https://static.igem.wiki/teams/5087/pics/protocols/rna-purification.pdf).

**c****syncrRNA****= 4530.9 ng/****ul**

### 15.04. - 21.04

### Buffer preparation and RNA electrophoresis

### Buffer preparation

1. A solution of**TBE Buffer
   (100ml)**was
   prepared:

* 10.8 g of Tris base
* 5.5 g of boric acid
* 4 ml EDTA (0.5M)
* water (enough to fill the mixture to obtain 100ml
  of solution).

2. A 50 ml solution of 10M urea buffer was prepared by
   dissolving 30 g of urea on a heat plate. The urea was first dissolved in a
   small volume (~10 ml) of deionized water, and then the solution was brought
   up to a final volume of 50 ml.

**Both buffers were filtered using a 0.45 nm
filter.**

### RNA electrophoresis

1. 20 ml of 5% polyacrylamide gel was prepared,
   according to the following instructions:

Table 4: Polyacrylamide gel
components:

|  |  |
| --- | --- |
| **Component** | **Volume [ml]** |
| 40 % acrylamide (Acrylamide/Bis-Acrylamide 19:1) | 2.5 |
| 10M Urea Buffer | 14 |
| 10 x TBE | 2 |
| 10% APS | 0.2 |
| H₂O | 1.3 |

2. The solution was lightly mixed, and 10 μl of
   TEMED was added; the solution was once again lightly mixed.
3. The gel was poured carefully and left to polymerize
   for 30 minutes.

**Sample preparation and
electrophoresis**

1. A dilution of the original syncrRNA solution was
   performed:

* 1 μl – RNA (c = 4530.9 ng/μl)
* 9 μl – DEPC H2O

2. The sample and marker mixes were prepared:

**SAMPLE**

* 1.1 μl – syncrRNA diluted solution (c =
  453.09 ng/μl)
* 4.9 μl – DEPC H2O
* 6 μl – RNA LB Buffer

**LADDER**

* 3 μl – RiboRuler High Range RNA Ladder (from
  TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific, lot.
  00150262)
* 3 μl – DEPC H2O
* 6 μl – RNA LB Buffer

3. RNA samples were heated at 70°C for 10 minutes, then
immediately chilled on ice.

4. 12 μl of each of the samples were loaded onto the
gel, alongside wells filled with LB Buffer, to maintain a straight line of sample
migration.

5. Electrophoresis was conducted for 45 minutes under 100
V.

**Staining the gel**

1. After the electrophoresis was completed, the gel
   was soaked for 15 minutes in 1x TBE buffer solution to remove the remaining
   urea.
2. The gel was stained in ethidium bromide solution (c
   = 0.5 μg/ml) for 15 minutes. The staining solution was prepared, by
   adding 3.5 μl of ethidium bromide (c = 10 mg/ml) to 70 ml of 1x TBE
   buffer.

![](https://static.igem.wiki/teams/5087/part-registry-images/syncrrnagel.webp)

Figure 4: Gel electrophoresis
of the obtained IVT reaction product

**Conclusions**: A clear and distinct RNA band was observed, confirming
that the**IVT reaction was
successful**. Although the RNA
ladder did not develop correctly, this does not impact the interpretation of the
results.

### Reagent preparation for the SHERLOCK assay

1. A 50 ml solution of **HEPES pH = 6.8** buffer was prepared, by dissolving 11.92 g of
   powdered HEPES substrate (Sigma Aldrich) in a sufficient amount of DEPC
   H2O. The pH was adjusted to 6.8 using a pH meter. The
   buffer was then sterile-filtered using a 0.22 μm filter under a laminar
   hood.
2. A 1M solution of **MgCl****2** was prepared by diluting a 3M stock with
   DEPC-treated water to a final volume of 500 μl.

**Cas13a dillutions**

Stocks of Cas13a of concentrations:

* Cas13a treated with SUMO protease (later
  called **Cas13aT**) = **0.87 mg/ml**
* Cas13a without SUMO protease treatment (later
  called **Cas13aNT**) = **0.73 mg/ml**

1. Dilutions were prepared with the SB buffer with
   freshly added DTT (2 μl 1M DTT and 998 μl SB) to
   obtain **63.3
   μl/ml**concentrations of the protein specified in Kellner
   et al.

* Cas13aT: 3.64 μl protein + 46.36 μl SB
  DTT
* Cas13aNT: 4.34 μl protein + 45.66 μl SB
  DTT

### SHERLOCK assay

**Reagent preparation**

1. A dilution of the syncrRNA was prepared, by
   combining the following, to obtain a concentration
   of **10
   ng/μl:**

* 1.1 μl of 453.1 ng/μl solution of
  syncrRNA
* 48.9 μl of ultra-pure H2O

2. Two aliquots of dry RNaseAlert were resuspended in
   25 μl 10X RNaseAlert® Lab Test Buffer, to a concentration
   of**2
   μM.**

**Assembling the assay**

The reactions were performed under 6 different
conditions:

1. Cas13aT (complete)
2. Cas13aNT (complete)
3. Using water instead of protein (negative
   control)
4. RNase A instead of protein (positive
   control)
5. Cas13aT without target and syncrRNA
6. Cas13aNT without target and syncrRNA

Table 5: The composition of
SHERLOCK reaction mixes:

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| **Component (volume in μl)** | **Sample number** | | | | | |
| **1** | **2** | **3** | **4** | **5** | **6** |
| UltraPure Water | 56.35 | 56.35 | 66.35 | 68.85 | 63.85 | 63.85 |
| HEPES, pH 6.8, 1M | 2 | 2 | 2 | 2 | 2 | 2 |
| MgCl2 | 0.9 | 0.9 | 0.9 | 0.9 | 0.9 | 0.9 |
| rNTP mix, 25mM each | 4 | 4 | 4 | 4 | 4 | 4 |
| Cas13a | 10 | 10 | - | - | 10 | 10 |
| Murine RNase inhibitor 40 U/μl | 5 | 5 | 5 | - | 5 | 5 |
| T7 RNA polymerase | 2.5 | 2.5 | 2.5 | - | - | - |
| crRNA (10 ng/μl) | 5 | 5 | 5 | - | - | - |
| RNase Alert | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 |

1. After assembling the reactions, the tubes were
   briefly vortexed and spinned down.
2. **4.6 μl of the PCR target
   DNA** product was added
   to tubes numbered **1, 2, 3,
   4**
3. **10 μl of RNaseA** from the RNase Alert kit was added to the tube
   number **4**.
4. The tubes were briefly vortexed and spun
   down.
5. 20 μl of the reaction mix from each of the tubes
   was transferred into a white 384-well plate. Each reaction was carried out
   with 4 technical replicates.
6. The plate was spun down and promptly put into a
   preheated fluorescence plate reader. The conditions were: 37°C, 490 nm
   excitation, 520 nm emission. Only the first, final read-out was
   captured.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/5-sher.png)

Figure 5: Bar plot illustrating
the final fluorescence intensity of the samples; numbers of the sample correspond to
the classification implemented in the table above

**Conclusions:** There was no significant difference in activity
between LwCas13a treated with SUMO protease and untreated LwCas13a. This suggests
that the **6xHis-TwinStrep-SUMO tag
does not** interfere with crRNA
and target RNA binding, and does not **affect the protein’s activity in the
assay**. Therefore, the tagged
protein can be effectively used in the assay, which helps reduce both purification
costs and labour.

### 20.05. - 26.05

### RPA of P. parvum DNA

An RPA reaction was performed using algal DNA
(the**K3
sample**) as the template, along
with a positive control. The reaction mixes were prepared as follows:

**Positive control:**

* 8 μl – positive control primer mix
* 29.5 μl – Primer Free Rehydration
  buffer
* 9 μl – water

**Mix for*****P. parvum*****:**

* 2.4 μl – primer mix (10 μM each;
  GalF & GalR)
* 29.5 μl – Primer Free Rehydration
  buffer
* 11.05 μl – water

1. 40 μl of reaction mixes were then transferred to
   Twist Amp enzyme mix pellets and pipetted thoroughly until the pellets
   dissolved completely.
2. 10 μl aliquots of the reaction mix were then
   distributed to PCR tubes and 1 μl of template DNA was added to each of
   the tubes. A negative control with water instead of the template solution
   was created.
3. 0.6 μl of MgAc was added to each of the
   tubes.
4. The tubes were placed into a thermocycler at
   37°C. To find the most efficient reaction run time, the following
   samples were prepared:

Table 7: Run times for RPA
rections

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | P. parvum | | | Positive control | | Negative control |
| Reaction run time [min] | 10 | 20 | 30 | 30 | 30 | 30 |

5. After 4 minutes, the samples were taken out of the
   thermocycler, vortexed, spinned down briefly and put back into the
   thermocycler, to increase the effectiveness of the RPA enzymes and help with
   the amplification of more complicated DNA regions.
6. After the incubation time was finished for each of
   the samples, the tubes were placed in –20°C to stop the
   reaction.

### Nonpurified amplification product electrophoresis

To verify if the correct product was obtained during
amplification, agarose gel electrophoresis was conducted. According to the available
literature, RPA amplification products typically need to be purified before running
on an agarose gel. To test this theory, a small amount of the unpurified product was
directly loaded onto the gel without any purification.

1. The gel was prepared according to the instructions
   from 8/04/2024
2. The electrophoresis was conducted under 90 V for 40
   minutes in TAE buffer.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/8-sher.png)

Figure 8: Gel electrophoresis
of the non-purified RPA amplification products; 10, 20, 30 –*P. parvum* products, (+) – positive control, (-) –
negative control

**Conclusions:** As anticipated, the electrophoresis results were
inconclusive, confirming that purification is a necessary step before analyzing RPA
amplification products.

### RPA product purification

1. Both test samples and positive control samples were
   pooled together, thus creating 15 μl and 10 μl of reaction mixes to be
   purified.
2. Samples were then purified using Syngen PCR/Gel ME
   Mini Kit (50), according to the protocol: “*DNA gel extraction protocol using the Syngen
   Gel/PCR ME Mini Kit*”

**Purified product electrophoresis**

1. The gel was prepared according to the instructions
   from 8/04/2024
2. The electrophoresis was conducted under 90 V for 40
   minutes in TAE buffer.

**![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/9-sher.png)**

Figure 9:  Gel
electrophoresis of the purified RPA amplification products

**Conclusions**: A product of the correct length (140 bp) was obtained for
the positive control. **However, no
product was detected for the test sample.**

### Annealing of PrymcrRNA template and T7-polymerase primer

**Two crRNA templates were
designed**, each targeting a
specific region of the *Prymnesium
parvum* genome. The sequence for
this region was obtained by sequencing the PCR product and amplifying it using the
Galuzzi\_FOR and Galuzzi\_REV primers.

Two DNA templates for crRNA were purchased. They will be
referred to as PrymcrRNA1 and PrymcrRNA2 from now on. These PrymCrRNAs are depostied
in the iGEM Registry under IDs: BBa\_K5087022 (PrymCrRNA1) and BBa\_K5087023
(PrymCrRNA2).

1. Both templates were diluted according to the
   instructions provided by the manufacturer, to obtain a concentration of 100
   μM.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/6-sher-1.png)

Figure 6: Manual detailing the
parameters of the PrymcrRNA1 and PrymcrRNA2

2. The annealing reaction for both samples was carried
   out as described on 10/04/2024.

### IVT reaction for PrymcrRNA1 and PrymcrRNA2

1. IVT reaction was performed for both of the samples.
   The reaction mix content was as follows:

Table 6: IVT Reaction Mix
components (for a single reaction):

|  |  |
| --- | --- |
| **Component** | **Volume [µl]** |
| H₂O | 13 |
| Annealing reaction mix | 10 |
| 5x Buffer (from TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific) | 4 |
| rNTP mix | 10 |
| Enzyme mix | 2 |
| **Total** | **39** |

The reaction ran for 4 hours at 37°C. The samples were
kept at -21°C until the next day.

### PrymcrRNA purification

The purification process was carried out as described on
12/04/2024.

Note: We encountered difficulties during the
phenol/chloroform extraction process described in subsection 3 of the protocol. For
PrymcrRNA1, the upper layer was challenging to retrieve, leading to a smaller volume
of the aqueous phase being collected. As a result, the concentration of the obtained
PrymcrRNA1 was lower than expected.

**PrymcrRNA1.1 = 431.3 ng/ul** (A260/A280=2.18 ; A260/A230 = 2.52)

**PrymcrRNA1.2 = 191.5 ng/ul** (A260/A280=2.14 ; A260/A230 = 2.51)

**PrymcrRNA2 = 7292.7 ng/ul** (A260/A280=2.08 ; A260/A230 = 2.41)

*For PrymcrRNA1, the sample was split into two Eppendorf
tubes during purification. Due to the difficulties mentioned earlier, the
purification method varied between the two samples, resulting in different
concentrations.*

### RNA Electrophoresis

The gel was prepared as described on 15/04/2024.

1. Samples were prepared accordingly:

**PrymcrRNA1.1**

* 1 μl – RNA
* 9 μl – DEPC H2O
* 10 μl – RNA LB Buffer

**PrymcrRNA1.2**

* 3 μl – RNA
* 7 μl – DEPC H2O
* 10 μl – RNA LB Buffer

**PrymcrRNA2 –** first, a dilution was prepared, by combining 1 μl
of the original sample with 14 μl of H2O, resulting in a concentration = **431 ng/μl**

* 1 μl – RNA
* 9 μl – DEPC H2O
* 10 μl – RNA LB Buffer

2. RNA samples were heated at 70°C for 10 minutes,
   and then immediately chilled on ice.
3. 20 μl of each of the samples was loaded onto the
   gel proceeded by 6 μl of RiboRuler High Range RNA Ladder (from
   TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific).
4. The electrophoresis ran under 100 V for 45 minutes
   in 1x TBE buffer.

**Staining the gel**

After the electrophoresis was completed, the gel was soaked
for 15 minutes in 1x TBE buffer solution to remove the remaining urea.

The gel was stained in ethidium bromide solution (c = 0.5
μg/ml) for 15 minutes. The staining solution was prepared, by adding 3.5 μl of
ethidium bromide (c = 10 mg/ml) to 70 ml of 1x TBE buffer.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/7-sher.png)

Figure 7:  Gel
electrophoresis of the obtained IVT reaction product; 1 – RNA ladder, 2,3
– PrymcrRNA1, 4 – PrymcrRNA2

**Conclusions:** The **product obtained appears to be the correct
length**when compared to
previous electrophoresis results. The additional bands observed may be due to
contamination or product degradation. Despite this, the samples were considered pure
enough to proceed with further experiments.

### 03.06. - 09.06

### Prymnesium genome and PCR product RPA

After initially failing to obtain the correct
post-amplification bands on the gel, we requested the AlgaLab team to isolate DNA
from a fresh culture. They confirmed the**presence of the Prymnesium
genome** in the sample
using **PCR according to the method
outlined by Galluzzi et al. [1].**

1. 8 RPA reactions were put together, using the
   following templates:

* (-) control: water (no genome)
* NOW5 genome *(the culture's origin is described in the
  AlgaLab's documentation)*
* DNA P genome *(the culture's origin is described in the
  AlgaLab's documentation)*
* NOW5 genome duplicate
* DNA P genome duplicate
* DNA P genome post PCR as in Galluzzi et.al
* NOW5 genome post PCR as in Galluzzi et.al

2. RPA reactions were assembled according to the
   protocol provided by Kellner et. al. [2] First, two master mixes were assembled
   on ice:

* Forward primer
  (GalF, **10
  µM**) – 2.4
  µl
* Reverse primer (Galuzzi
  REV, **10
  µM**) – 2.4
  µl
* TwistAmp Rehydration Buffer (from the TwistAmp
  Basic kit) – 29.5 µl
* H2O – 8.65 µl

3. 40 µl of master mix were added to a single
   enzyme pellet aliquot and the pellet was carefully resuspended
4. 2.5 µl of 280 mM TwistAmp magnesium acetate
   was added to each of the tubes.
5. 10 µl aliquots of the reaction mix were
   distributed into 7 PCR tubes. 1 µl of template DNA was added to 6 of
   them. A water-only negative control was included.
6. Reactions run for **30 minutes** in a thermocycler preheated to 37°C. After
   the reaction was finished, samples were stored in
   –20°C.

The samples were then divided:

* 1, 2, and 3 were purified as a whole
* 6,7 were divided into two, one half was
  purified.
* 4,5 and the other halves of 6,7 were
  stored.

The purification protocol was as described in
“*DNA gel extraction protocol using
the Syngen Gel/PCR ME Mini Kit*”

**Electrophoresis**

1. The gel was prepared according to the instructions
   from 8/04/2024
2. The electrophoresis was conducted under 90 V for 40
   minutes in the TAE buffer.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/10-sher.png)

Figure 10: Gel electrophoresis
of the purified RPA amplification products

When the photo was overexposed, the correct bands appeared
in wells containing samples 6 and 7. A faint shadow of the product was also visible
in well 2, though it was unclear.

In the future, running electrophoresis after amplification
should not be necessary, as the SHERLOCK method can detect much smaller amounts of
DNA than what can be identified using standard agarose gel electrophoresis.

### SHERLOCK on P. parvum – PrymcrRNA1&2 testing

5/06/2024

We decided to proceed with the SHERLOCK reaction despite
not observing any bands on the gel. According to Kellner et al. [1], gel visualization
isn’t typically required after the RPA reaction. While we checked the gel for
confirmation, we ultimately concluded it wasn't a necessary step to move
forward.

In this SHERLOCK test, we employed two types of
amplification: **first PCR, followed
by RPA**. Our ultimate goal is to
rely **solely on
RPA**, but for this experiment, we
aimed to maximize amplification to ensure we had sufficient DNA to validate our
PrymcrRNAs design.

1. The following reaction combinations have been
   prepared in duplicates:

**DNA P genome post PCR concentration: 280
ng/µl**

Table 8: SHERLOCK reactions
components

|  | **Template DNA used for RPA** | **crRNA** |
| --- | --- | --- |
| 1 | DNA P post PCR (GalF & GalR) | PrymcrRNA1 |
| 2 | DNA P post PCR (GalF & GalR) | PrymcrRNA2 |
| 3 | DNA P post PCR (GalF & GalR) | syncrRNA |
| 4 | SynDNA1 post PCR | PrymcrRNA1 |
| 5 | SynDNA1 post PCR | PrymcrRNA2 |
| 6 | SynDNA1 post PCR | syncrRNA |
| 7 | Positive control (RNase) | - |

The 7th reaction was a positive control with RnaseA

2. The volumes of each reagent used for the reaction
   are listed below:

Table 9: SHERLOCK samples
content

|  |  |  |
| --- | --- | --- |
| **Component** | **Volume for regular reactions [µl]** | **Volume for positive control [µl]** |
| UltraPure Water | 28.18 | 36.43 |
| HEPES, pH 6.8, 1M | 1 | 1 |
| MgCl2 | 0.45 | 0.45 |
| rNTP mix, 25mM each | 2 | - |
| Cas13a | 5 | - |
| Murine RNase inhibitor 40 U/μl | 2.5 | - |
| T7 RNA polymerase | 1.25 | - |
| crRNA (10 ng/μl) (interchangable) | 2.5 | - |
| RNase Alert | 3.13 | 3.13 |

3. 2,3 μl of the template (interchangeable, as
   described above) were added to each reaction mix.
4. 20 μl of the reaction mix from each tube was
   transferred into a white 384-well plate. Each reaction was carried out with
   2 technical replicates.
5. The plate was spun down and promptly put into a
   pre-heated fluorescence plate reader. The conditions were: 37°C, 490 nm
   excitation, 520 nm emission.

Note: To determine if the result of the assay is positive
or negative, we compare the fluorescence intensity of the negative control to the
fluorescence intensity of the tested sample. If the fluorescence intensity of the
tested sample exceeds that of the negative control, we conclude that the assay
result is positive.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-1.webp)

**Conclusions**: **Both of
the PrymcrRNAs designs proved to be successful in algal DNA
detection.** Further testing is
required to gather more proof and learn more about both of the PrymcrRNAs ability to
bind to Cas13a protein and to recognise the target region in the template
DNA.

### SHERLOCK PrymcrRNA combination testing

7/06/2024

**This assay aimed to check for the detection
of*****Prymnesium*****genomic DNA without prior PCR
amplification.**

DNA template: **DNA P: 280ng/µl**(as obtained on 3.06. sample number 4) and synDNA: 8.97
ng/µl used for the reaction.

**Note:** The exact proportion of the 280 ng/µl genomic
DNA that belongs to *Prymnesium
parvum* is uncertain due to the
impurity of the culture. It is likely that the *Prymnesium parvum* DNA constitutes only a small fraction of the total DNA
concentration in the sample.

Table 10: SHERLOCK reactions
components

|  | **Template DNA used for RPA** | **crRNA** |
| --- | --- | --- |
| 1 | DNA P genome (GalF & GalR) | PrymcrRNA1 |
| 2 | DNA P genome (GalF & GalR) | PrymcrRNA2 |
| 3 | DNA P genome (GalF & GalR) | PrymcrRNA1 + PrymcrRNA2 |
| 4 | SynDNA | PrymcrRNA1 + PrymcrRNA2 |
| 5 | SynDNA | syncrRNA |
| 6 | Positive control (RNase) | - |

The volumes of reagents used for each of the reactions were
as the one specified on 5/06/2024.

All other procedures regarding the assay were carried out
in the exact same manner.

![Sherlock 2 07.06.2024 without PCR](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-2.webp)

**Conclusions:**

***Prymnesium
parvum*****genomic
DNA was detected**, but the overall
fluorescence intensity of the samples was
lower than in the previous assay. This reduced intensity may be due to the DNA
concentration being too low or potential issues with the RPA primers.

### 10.06. - 16.06

### SHERLOCK — limit of detection (Post PCR + RPA template)

12/06/2024

Since the SHERLOCK assay did not perform efficiently with
the genomic DNA, **PCR amplification
was employed to further test the PrymcrRNA molecules, determine the limit of
detection, and obtain additional template material.**

1. DNA template (**DNA PCR P+Maj2**) dilutions used for the reactions:

* 19.5 ng/µl
* 1.95 ng/µl
* 0.195 ng/µl
* 0.0195 ng/µl
* 0.00195 ng/µl.

2. The following reactions were
   assembled:

Table 11: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1.1 | DNA PCR P+Maj2 (19.5 ng/µl; GalF & GalR) | PrymcrRNA1 |
| 2.1 | DNA PCR P+Maj2 (1.95 ng/µl; GalF & GalR) |
| 3.1 | DNA PCR P+Maj2 (0.195 ng/µl; GalF & GalR) |
| 4.1 | DNA PCR P+Maj2 (0.0195 ng/µl; GalF & GalR) |
| 5.1 | DNA PCR P+Maj2 (0.00195 ng/µl; GalF & GalR) |
| (-).1 | Negative control (Water in RPA instead of the template) |
| 1.2 | DNA PCR P+Maj2 (19.5 ng/µl; GalF & GalR) | PrymcrRNA2 |
| 2.2 | DNA PCR P+Maj2 (1.95 ng/µl; GalF & GalR) |
| 3.2 | DNA PCR P+Maj2 (0.195 ng/µl; GalF & GalR) |
| 4.2 | DNA PCR P+Maj2 (0.0195 ng/µl; GalF & GalR) |
| 5.2 | DNA PCR P+Maj2 (0.00195 ng/µl; GalF & GalR) |
| (-).2 | Negative control (Water in RPA instead of the template) |
| (+) | Positive control (SynDNA) | - |

3. There was a change in the RNase inhibitor used,
   thus its volume was adjusted to retain the same
   activity. **From this
   moment onward, the same volumes of all the reagents were used in all
   of the assays– see [SHERLOCK–
   protocol
   2](https://static.igem.wiki/teams/5087/pics/protocols/sherlock02.pdf)**.

Table 12: SHERLOCK samples
content

|  |  |  |
| --- | --- | --- |
| **Component** | **Volume for regular reactions (µl)** | **Volume for positive control (µl)** |
| UltraPure Water | 28.68 | 36.43 |
| HEPES, pH 6.8, 1M | 1 | 1 |
| MgCl2 | 0.45 | 0.45 |
| rNTP mix, 25mM each | 2 | - |
| Cas13a | 5 | - |
| EURX Ribonuclease Inhibitor 50U/ul | 2 | - |
| T7 RNA polymerase | 1.25 | - |
| crRNA (10 ng/μl) (interchangable) | 2.5 | - |
| RNase Alert | 3.13 | 3.13 |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-3-crrna1.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-3-crrna2.webp)

**Conclusions**:
The high signal from the negative control samples for
both PrymcrRNA1 and PrymcrRNA2 indicated possible contamination - no results
regarding the LOD can be drawn. This result necessitated further testing.

### 17.06. - 23.06

### Repetition of the previous SHERLOCK assay

21/06/2024

Goal: Determine the limit of detection (LOD) for the
detection of PCR products.

1. The same post-PCR DNA dilutions were used for the
   RPA reactions.
2. Compared to the previous assay, there was an
   additional negative control for synDNA included (an RPA reaction with water
   instead of template DNA).

Table 13: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1.1 | DNA PCR P+Maj2 (19.5 ng/µl; GalF & GalR) | PrymcrRNA1 |
| 2.1 | DNA PCR P+Maj2 (1.95 ng/µl; GalF & GalR) |
| 3.1 | DNA PCR P+Maj2 (0.195 ng/µl; GalF & GalR) |
| 4.1 | DNA PCR P+Maj2 (0.0195 ng/µl; GalF & GalR) |
| 5.1 | DNA PCR P+Maj2 (0.00195 ng/µl; GalF & GalR) |
| (-).1 | Negative control (water in RPA mix instead of template) |
| 1.2 | DNA PCR P+Maj2 (19.5 ng/µl; GalF & GalR) | PrymcrRNA2 |
| 2.2 | DNA PCR P+Maj2 (1.95 ng/µl; GalF & GalR) |
| 3.2 | DNA PCR P+Maj2 (0.195 ng/µl; GalF & GalR) |
| 4.2 | DNA PCR P+Maj2 (0.0195 ng/µl; GalF & GalR) |
| 5.2 | DNA PCR P+Maj2 (0.00195 ng/µl; GalF & GalR) |
| (-).2 | Negative control (water in RPA mix instead of template) |
| (-).1/2 | Negative control (water in RPA mix instead of template) | PrymcrRNA1 + PrymcrRNA2 |
| (+) | Positive control (SynDNA) | - |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-4-crrna1.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-4-crrna2.webp)

**Conclusions:**

Unfortunately, the results
indicated **contamination** in two of the three negative control samples: (-).1
and (-).2. No signal was observed in the wells containing the (-).1/2 sample. It was
concluded that the contamination likely resulted from a faulty RPA reaction, most
probably due to contaminated primer solutions.

### 24.06. - 30.06

### SHERLOCK on Prymnesium (post PCR + genomic)

24/06/2024

**Goal:** assessing preliminary limit of detection

1. RPA reactions were conducted according to the
   Kellner et al protocol. The following target DNA dilutions were used:

* post PCR– **200 nM**(that is equal to the previously used 19.5
  ng/µl concentration)**, 200
  pM, 200 fM, 200 aM**
* genomic sample (Maj1)

2. The SHERLOCK assay was assembled:

Table 14: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1.1 | DNA PCR P+Maj2 (**200 nM**; GalF & GalR) | PrymcrRNA1 |
| 2.1 | DNA PCR P+Maj2 (**200 pM**; GalF & GalR) |
| 3.1 | DNA PCR P+Maj2 (**200 fM**; GalF & GalR) |
| 4.1 | DNA PCR P+Maj2 (**200 aM**; GalF & GalR) |
| 5.1 | P. parvum genome isolate (GalF & GalR) |
| (-).1 | Negative control (water in RPA mix instead of template DNA) |
| 1.2 | DNA PCR P+Maj2 (**200 nM**; GalF & GalR) | PrymcrRNA2 |
| 2.2 | DNA PCR P+Maj2 (**200 pM**; GalF & GalR) |
| 3.2 | DNA PCR P+Maj2 (**200 fM**; GalF & GalR) |
| 4.2 | DNA PCR P+Maj2 (**200 aM**; GalF & GalR) |
| 5.2 | P. parvum genome isolate (GalF & GalR) |
| (-).2 | Negative control (water in RPA mix instead of template DNA) |
| (-).1/2 | Negative control (water in RPA mix instead of template DNA) | PrymcrRNA1 + PrymcrRNA2 |
| (+) | Positive control | - |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-5-crrna1.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-5-crrna2.webp)

**Conclusions:**

The high signal from the negative control makes it
impossible to draw any definitive conclusions from the test. Several potential
explanations for this result include:

1. **Contamination with
   RNases:** Although this
   is unlikely since the negative control for PrymcrRNA2 was indeed
   negative.
2. **PrymcrRNA1 Binding
   Issue:** There may be a
   problem with PrymcrRNA1, causing it to activate huLwCas13a even in the
   absence of target RNA.

### RNase contamination check

**Goal:** Our instructors advised us to check for the presence
of RNases in the reagents to determine if they might be responsible for the high
signal observed in the PrymcrRNA1 negative control from the previous test.

1. RNase alert and reagents were mixed in the
   proportions as in the SHERLOCK reaction.
2. The mixes were placed on a plate, incubated for 30
   minutes in 37 °C and put in a fluorescence reader. The results obtained are
   listed below:

Table 15: Fluorescence of
SHERLOCK reagents

|  |  |
| --- | --- |
| **Reagent** | **Fluorescence [a.u.]** |
| Water | 1598 |
| HEPES | 1492 |
| MgCl2 | 3069 |
| rNTP mix | 1967 |
| huLwCas13a | 2204 |
| RNase inhibitor | 2701 |
| T7 RNA polymerase | 4407 |
| PrymcrRNA1 | 1737 |
| PrymcrRNA2 | 2353 |
| RPA buffer | 1847 |
| MgAc | 1983 |
| DTT | 3855 |

**Conclusions:**

The highest fluorescence signals were observed for T7
polymerase, DTT, and MgCl2. However, **none of these signals matched the high levels seen in
the negative control** for
PrymcrRNA1 from Monday (24.06). Therefore, we doubt that RNase contamination is the
cause of the elevated signal.

### SHERLOCK repetition

26/06/2024

1. The samples were prepared by using the template DNA
   obtained from the RPA reactions carried out on 24/06/2024.
2. A new 1M MgCl2 solution was prepared to be sure that no RNase
   contamination was present.

**Important**! A new 384-well plate was used to eliminate potential
contamination that might have occurred from reusing the same plate multiple
times.

3. The SHERLOCK assay was assembled in the exact same
   manner as on 24/06/2024.

**The assay was inconclusive and faulty due to
improper calibration of the fluorescence reader settings for the new
plate.**

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-6-crrna1.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-6-crrna2.webp)

**Conclusions:**

No conclusions can be drawn from the results due to the low
fluorescence signal, which appears to be noise. The plate reader settings need to be
adjusted for the new plate.

### SHERLOCK assay

28/06/2024

This assay is a repetition of the SHERLOCK test conducted
on 24/06/2024. A new negative control (-).1/2.prym was included: RPA was performed on
synthetic
target RNA using both synthetic primers and primers specific
for *Prymnesium*. This addition aimed to determine if
the ***Prymnesium*****RPA primers might have inadvertently acted as a
target for PrymcrRNA1**, potentially
explaining the observed signal in the PrymcrRNA1 negative control (in RPA: water +
GalF & GalR). The details of the RPA reactions are listed below:

Table 16: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1.1 | DNA PCR P+Maj2 (**200 nM**; GalF & GalR) | PrymcrRNA1 |
| 2.1 | DNA PCR P+Maj2 (**200 pM**; GalF & GalR) |
| 3.1 | DNA PCR P+Maj2 (**200 fM**; GalF & GalR) |
| 4.1 | DNA PCR P+Maj2 (**200 aM**; GalF & GalR) |
| 5.1 | P. parvum genome isolate (GalF & GalR) |
| (-).1 | Negative control (water in RPA mix instead of template DNA) |
| 1.2 | DNA PCR P+Maj2 (**200 nM**; GalF & GalR) | PrymcrRNA2 |
| 2.2 | DNA PCR P+Maj2 (**200 pM**; GalF & GalR) |
| 3.2 | DNA PCR P+Maj2 (**200 fM**; GalF & GalR) |
| 4.2 | DNA PCR P+Maj2 (**200 aM**; GalF & GalR) |
| 5.2 | P. parvum genome isolate (GalF & GalR) |
| (-).2 | Negative control (water in RPA mix instead of template DNA) |
| (-).1/2 | Negative control (water in RPA mix instead of template DNA) | PrymcrRNA1 + PrymcrRNA2 |
| (-).1/2.prym | Negative control (GalF & GalR + synDNA primers) | PrymcrRNA1 + PrymcrRNA2 |
| (+) | Positive control | - |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-7-crrna1.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-7-crrna2.webp)

No signal was observed in either the negative control or
the test samples. Something **inexplicable is occurring with all samples containing
PrymcrRNA1, so we have decided to remove PrymcrRNA1 from further tests for
now.**

The negative control showed a low fluorescence signal. The
current limit of detection for GalF-GalR-PrymCrRNA2 is 200 pM.

### 08.07. - 14.07

### RPA & SHERLOCK with new RPA primers (Mod and Alt)

1. RPA was performed only on the genomic DNA sample
   (**MAJ1; 39.3
   ng/µl**) to determine
   if new primers would enable amplification of the target sequence to a
   detectable level.
2. The SHERLOCK assay was conducted on the following
   samples:

Table 17: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1 | Positive control (SynDNA) | syncrRNA |
| 2 | Negative control (for SynDNA primers– water instead of template DNA) | syncrRNA |
| 3 | MAJ1 (**AltF & AltR**) | PrymcrRNA2 |
| 4 | Negative control (**AltF & AltR**) | PrymcrRNA2 |
| 5 | MAJ1 (**ModF & ModF**) | PrymcrRNA2 |
| 6 | Negative control (**ModF & ModF**) | PrymcrRNA2 |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-8.webp)

The **Alt** primers seem to be
**effective** in detecting the genomic DNA. In case of **Mod**
primers, the negative control emitted strong fluorescence singal but **no
signal** was observed in the sample containing genomic DNA. Also no signal
was observed in some previous experiments when using **Gal** primers. We
have two hypotheses to explain why this is possible:

1. **Low Genomic DNA
   Concentration:** The
   genomic DNA concentration might be below the limit of detection for our
   test.
2. **Tightly Packed DNA:** The DNA in the region targeted by the primers
   could be more tightly packed. This is unlikely, as ribosomal genes are
   housekeeping genes and the chromatin should remain active at all times. We
   will nevertheless test if thermal denaturation can help generate any
   signal.

### SHERLOCK repetition - Galuzzi RPA primers

9/07/2024

1. 3 post-PCR DNA samples were cleaned using Monarch
   PCR&DNA CleanUp Kit (NEB). The obtained concentrations for the sample
   were as follows:

* **Maj2– 25.9
  ng/µl;** A260/A280=1.66
* **Now5.1– 15.5
  ng/µl**; 1.89
* **Maj1 – 35.4
  ng/µl**; 1.78

2. The sample “**Maj1**” was used for further testing. Dilutions of
   the sample were created: to obtain the following
   concentrations: **200 nM,
   200 pM, 200 fM, 200 aM**.
3. Genomic DNA sample (**Maj1, 24.1 ng/μl**) was thermally denatured for 10 minutes at
   100°C, to check the hypothesis about **faulty genomic DNA detection due to tightly
   packed chromatin**.
4. The SHERLOCK assay was conducted on the following
   samples:

Table 18: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1 | DNA PCR Maj1 (**200 nM**; GalF & GalR) | PrymcrRNA2 |
| 2 | DNA PCR Maj1 (**200 pM**; GalF & GalR) |
| 3 | DNA PCR Maj1 (**200 fM**; GalF & GalR) |
| 4 | DNA PCR Maj1 (**200 aM**; GalF & GalR) |
| 5 | P. parvum genome isolate after thermal denaturation (GalF & GalR) |
| 6 | Negative control (negative control from the PCR reaction used instead of template in RPA; we wanted to check whether the PCR mix itself does not cause the SHERLOCK reaction to be positive, even without the target RNA) |
| 7 | Negative control (water in RPA mix instead of template DNA) |
| 8 | Positive control (SynDNA) | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-9-fix.webp)

**Conclusions:**

Galuzzi primers proved to be ineffective – a very
weak signal was observed in the 200 nM sample, with no signal in the fM, pM and aM
samples. We suspect that the **PCR
product may be incorrect or non-specific, resulting in undetectability by Cas13a
protein (in this test we used the Maj1 template, while on 24 and 28.06 Maj2
template)**.

PCR on the target DNA, which proved to be successfully
detected previously was carried out to obtain more template for the test
repetition.

The lack of signal in the genomic sample
is **not due to tightly condensed
chromatin** (as there was no
signal in sample 5). It is likely that the **concentration of the genomic DNA is below the limit of
detection**. This conclusion is
supported by the following calculations:

***Prymnesium
parvum*****genome
length**

Based on this article: <https://orbit.dtu.dk/en/publications/long-read-genome-sequencing-provides-novel-insights-into-the-harm>, the *P.
parvum* genome is somewhere
between 97.56 and 107.32 Mb. For the clarity sake, the average length used for the
calculations was 100 Mb.

The average weight
of a single DNA bp is 650 daltons. So the molecular weight of the
whole *P.
parvum* genome
is:

100 × 106 × 650 Da = **6.5 × 10****10****Da**

Based on that, we calculate the molar concentration:

(24.1 ng/μl / 6,5 ×
1010Da ) × 106= 0,000370769 nM = **370.8 fM**

However, taking into account the analysis conducted on
06.08 by Filip in the GenomeLab we know that on average 12 copies of ITS are present
in the algal genome (25% of cells have 8 ITS copies, 50% have 12 copies, 25% have 16
copies)

Therefore, we multiply the concentration above by
12:

370.8 fM × 12 = 4449.6 fM
= 4,45 pM

Such low concentration **may be below the limit of detection for our test (most
likely it is between 200 pM and 200 fM)**. The LOD for our test needs to be set more precisely in
further tests.

### PCR product purification

The PCR for 0.5 μl of the target DNA from 24/06 and
28/06 (P+Maj2 sample) was successful, as confirmed by electrophoresis. The PCR and
electrophoresis were conducted by Dominika and Karolina at AlgaLab.

9 μl of the PCR product was purified using Monarch
PCR&DNA CleanUp Kit (NEB). The obtained concentration
was **27.4
ng/μl**.

### RPA reaction on P+Maj2 PCR sample

1. Samples were diluted to the following
   concentrations: **200 nM,
   200 pM, 20 pM, 2 pM, 200 fM, 200 aM**. The addition of two new dilutions (20 pM and 2 pM)
   was caused by the fact, that the **PrymFlow lab and our test from 28/06 indicated
   that
   the limit of detection is somewhere between 200 pM and 200
   fM.**
2. RPA reactions were conducted for 20 minutes and
   then stored at –20°C.

### SHERLOCK – repetition

11/07/2024

Repetition of the previous assay.

1. The SHERLOCK assay was conducted on the following
   samples:

Table 19: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1 | DNA PCR Maj2 (**200 nM**; GalF & GalR) | PrymcrRNA2 |
| 2 | DNA PCR Ma2 (**200 pM**; GalF & GalR) |
| 3 | DNA PCR Maj2 (**200 fM**; GalF & GalR) |
| 4 | DNA PCR Maj2 (**200 aM**; GalF & GalR) |
| 5 | Negative control (water in RPA mix instead of template DNA) |
| 6 | Positive control (SynDNA) | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-10-fix.webp)

The PCR amplified template was suitable for SHERLOCK tests. It was
again confirmed that LOD for GalF-GalR-PrymCrRNA2 is between 200 pM and 200 fM.

### SHERLOCK – limit of detection testing

11/07/2024

**We aimed to assess the limit of detection (LOD)
more precisely, which is likely between 200 pM and 200 fM.**

1. The SHERLOCK assay was conducted on the following
   samples:

Table 20: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 2 | DNA PCR Maj2 (**200 pM**; GalF & GalR) | PrymcrRNA2 |
| 3 | DNA PCR Maj2 (**20 pM**; GalF & GalR) |
| 4 | DNA PCR Maj2 (**2 pM**; GalF & GalR) |
| 5 | DNA PCR Maj2 (**200 fM**; GalF & GalR) |
| 7 | Negative control (water in RPA mix instead of template DNA) |
| 8 | Positive control (SynDNA) | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-11.webp)

**Conclusions:**

The initial template DNA concentration does not show a
linear relation with fluorescence intensity. **Optimizing primer concentration may solve the
issue.** The **detection of 200 fM** by the assay is a promising sign for the LOD
value.

From the first SHERLOCK test, it was concluded that the LOD
is between 200 pM and 200 fM. The second SHERLOCK test was performed to assess the
LOD more precisely, and it was unexpectedly able to detect a 200 fM
concentration.

### 15.07. - 21.07

### SHERLOCK on Mod and Alt primers

16/07/2024

**After the first test of Mod and Alt primers on 08.07 we
wanted to evaluate their performance more deeply. We aimed to determine if the Mod
or Alt primers
used for RPA could detect a 200 fM sample of target DNA. Both primer sets were
tested separately with PrymcrRNA1 and PrymcrRNA2.**

1. Adequate RPA reactions were assembled, using the
   KAC PCR product. The reaction ran for 20 minutes.
2. The SHERLOCK assay was conducted on the following
   samples:

Table 21: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1 | DNA PCR KAC (**530 nM**; GalF & GalR) | PrymcrRNA2 |
| 2 | DNA PCR KAC (**200 fM**; GalF & GalR) | PrymcrRNA2 |
| 3 | Negative control (water in RPA GalF & GalR) | PrymcrRNA2 |
| 4 | DNA PCR KAC (**530 nM**; AltF & AltR) | PrymcrRNA1 |
| 5 | DNA PCR KAC (**530 nM**; AltF & AltR) | PrymcrRNA2 |
| 6 | DNA PCR KAC (**200 fM**; AltF & AltR) | PrymcrRNA1 |
| 7 | DNA PCR KAC (**200 fM**; AltF & AltR) | PrymcrRNA2 |
| 8 | Negative control (water in AltF & AltR) | PrymcrRNA1 |
| 9 | Negative control (water in AltF & AltR) | PrymcrRNA2 |
| 10 | DNA PCR KAC (**530 nM**; ModF & ModF) | PrymcrRNA1 |
| 11 | DNA PCR KAC (**530 nM**; ModF & ModF) | PrymcrRNA2 |
| 12 | DNA PCR KAC (**200 fM**; ModF & ModF) | PrymcrRNA1 |
| 13 | DNA PCR KAC (**200 fM**; ModF & ModF) | PrymcrRNA2 |
| 14 | Negative control (water in ModF & ModF) | PrymcrRNA1 |
| 15 | Negative control (water in ModF & ModF) | PrymcrRNA2 |
| 16 | Positive control (SynDNA) | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-12-200fm.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-12-530nm.webp)

**Conclusions:**

The best primer-PrymcrRNA pair appears to
be**Mod-PrymcrRNA1**, as it achieved high fluorescence intensity for
both**530 nM** and **200
fM** samples. **No other combination** was effective in detecting the 200 fM target
concentration.

### SHERLOCK – repetition of the previous assay

17/07/2024

To confirm that the obtained results were not a
coincidence, the SHERLOCK assay was repeated. The procedures and descriptions from
the previous assay were applied.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-13-200fm.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-13-530nm.webp)

**Conclusions:**

Results show **consistent detection** of target DNA concentration,
with **slight variability in
fluorescence reads** possibly
due to **non-optimized RPA primer
concentration**.

### SHERLOCK – primer/PrymcrRNA mixing screening

17/07/2024

The purpose of this test was to determine
if **mixing
PrymcrRNAs**or **forward
and reverse primers** between
the sets could achieve a **lower
limit of detection**than the
previously established 200 fM (for Mod+PrymcrRNA1 and occasionally
Gal+PrymcrRNA2).

1. Adequate RPA reactions were assembled, using the
   KAC PCR product. The reaction ran for 20 minutes. Galuzzi primers were not
   used for the test with PrymcrRNA1+2 because it is known from the previous
   tests that Gal primers + PrymcrRNA1 give us false-positive
   results.
2. The SHERLOCK assay was conducted on the following
   samples:

Table 22: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1 | DNA PCR KAC (**480 nM**; ModF & ModF) | PrymcrRNA1 PrymcrRNA2 |
| 2 | DNA PCR KAC (**200 fM**; ModF & ModF) | PrymcrRNA1 PrymcrRNA2 |
| 3 | Negative control (water in RPA ModF & ModF) | PrymcrRNA1 PrymcrRNA2 |
| 4 | DNA PCR KAC (**480 nM**; AltF & AltR) | PrymcrRNA1 PrymcrRNA2 |
| 5 | DNA PCR KAC (**200 fM**; AltF & AltR) | PrymcrRNA1 PrymcrRNA2 |
| 6 | Negative control (water in AltF & AltR) | PrymcrRNA1 PrymcrRNA2 |
| 7 | DNA PCR KAC (**480 nM**; ModF & GalR) | PrymcrRNA1 |
| 8 | DNA PCR KAC (**200 fM**; ModF & GalR) | PrymcrRNA1 |
| 9 | Negative control (water in ModF & GalR) | PrymcrRNA1 |
| 10 | DNA PCR KAC (**480 nM**; ModF & GalR) | PrymcrRNA2 |
| 11 | DNA PCR KAC (**200 fM**; ModF & GalR) | PrymcrRNA2 |
| 12 | Negative control (water in ModF & GalR) | PrymcrRNA2 |
| 13 | DNA PCR KAC (**480 nM**; ModF & AltR) | PrymcrRNA1 |
| 14 | DNA PCR KAC (**200 fM**; ModF & AltR) | PrymcrRNA1 |
| 15 | Negative control (water in ModF & AltR) | PrymcrRNA1 |
| 16 | DNA PCR KAC (**480 nM**; ModF & AltR) | PrymcrRNA2 |
| 17 | DNA PCR KAC (**200 fM**; ModF & AltR) | PrymcrRNA2 |
| 18 | Negative control (water in ModF & AltR) | PrymcrRNA2 |
| 19 | DNA PCR KAC (**480 nM**; AltF & ModF) | PrymcrRNA1 |
| 20 | DNA PCR KAC (**200 fM**; AltF & ModF) | PrymcrRNA1 |
| 21 | Negative control (water in AltF & ModF) | PrymcrRNA1 |
| 22 | DNA PCR KAC (**480 nM**; AltF & ModF) | PrymcrRNA2 |
| 23 | DNA PCR KAC (**200 fM**; AltF & ModF) | PrymcrRNA2 |
| 24 | Negative control (water in AltF & ModF) | PrymcrRNA2 |
| 25 | DNA PCR KAC (**480 nM**; AltF & GalR) | PrymcrRNA1 |
| 26 | DNA PCR KAC (**200 fM**; AltF & GalR) | PrymcrRNA1 |
| 27 | Negative control (water in AltF & GalR) | PrymcrRNA1 |
| 28 | DNA PCR KAC (**480 nM**; AltF & GalR) | PrymcrRNA |
| 29 | DNA PCR KAC (**200 fM**; AltF & GalR) | PrymcrRNA2 |
| 30 | Negative control (water in AltF & GalR) | PrymcrRNA2 |
| 31 | DNA PCR KAC (**480 nM**; GalF & ModF) | PrymcrRNA1 |
| 32 | DNA PCR KAC (**200 fM**; GalF & ModF) | PrymcrRNA1 |
| 33 | Negative control (water in GalF & ModF) | PrymcrRNA1 |
| 34 | DNA PCR KAC (**480 nM**; GalF & ModF) | PrymcrRNA2 |
| 35 | DNA PCR KAC (**200 fM**; GalF & ModF) | PrymcrRNA2 |
| 36 | Negative control (water in GalF & ModF) | PrymcrRNA2 |
| 37 | DNA PCR KAC (**200 fM**; GalF & AltR) | PrymcrRNA1 |
| 38 | Negative control (water in GalF & AltR) | PrymcrRNA1 |
| 39 | DNA PCR KAC (**200 fM**; GalF & AltR) | PrymcrRNA2 |
| 40 | Negative control (water in GalF & AltR) | PrymcrRNA2 |
| 41 | Positive control (SynDNA) | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-14-200fm.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-14-480nm.webp)

**Conclusions:**

The best combination appears to
be **ModF + GalR +
PrymcrRNA1** because it yielded
high signals for both 480 nM and 200 fM target DNA concentrations. No other sample
produced a signal for 200 fM initial target concentration.

### SHERLOCK – choosing the best primer combination for further optimization

19/07/2024

**To ultimately select the most promising primer pair
for optimization, an assay was conducted using the primers that produced the
most satisfactory results in the previous test.**

1. **ModF & GalR (17.07)**
2. **ModF & ModF
   (16&17.07)**
3. **AltF & GalR (17.07)**

1. Additional RPA reactions were set up for target
   concentrations of **200
   aM**and **200 zM**, as the high fluorescence readouts suggested that
   the limit of detection (LOD) might be in that range. A genomic
   sample **L1**was also included to test whether these
   primer-PrymcrRNA pairs could detect genomic DNA without prior PCR. New
   negative control samples were prepared for use in the assay.
2. The SHERLOCK assay was conducted on the following
   samples:

Table 23: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1 | DNA PCR KAC (**200 nM**; ModF & GalR) | PrymcrRNA1 |
| 2 | DNA PCR KAC (**200 fM**; ModF & GalR) | PrymcrRNA1 |
| 3 | DNA PCR KAC (**200 aM**; ModF & GalR) | PrymcrRNA1 |
| 4 | DNA PCR KAC (**200 zM**; ModF & GalR) | PrymcrRNA1 |
| 5 | DNA PCR KAC (**L1**; ModF & GalR) | PrymcrRNA1 |
| 6 | Negative control (water in ModF & GalR) | PrymcrRNA1 |
| 7 | DNA PCR KAC (**200 nM**; ModF & ModF) | PrymcrRNA1 |
| 8 | DNA PCR KAC (**200 fM**; ModF & ModF) | PrymcrRNA1 |
| 9 | DNA PCR KAC (**200 aM**; ModF & ModF) | PrymcrRNA1 |
| 10 | DNA PCR KAC (**200 zM**; ModF & ModF) | PrymcrRNA1 |
| 11 | DNA PCR KAC (**L1**; ModF & ModF) | PrymcrRNA1 |
| 12 | Negative control (water in ModF & ModF) | PrymcrRNA1 |
| 13 | DNA PCR KAC (**200 nM**; AltF & GalR) | PrymcrRNA1 |
| 14 | DNA PCR KAC (**200 fM**; AltF & GalR) | PrymcrRNA1 |
| 15 | DNA PCR KAC (**200 aM**; AltF & GalR) | PrymcrRNA1 |
| 16 | DNA PCR KAC (**200 zM**; AltF & GalR) | PrymcrRNA1 |
| 17 | DNA PCR KAC (**L1**; AltF & GalR) | PrymcrRNA1 |
| 18 | Negative control (water in AltF & GalR) | PrymcrRNA1 |
| 19 | Positive control (SynDNA) | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-15.webp)

**Conclusions:**

Based on the results from that day and the previous assay
(17/07/2024), the best primer combination is **ModF & GalR with PrymcrRNA1**. This combination provides the highest fluorescence
intensity and effectively detects a 200 fM target DNA concentration, as demonstrated
in the previous test. Unfortunately the L1 genomic sample fro algal culture was not
detected in any RPA primers-PrymCrRNA1 combination.

### 22.07. - 28.07.

### SHERLOCK – optimisation of the primer concentration

23/07/2024

Based on the previous assays (from 17/07/24 and 19/07/24)
the **best primer pair –
PrymcrRNA combination proved to be ModF & GalR +
PrymcrRNA1**.

Optimization of the RPA primer concentration enables the
assay to **potentially become
quantifiable**, where the amount
of **input DNA correlates with
fluorescence intensity**. To achieve
this, an assay was conducted to compare how varying**primer concentrations impact the amplification
rate and fluorescence intensity**.
The method used is based on the approach presented in the following
article:Jonathan S. Gootenberg et al. [3]

1. RPA reactions were set up, with different primer
   and input DNA concentrations, alongside a positive control with SynDNA. For
   template dilutions, a PCR product coming from **NOW5.1** algal culture was used. Dilutions
   of **200 nM, 2nM, 20 pM and
   200 fM** were tested,
   alongside the following primer concentrations: **960 nM, 480 nM, 240 nM and 120
   nM**.
2. The SHERLOCK assay was conducted on the following
   samples:

Table 24: SHERLOCK reactions
components

|  |  |  |  |
| --- | --- | --- | --- |
|  | **Template DNA used for RPA** | **The primer concentration used for RPA** | **crRNA** |
| 1 | DNA PCR KAC (**200 nM**; ModF & GalR) | 960 nM | PrymcrRNA1 |
| 2 | DNA PCR KAC (**2 nM**; ModF & GalR) | 960 nM | PrymcrRNA1 |
| 3 | DNA PCR KAC (**20 pM**; ModF & GalR) | 960 nM | PrymcrRNA1 |
| 4 | DNA PCR KAC (**200 fM**; ModF & GalR) | 960 nM | PrymcrRNA1 |
| 5 | Negative control (water in ModF & GalR) | 960 nM | PrymcrRNA1 |
| 6 | DNA PCR KAC (**200 nM**; ModF & GalR) | 480 nM | PrymcrRNA1 |
| 7 | DNA PCR KAC (**2 nM**; ModF & GalR) | 480 nM | PrymcrRNA1 |
| 8 | DNA PCR KAC (**20 pM**; ModF & GalR) | 480 nM | PrymcrRNA1 |
| 9 | DNA PCR KAC (**200 fM**; ModF & GalR) | 480 nM | PrymcrRNA1 |
| 10 | Negative control (water in ModF & GalR) | 480 nM | PrymcrRNA1 |
| 11 | DNA PCR KAC (**200 nM**; ModF & GalR) | 240 nM | PrymcrRNA1 |
| 12 | DNA PCR KAC (**2 nM**; ModF & GalR) | 240 nM | PrymcrRNA1 |
| 13 | DNA PCR KAC (**20 pM**; ModF & GalR) | 240 nM | PrymcrRNA1 |
| 14 | DNA PCR KAC (**200 fM**; ModF & GalR) | 240 nM | PrymcrRNA1 |
| 15 | Negative control (water in ModF & GalR) | 240 nM | PrymcrRNA1 |
| 16 | DNA PCR KAC (**200 nM**; ModF & GalR) | 120 nM | PrymcrRNA1 |
| 17 | DNA PCR KAC (**2 nM**; ModF & GalR) | 120 nM | PrymcrRNA1 |
| 18 | DNA PCR KAC (**20 pM**; ModF & GalR) | 120 nM | PrymcrRNA1 |
| 19 | DNA PCR KAC (**200 fM**; ModF & GalR) | 120 nM | PrymcrRNA1 |
| 20 | Negative control (water in ModF & GalR) | 120 nM | PrymcrRNA1 |
| 21 | Positive control (SynDNA) | - | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-16-120nm-nopoz.webp)

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-16-240nm-nopoz.webp)

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-16-480nm-nopoz.webp)

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-16-960nm-nopoz.webp)

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/histogram-120nm.webp)

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/histogram-240nm.webp)

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/histogram-480nm.webp)

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/histogram-960nm.webp)

![](https://static.igem.wiki/teams/5087/pics/measurement/figure-12.png)

![](https://static.igem.wiki/teams/5087/pics/measurement/figure-9.png)

![](https://static.igem.wiki/teams/5087/pics/measurement/figure-6.png)

![](https://static.igem.wiki/teams/5087/pics/measurement/figure-3.png)

**Conclusions:**

Based on the bar graphs and the **R² value** calculated for the final fluorescence concentration
(background subtracted), the **optimal
primer concentration** appears to
be **960 nM**.
This concentration provides the most proportional relationship between the DNA
target sample concentration used for RPA and the final fluorescence
intensity.

An additional test will be conducted to further validate
this finding and to determine the range of DNA concentrations for which the
relationship between log₂[DNA] and fluorescence intensity is linear.

The assay needed to be manually extended, which caused a
bump on the graph, but this does not impact the final results.

### SHERLOCK – limit of detection tested on the optimised primer concentration

25/07/2024

Based on the optimal primer concentration established in
the previous assay, an additional assay was conducted to:

1. Determine the **final LOD** for our test.
2. Establish the **range of DNA concentrations** for which the relationship
   between **log₂[target DNA]
   and fluorescence intensity** is **linear**, allowing for quantification of target DNA
   concentration in the test sample.
3. Test two **genomic samples** to verify if genomic DNA isolated from the
   cultures can be detected using the chosen primer-PrymcrRNA pair (following
   the failed attempt with the L1 culture on 19/07).

To verify if RPA combined with SHERLOCK can be used for
quantitative sample testing, we designed tests on 23/07 and 25/07 to assess the
repeatability of both reactions:

* **SHERLOCK Reaction:** On 25/07, we conducted the SHERLOCK assay in
  two separate runs (test duplicates). Both runs used the same RPA mixes from
  25/07, so any differences in the fluorescence signal would be due to the
  kinetics of the SHERLOCK reaction. This test aimed to determine if the
  average final fluorescence intensity signal corresponding to a particular
  target DNA concentration can be used to quantify target DNA in test samples,
  potentially eliminating the need for a standard curve in every test.
* **RPA:** On 25/07, we used a separate RPA reaction (but
  on the same algal template, NOW5.1) compared to the test from 23/07. This
  was done to check if RPA reactions conducted under the same conditions
  produce a comparable amount of target DNA for subsequent SHERLOCK
  reactions.

Methods:

1. RPA reactions were set up, with different template
   DNA concentrations: 200 nM, 20 nM, 2 nM, 200 pM, 40 pM, 20 pM, 10 pM, 5 pM,
   2 pM, 1 pM, 200 fM, 20 fM and 2 fM, as well as genomic DNA isolates, coming
   from liquid cultures **L1
   (80.6 ng/μl)** and **Szczecin (7.3 ng/μl)**. For template dilutions, a PCR product coming
   from **NOW5.1** algal culture was used.
2. The SHERLOCK assay was conducted on the following
   samples in 2 separate runs (duplicate of the test):

Table 25: SHERLOCK reactions
components

|  |  |  |
| --- | --- | --- |
|  | **Template DNA used for RPA** | **crRNA** |
| 1 | DNA PCR KAC (**200 nM**; ModF & GalR) | PrymcrRNA1 |
| 2 | DNA PCR KAC (**20 nM**; ModF & GalR) | PrymcrRNA1 |
| 3 | DNA PCR KAC (**2 nM**; ModF & GalR) | PrymcrRNA1 |
| 4 | DNA PCR KAC (**200 pM**; ModF & GalR) | PrymcrRNA1 |
| 5 | DNA PCR KAC (**40 pM**; ModF & GalR) | PrymcrRNA1 |
| 6 | DNA PCR KAC (**20 pM**; ModF & GalR) | PrymcrRNA1 |
| 7 | DNA PCR KAC (**10 pM**; ModF & GalR) | PrymcrRNA1 |
| 8 | DNA PCR KAC (**5 pM**; ModF & GalR) | PrymcrRNA1 |
| 9 | DNA PCR KAC (**2 pM**; ModF & GalR) | PrymcrRNA1 |
| 10 | DNA PCR KAC (**1 pM**; ModF & GalR) | PrymcrRNA1 |
| 11 | DNA PCR KAC (**200 fM**; ModF & GalR) | PrymcrRNA1 |
| 12 | DNA PCR KAC (**20 fM**; ModF & GalR) | PrymcrRNA1 |
| 13 | DNA PCR KAC (**2 fM**; ModF & GalR) | PrymcrRNA1 |
| 14 | DNA PCR KAC (**L1**; ModF & GalR) | PrymcrRNA1 |
| 15 | DNA PCR KAC (**Szczecin**; ModF & GalR) | PrymcrRNA1 |
| 16 | Negative control (water in ModF & GalR) | PrymcrRNA1 |
| 17 | Positive control (SynDNA) | syncrRNA |

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-1-higher-nopoz.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-1-lower-nopoz.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-2-higher-nopoz.webp)
![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-2-lower-nopoz.webp)

**Conclusions:**

1. **Determining the Range of DNA
   Concentrations:** Despite
   optimizing the RPA primer
   concentration,**it is not
   possible** to define a
   range of target DNA concentrations that are proportional to the final
   fluorescence intensity.
2. **SHERLOCK Reaction
   Repeatability:** The
   SHERLOCK reaction proved to be **unrepeatable**. Different fluorescence intensities were observed
   for samples from the same RPA, and varying limits of detection (LOD) were
   determined for test duplicates: 1 pM for the first test and 200 fM for the
   second. This inconsistency undermines the ability to compare fluorescence
   intensities between tests from 23/07 and 25/07.

Given these observations, the **test does not appear suitable for quantitative
measurements**. A range of
concentrations
proportional to fluorescence intensity could be established (point 1), then creating
a standard curve for each test would provide reference points for quantifying DNA
concentration.

However, we can conclude that the **limit of detection (LOD) of our test is 1
pM**. This concentration corresponds
to 5 × 10¹⁰ *Prymnesium
parvum* cells per liter of water
(see calculations below). Additionally, we successfully
detected *Prymnesium
parvum* in
the **genomic
sample**isolated from the
“Szczecin” culture. These findings suggest
that **SHERLOCK can be used for
screening the presence of*****Prymnesium parvum*****in water with a detection limit of 1
pM**.

Based on the analysis conducted on 06.08 in GenomeLab by
Filip, we have determined that *Prymnesium
parvum's*  ITS1-5.8S-ITS2 sequence is
found exclusively on chromosome
20 in two haplotypes: one with 8 repetitions of the and
another with 4 repetitions of the sequence. Statistically, 25% of
the *Prymnesium parvum* population has 8 copies of the ITS1-5.8S-ITS2 sequence
(haplotypes 4 + 4), 50% has 12 copies (8 + 4), and 25% has 16 copies (8 + 8).

Here we can make a simulation for further
calculations:

Assuming there are 4 *Prymnesium parvum*cells in 1 L of water there should be 48 copies of the
ITS1-5.8S-ITS2 sequence (1 cell with 8 copies, 2 with 12 and 1 with 16).

To convert this to moles:

48 ITS1-5.8S-ITS2 copies
—- x mole

6.02 × 1023 —-- 1 mole

X = 7.97 × 10-23 mole

7.97 × 10-23 mole of ITS1-5.8S-ITS2 —-
4 *Prymnesium
parvum*in 1 L of water

To detect 1 pM of
ITS1-5.8S-ITS2:

10-12 —--- y mole

Y = 5 × 1010 = **50
billion of*****Prymnesium
parvum*****cells
in 1 L of water**

These calculations indicate that our SHERLOCK assay, with
an LOD of 1 pM, is capable of detecting *Prymnesium parvum* concentrations as low as 5 × 10¹⁰ cells
per liter, making it **a useful tool
for screening water for the presence of this algae. To make the method more
sensitive centrifugation of water samples (there are some that can be used in the
field) or using syringes with filter can be applied to make water samples more
condensed.**

### References

[1] M. J. Kellner, J. G. Koob, J. S. Gootenberg, O. O. Abudayyeh, and F. Zhang,
“SHERLOCK: nucleic acid detection with CRISPR nucleases.,” Nat Protoc, vol.
14, no. 10, pp. 2986–3012, Oct. 2019, doi: 10.1038/s41596-019-0210-2. [2]
Galluzzi, L., Bertozzini, E., Penna, A., Perini, F., Pigalarga, A., Graneli, E. and
Magnani, M. (2008), Detection and quantification of Prymnesium parvum (Haptophyceae) by
real-time PCR. Letters in Applied Microbiology, 46: 261-266.

[2] Galluzzi, L., Bertozzini, E., Penna, A., Perini, F., Pigalarga, A., Graneli, E. and
Magnani, M. (2008), Detection and quantification of Prymnesium parvum (Haptophyceae) by
real-time PCR. Letters in Applied Microbiology, 46: 261-266.

[3] Jonathan S. Gootenberg et al., Multiplexed and portable nucleic acid detection
platform with Cas13, Cas12a, and Csm6.
Science360,439-444(2018).DOI:10.1126/science.aaq0179

## SynLOCK Lab

---

### 10.06. - 16.06

### A list of all abbreviations

Table 1. A list of all
abbreviations and their meanings used throughout this notebook.

|  |  |
| --- | --- |
| G7 plasmid | PSB1C5C plasmid backbone carrying the BBa\_K2910000 part |
| System Vector Cassette (SVC) | The SynLOCK Cassette without the reporter device in the PSB1C5C plasmid backbone |
| System Vector Reporter (SVR) | The SynLOCK Cassette in the PSB1C5C plasmid backbone |
| Ultimate System Vector (USV) | The SynLOCK Cassette in the pSB1C3 plasmid backbone |
| G5 | BBa\_J23110 |
| K5 | BBa\_J23116 |
| O5 | BBa\_J428032 |
| G1 | BBa\_K1033906 (tsPurple) |
| G3 | BBa\_J97003 (TannenRFP) |
| K1 | BBa\_K1033919 (gfasPurple) |
| C1 | BBa\_J428092 |
| A1 | BBa\_J428091 |
| A11 | PSB1C3 plasmid backbone  with the BBa\_I20270 part |
| G8 | pJUMP29-1A(sfGFP) plasmid   BBa\_J428341 part |
| A10 | pJUMP28-1A(sfGFP) plasmid with   Bba\_J428353 part |

### Transformation of E.coli with PSB1C5C plasmid carrying the BBa\_K2910000 part

1. Two Petri dishes with chloramphenicol (170
   µg/ml [1]) were warmed up in the incubator at 37°C.
2. 10 µl of H₂O was added to
   the **G7 well in Kit Plate 1
   of the iGEM Distribution Kit 2024**. The mixture was pipetted and left to incubate for
   5 minutes. The liquid was observed turning red, as expected.
3. The transformation was conducted using Top10
   chemically competent *E.
   coli*cells (ThermoFisher)
   according to “Transformation Protocol I”.

***Plating
modifications:*** 75
µl of the culture was spread on one plate. The remaining culture was
centrifuged for 1 minute at maximum speed. 300 µl of the supernatant was
discarded, the pellet was resuspended in the remaining liquid, and it was spread on
the second plate.

[1] QIAGEN. Growth of bacterial cultures. QIAGEN.<https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/technology-and-research/plasmid-resource-center/growth-of-bacterial-cultures>. Accessed June 6, 2024.

### Observations - Transformation with the G7 plasmid

Bacteria only grew on these plates on which concentrated
samples were seeded.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image09.png "image1.png")

**Figure
1**: Transformation results with the
G7 plasmid.

### Overnight culture preparation

A single colony was taken from the plate with a pipette tip
and placed in a flask with 12 ml LB and 60 μl of chloramphenicol (final
concentration: 170 mg/μl) and left to incubate overnight in 37°C.

### Plasmid isolation

The G7 plasmid was isolated according to the “Plasmid
Isolation Protocol”.

**Obtained concentration:**353 ng/μl.

### 17.06. - 23.06

### BsaI digestion of the G7 plasmid

***Note:****The goal of this digestion is to remove the unwanted
coding sequence from the BBa\_K2910000 part, leaving us with the PSB1C5C plasmid
backbone. We'll then use this backbone to host the components of our
SynLOCK system.*

1. The G7 plasmid in concentration 353 ng/μl was
digested according to the table below:

Table 2. Digestion reaction
composition of the G7 plasmid.

|  |  |
| --- | --- |
| Reaction Component | Volume |
| plasmid | 20.5 μl |
| CutSmart x10 buffer | 2.5 μl |
| BsaI (10U/μlNEB) | 2 μl |

2. The whole reaction mix was vortexed and
short-spinned.

3. The samples were left overnight in an incubator at
37°C

### Preparing the PSB1C5C plasmid backbone for further experimentation

1. A 1% agarose gel was prepared, and the reaction
   products were visualised:

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image15.png)

**Figure
2**.: Results of the G7 plasmid
digestion using BsaI enzyme.

***Conclusion***: Correct bands have been obtained at 2045 bp and at 892
bp.

2. The G7 upper band was then cut out using a scalpel and
transferred to an Eppendorf tube. The isolation of the DNA from the gel fragment was
conducted according to the “DNA gel Extraction Protocol”.

The G7 upper band weighed**97 mg**.
The measured concentration of isolated DNA
was **28.2
 ng/μl.**

### 24.06. - 30.06

### Oligo annealing to build the SynLOCK Cassette spine.

Table 3: Sequences of the
oligos ordered to form the spine.

|  |  |
| --- | --- |
| System\_oligoF | AATGGAAATTAATACGACTCACTATAGGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACCGAAGAGCGAGCTCTTCCATGTCTTC |
| System\_oligoR | AAGCGAAGACATGGAAGAGCTCGCTCTTCGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC |

1. The oligos were suspended in water to obtain 100
   μM concentrations as in the manufacturer's manual.
2. The Oligos were then annealed according to the
   “Oligo Annealing and Ligation Protocol”.

***Note****: The*PSB1C5C
plasmid backbone*digested with BsaI was
used for the ligation
reaction. The amount of plasmid DNA should be 50 ng so 2 μl of 28.2 ng/μl
concentration of the backbone was used.*

3. Top 10 Competent *E. coli*(ThermoFisher) were transformed with the ligation
   reaction according to “Transformation Protocol I”.

**Note**:
We plated 100 μl of the outgrown culture, then spinned it down, removed most of
the supernatant and plated the remaining 70 μl of the resuspended pellet on a
separate plate.

### Observations: The SynLOCK Cassette Spine

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image16.png)

**Figure
8**: Transformation results of
ligating the SynLOCK Cassette spine into the PSB1C5C plasmid backbone, and the
negative control. The plasmid obtained from this ligation was
called **System Vector Cassette
(SVC)**. The black circles indicate
colonies picked for overnight culture preparation.

### Overnight culture preparation

Three single colonies from the transformation plates from
the previous day were used to prepare three liquid cultures which were shaken at
37°C overnight.

### SVC plasmid isolation and digestion

1. Six samples of the SVC plasmids were isolated
   according to the “Plasmid Isolation Protocol” from the overnight
   cultures prepared the previous day.

Table 4: Nanodrop measurement
results:

|  |  |  |
| --- | --- | --- |
| plasmid | A260/280 | A260/230 |
| SVC1 | 1.86 | 2.04 |
| SVC2 | 1.73 | 0.96 |
| SVC3 | 1.86 | 2.34 |
| SVC4 | 1.88 | 2.35 |
| SVC5 | 1.82 | 1.56 |
| SVC6 | 1.88 | 2.30 |

***Note****: DNA generally accepted as "pure" has a
A260/280 ratio about 1.8 and A260/230 about 2.0-2.2.*

***Conclusion***: Analysis of the spectrophotometric data reveals that
samples 2 and 5 demonstrate the lowest purity among the tested samples. The observed
absorbance values, which are lower than anticipated, suggest the presence of
contaminants with absorption maxima at 230 nm. The quantified concentrations are
high.

2. Plasmids 2-6 were digested overnight
   (37oC) to ensure that the first stage of cloning was
   carried out successfully.

Table 5: Components of the
digestion reaction mixture:

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| **Plasmid sample** | **SVC1** | **SVC2** | **SVC3** | **SVC4** | **SVC5** | **SVC6** |
| **Concentration [ng/μl]** | **112** | **365** | **183** | **366** | **235** | **205** |
| Plasmid [ul] | - | 11 | 22 | 11 | 17 | 20 |
| CutSmart buffer x10 | - | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| SapI | - | 2 | 2 | 2 | 2 | 2 |
| water | - | 9.5 | - | 9.5 | 3.5 | 0.5 |

### Electrophoresis of the digested SVC plasmids

**![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image33.png)

Figure
9:
Results of the digestion of the SVC plasmids with SapI. The non-digested
versions were visualised for comparison.

Conclusions:
A single band on the gel indicates that the plasmids were successfully linearized.
The band positioned slightly above 2000 bp matches the expected
size of 2136 bp.**

### Gel isolation of correct plasmid bands

1. The bands of digested SVC3 and SVC4 were isolated
   from the gel following the “DNA gel Extraction Protocol”.
2. The DNA was eluted in 10 µl of water.

The final concentrations were measured using a NanoDrop.
The concentration for **SVC3**was **20.5
ng/µl,**and
for**SVC4,**it was **24
ng/µl**.

### 1.07. - 7.07

### Restriction analysis of the SVC plasmids

1. To confirm that the SynLOCK Cassette spine was
   cloned inside the plasmids, a restriction analysis was planned. Two
   different analyses were conducted:

   1. **SacI**digestion:
   2. If the cassette is correctly ligated, the
      expected DNA fragments should be 1152 bp and 984 bp.
   3. If the cassette is not present, the
      linearized plasmid size should be 2136 bp.
   4. **BsaI**digestion:
   5. If the cassette is present, the expected
      DNA fragments should be 2045 bp and 111 bp.
   6. If the cassette is not present, the
      expected DNA fragments should be 2045 bp and 892 bp.
   7. To digest the plasmids, master mixes were
      prepared (6 µl per sample):

   Table 6: The master mix
   components for the restriction analysis:

   |  |  |
   | --- | --- |
   | Reaction Component | Volume |
   | Enzyme volume (SacI/BsaI) | 1 μl |
   | Water | 24 μl |
   | Cut Smart buffer 10x | 5 μl |
   | Total volume | 30 μl |

   Then 4 µl of SVC 3, 4, and 5 were added. An
   additional control with the complete G7 plasmid was prepared in the same
   way.

   ![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image23.png)

   **Figure
   10:**Results of the
   restriction analysis of SVC plasmids using gel
   electrophoresis. (a) The
   corresponding samples and enzymes are described, with G7 serving as the
   control. (b) A longer exposure
   time revealed the smallest DNA fragment (111 bp) produced by BsaI
   digestion.

   ***Conclusions***: The results confirmed that all three plasmids
   carry the SynLOCK Cassette spine.

### SVC plasmid sequencing

To ensure that the DNA sequences were consistent with the
expected ones and free from mutations, we decided to send the plasmids for
sequencing.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image5.png)

**Figure
11**: Sequencing data confirming the
successful integration of the Cassette spine into the SVC3, SVC4, and SVC5 plasmids,
with no detected mutations.

### Concentration of chloramphenicol adjustment

***Note:***We
concluded that the 170 μg/ml concentration of chloramphenicol was too high and
hindered the growth of our bacteria.

 Table 11: Revised
antibiotic concentrations used for further experimentation.

|  |  |  |
| --- | --- | --- |
| Antibiotic | Abbreviation | Concentration |
| Chloramphenicol | Cm | 34 μg/ml |
| Kanamycin | Kan | 50 μg/ml |
| Ampicillin | Amp | 100 μg/ml |

### Preparation of Parts from the iGEM Distribution Kit

***Note****: After consulting with experts, we decided to add
a***Reporter Transcription
Unit (TU)** *to our system to simplify the screening of correct
colonies. Additionally, we chose to change the plasmid backbone to enable the
transfer of our system into other backbones using BioBrick assembly.*

Table 12: Parts from the
distribution kit (Kit Plate 1) that were used to transform *E.coli*.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Well | Part Name | Part Type | Plasmid Resistance | Purpose in the system |
| G5 | BBa\_J23110 | Promoter | Cm | Reporter TU component |
| K5 | BBa\_J23116 | Promoter | Cm | Reporter TU component |
| O5 | BBa\_J428032 | RBS | Cm | Reporter TU component |
| G1 | BBa\_K1033906 (tsPurple) | CDS | Cm | Reporter TU component |
| G3 | BBa\_J97003 (TannenRFP) | CDS | Cm | Reporter TU component |
| K1 | BBa\_K1033919 (gfasPurple) | CDS | Cm | Reporter TU component |
| C1 | BBa\_J428092 | Terminator | Cm | Reporter TU component |
| A1 | BBa\_J428091 | Terminator | Cm | Reporter TU component |
| A11 | BBa\_I20270 | Device | Cm | BioBrick compatible vector |
| G8 | BBa\_J428341 | Device | Kan | Reporter TU Acceptor |
| A10 | Bba\_J428353 | Device | Kan | Reporter TU Acceptor |

 All transformations were performed according to the
“Transformation Protocol I”.

### Observations: Distribution Kit Parts

1. The transformation plates were taken out of the
   incubator, looking like this:

 ![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/obs.png "Tranformation results")

**Figure
12**: Results of the transformation
with DNA from different wells from the iGEM 2024 distribution kit. The respective
wells are indicated in the image.

2. The plates containing fluorescent reporter devices
   were visualised under UV light:

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image3.jpg "Tranformation results of A10, A11 and G8 plasmids  under UV light")

**Figure 13**: Visualisation of the colonies carrying plasmids with
fluorescent modules under the UV light.

***Conclusion***: The transformation was successful. The reduced
concentration of chloramphenicol (Cm) contributed to the enhanced transformation
efficiency.

### 8.07. - 14.07

### Cultures and transfromations

1. Overnight cultures from one colony picked from
   every plate (C1, K1, G3, K5, G5, G1, O5, A10, G8, A1, A10) were prepared in
   12 ml LB with corresponding antibiotics. Additionally, the picked colonies
   were stored for later on agar plates.
2. Four transformations were performed with the DNA
    taken from wells H1, M1, O1 and O13, according to the
   “Transformation Protocol II”.

### Observations: Distribution Kit Parts

* All overnight liquid cultures grew correctly,
  except for A10.
* All transformations from the previous day were
  successful.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image17.png "Transformation results 09/07")

**Figure
14:** Results of the
transformation with DNA from different wells from the iGEM 2024 distribution kit.
The respective wells are indicated in the image.

It was possible to successfully grow the clones picked
yesterday on separate agar plates for storage:

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image4.png "Storage of clones used for overnight cultures")

**Figure
16:** Plates storing the clones
picked for further experiments.

### Overnight culture preparation

Overnight liquid cultures from colonies picked from all
four plates were prepared. Additionally, picked clones were stored on a separate
agar plate. For **O13** a
red colony was picked:

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image1.png "Zoom on O13 transformation result")

**Figure 15**: An agar plate showing the transformation results with DNA
from the O13 well. The arrow points to the red colony that was picked as the correct
one, since it carried the mRFP1.

### Plasmid Isolation

Plasmids from overnight cultures of C1, K1, G3, K5, G5, G1,
O5, G8, A1 were isolated according to “Plasmid Isolation Protocol
II”.

Table 13: The concentrations
and purity of the obtained plasmids.

|  |  |  |  |
| --- | --- | --- | --- |
| Plasmid | Concentration [ ng/μl ] | A260/A280 | A260/A230 |
| G1 | 146.4 | 1.88 | 2.25 |
| G3 | 259.0 | 1.88 | 2.23 |
| G8 | 59.2 | 1.82 | 1.73 |
| K1 | 185.9 | 1.83 | 1.83 |
| K5 | 235.2 | 1.75 | 1.22 |
| G5 | 204.5 | 1.80 | 1.49 |
| C1 | 163.5 | 1.87 | 2.13 |
| O5 | 172.0 | 1.87 | 2.21 |
| A11 | 222.6 | 1.88 | 2.28 |

### Golden Gate Reaction I

In order to obtain the reporter transcription unit for our
system we set up a Golden Gate reaction according to the “Golden Gate Assembly
of Four Parts: Protocol”.

***Notes to the protocol mentioned above, as the
reaction was performed:***

Three different transcription units (TUs) were constructed
to identify the one that exhibits the fastest and most intense colour.

Table 14: Components of the
constructed transcription units.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| TU index | Promoter | RBS | CDS | Terminator | Destination Vector |
| TU1 | G5 | O5 | G1 | C1 | A11 |
| TU1 | G5 | O5 | K1 | C1 | A11 |
| TU3 | G5 | O5 | G3 | C1 | A11 |

The transformation step was performed according to the
“Transformation Protocol II”.

### Observations: Golden Gate Reaction I

None of the colonies showed any color. On top of that, the
negative control had a lot of colonies. Nearly all the colonies glowed green under
UV light.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image36.png "Transformation reactions with the Golden Gate mixture")

**Figure
16**: Results of the transformation
with the Golden Gate Reaction I mixtures. The respective transcription units are
indicated in the image.

***Realisation and
Conclusion:***There
was an **error**in
the reaction involving the choice of the destination
plasmid. The plasmid used was A11, which isn't compatible with Type IIS
assembly. Additionally, this plasmid has Cm resistance, allowing bacteria carrying
it to grow on the negative control plate. In the negative control, bacteria with
uncut plasmids carrying different parts could also grow because these part plasmids
also had Cm resistance. The Golden Gate reaction failed due to the incompatible
sticky ends between the A11 destination vector and the parts. The correct plasmid
backbone should be **G8.**

### Plasmid Isolation

Plasmids from overnight cultures of O13, H1, O1 and M1 were
isolated according to the “Plasmid Isolation Protocol II” using and
eluted in 30 μl of water.

Table 15: The concentrations
and purity of the obtained plasmids.

|  |  |  |  |
| --- | --- | --- | --- |
| Plasmid | Concentration [ ng/μl ] | A260/A280 | A260/A230 |
| O12 | 256.1 | 1.82 | 1.75 |
| H1 | 216.8 | 1.7 | 0.95 |
| O1 | 184.2 | 1.77 | 1.23 |
| M1 | 149.2 | 1.86 | 1.87 |

### Golden Gate Reaction II

The Golden Gate reaction was repeated, according to the
“Golden Gate Assembly of Four Parts: Protocol”. Four different
Transcription Units (TUs) were constructed.

Table 16:  Components of
the constructed transcription units.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| TU index | Promoter | RBS | CDS | Terminator | Destination Vector |
| TU 4 | K5 | H1 | G1 | C1 | G8 |
| TU 5 | G5 | O5 | G1 | C1 | G8 |
| TU 6 | G5 | O5 | G3 | C1 | G8 |
| TU 7 | G5 | O5 | O1 | C1 | G8 |

The transformation step was performed according to the
“Transformation Protocol II”.

### Observations after 17 hours after plating

* The negative control plate had a few colonies with
  green fluorescence, which was anticipated. The G8 destination vector
  contained a GFP expression module, so some of the vectors that remained
  closed or were not digested could have been uptaken by bacteria and
  consequently displayed green fluorescence.

* None of the colonies on the other plates were
  fluorescent. TU5 colonies were slightly purple, making them barely visible
  against the purple background, while TU7 colonies were very lightly blue.
  Other TU colonies appeared completely white.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image28.png)

**Figure 17:** Results of the transformation with the Golden Gate
Reaction II mixtures after 17h incubation at 37°C. The respective transcription
units are indicated in the image.

***Hypothesis***: The G8 plasmid is a medium copy plasmid, which is why the
color is developing slower.

***Information***: The available data indicated that a 24-48 hour incubation
may be needed for the color to be fully developed [2].

**Conclusions**:

* A higher copy plasmid would improve color
  formation.
* TU5 was the unit that developed colour the fastest
  during incubation and produced the highest intensity among all the units we
  tested. TU7 also showed potential.

### Overnight culture preparation

1. Overnight cultures of TU5 and TU7 were prepared in
   LB with Cm.
2. The plates were left to incubate overnight at room
   temperature, as recommended [1].

[1] Liljeruhm J, Funk SK, Tietscher S, et al. Engineering a
palette of eukaryotic chromoproteins for bacterial synthetic biology. J Biol Eng.
2018;12:8. Published 2018 May 10. doi:10.1186/s13036-018-0100-0.

### Observations 30 hours after plating

* Colonies carrying TU5 showed a visible light-purple
  color.
* Colonies carrying TU7 were greyish-blue.
* Colonies carrying TU6 showed a barely visible pink
  color.
* Colonies carrying TU4 remained white.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image20.png)

**Figure
18**: Plates with colonies carrying
different transcription units (indicated in the picture) 30h after
plating.

1. All four overnight cultures were
   centrifuged.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image8.png)

**Figure
19**: Pellets formed after
centrifuging the overnight cultures carrying various transcription units (indicated
in the picture).

### Reporter Plasmid Isolation

1. Plasmid isolation from the overnight cultures of TU5 and
TU7 (selected due to their strong colour visibility) was carried out according to
“Plasmid Isolation Protocol I”.

The overnight cultures for each transcription unit were
split into two, and plasmids were isolated using separate columns, resulting in four
samples in total. Each plasmid was labelled according to the transcription unit it
contained.

Table 17: TU5 and TU7 plasmid
concentrations after isolation.

|  |  |
| --- | --- |
| **Plasmid** | **Concentration [ ng/μl ]** |
| TU5 | 254 and 89.5 |
| TU7 | 201.2 and 154.2 |

2. After centrifugation, the remaining pellets from the TU5
and TU7 clones were plated on separate agar plates containing kanamycin for storage.
These plates were then left on the bench over the weekend.

### 15.07. - 21.07

### Observations: TU5 and TU7 reporters

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image35.jpg)

**Figure
20**: The saved clones carrying
expression modules TU7 and TU5 grew over the weekend, successfully developing
colour.

***Conclusion***: After last week's experiments, we concluded
that **TU5 is the best
choice** as the reporter for
our system. It was the fastest to develop a visible color, even in a medium-copy
plasmid, and it also matches our team's color theme beautifully!

### PCR amplifying the TU5 reporter module

**Purpose:** Our goal was to insert the reporter expression module
into the cassette spine to create the complete SynLOCK Cassette, while preserving
the SapI recognition sites. These sites would later enable the reporter to be
excised and replaced with the crRNA spacer. Additionally, the final product needed
to have compatible sticky ends after SapI digestion, allowing it to be ligated into
the SVC plasmid.

To do this, we designed and ordered two primers, which
introduced SapI recognition sites into the TU5 fragment.

Table 18: Sequences of the
primers introducing the SapI restriction sites.

|  |  |
| --- | --- |
| **Primer name** | **Primer Sequence** |
| SapF | AAACCGAAGAGCCGTCTCTGGAGTTTACGGCTAGC |
| SapR | CGCTACTAGTAAAGCGAAGACATGGAAGAGCCG |

**PCR Setup:**

1. A dilution of the matrix DNA for the PCR was
   prepared by mixing 2 µl of isolated TU5 plasmid (254 ng/µl) with
   38 µl H₂O.

Table 19: PCR Reaction Master
Mix components (for a single reaction):

|  |  |
| --- | --- |
| **Component** | **Volume (µl)** |
| Q5 mix | 12.5 |
| SapF primer (10 µM) | 1.25 |
| SapR primer (10 µM) | 1.25 |
| H₂O | 9.0 |
| **Total** | **24** |

2. After pipetting the master mix into PCR tubes, 1
   µl of the TU5 plasmid dilution was added to all samples except for the
   negative control (substituted with H₂O).

**Thermocycler Program:**

1. Initial denaturation: 98°C for 30s
2. Denaturation: 98°C for 10s
3. Annealing: 70°C (for negative control and one
   sample), 65°C (for another sample) for 15s
4. Extension: 72°C for 25s
5. Final extension: 72°C for 2min
6. Hold: 4°C indefinitely

Steps 2, 3, and 4 were looped 30 times.

**Note:** The annealing temperature was calculated using the NEB
Tm  Calculator tool and was estimated at 72°C.

### Gel electrophoresis of the PCR product

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image24.png)

**Figure
21**: Gel electrophoresis of the TU5
module amplified with SapF and SapR primers.

**Conclusion**: An anticipated **939 bp** band was obtained. As expected, the 72°C annealing
temperature yielded higher reaction efficiency, as indicated by the brighter band on
the gel. However, the product was also successfully created at 65°C.

The PCR products of both reactions were pooled into one
tube.

### DpnI digestion and clean-up

20 µl of the pooled PCR product was digested with 0.5
μl DpnI to ensure no matrix DNA remained in the sample. The reaction was placed
at 37°C for 1h.

***Note:*** This step could have been avoided because, even if the
matrix plasmid prevailed in the sample, it carried Kan resistance, while the vector
intended for ligating the amplified PCR product later on carries Cm resistance.
Therefore, colonies that took up the matrix plasmid would not grow.

### Post enzymatic reaction clean-up

1. The clean-up was performed according to the
   “Post Enzymatic Reaction Clean-Up Protocol”.
2. The elution step was completed by adding 20μl of
   water.

The**concentration**of the clean-up PCR product was**45.2  ng/μl.**

### SapI digestion of TU5 PCR product

**Purpose**:
In order to later ligate the TU5 module into the SynLOCK
Cassette Spine in the SVC plasmid, it was necessary to create compatible sticky ends
flanking our PCR product.

Table 20: Components used for
enzymatic digestion using SapI.

|  |  |
| --- | --- |
| **Reaction Component** | **Volume (µl)** |
| Cleaned up TU5 PCR product | 20 |
| SapI | 1 |
| Cut Smart Buffer | 2 |

The reaction was incubated at 37°C overnight.

### Post enzymatic reaction clean-up

1. TU5 PCR product was cleaned up after being digested
   with SapI according to the  The clean-up was performed according to the
   “Post Enzymatic Reaction Clean-Up Protocol”.
2. The obtained **concentration**was **81.6  ng/μl**

### Ligation reaction: Creation of System Vector Reporter (SVR) by inserting TU5 into the SVC plasmid

1. The reaction was carried out following the
   “DNA Ligation Protocol For Cohesive Ends” protocol.

***Notes:*** 67 ng of the insert were used in the reaction, based
on calculations from the provided protocol equation. As for the vector, the SVC3
sample extracted from gel after digestion with SapI was used.

### Observations: SVR plasmids

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image22.png)

**Figure
22**: Results of obtaining SVR
plasmid-carrying colonies.

**Conclusions**: All colonies were visibly violet. The used plasmid
backbone (**PSB1C5C**) is a high copy plasmid, therefore intense colour was
visible in the morning after just one night of incubation.

### Overnight culture preparation

Overnight cultures were prepared from two colonies picked
from the SVR plate (labelled SVR1 and SVR2). Additionally, those clones were stored
on a separate plate.

### crRNA spacer oligo annealing

1. All crRNA spacer oligonucleotide samples were
   diluted in water according to the manufacturer’s manual.

Table 21: Sequences of all
oligos used to form crRNA spacer templates

|  |  |
| --- | --- |
| Oligo name | Sequence |
| PrymCrRNA1\_coding\_oligo | CATAGGCGACGCTCGAGCCTTGATCTGGCGC |
| PrymCrRNA1\_matrix\_oligo | AACGCGCCAGATCAAGGCTCGAGCGTCGCCT |
| PrymCrRNA2\_coding\_oligo | CATGAGGATCCTCCCGTGCAACGCTGCCCTC |
| PrymCrRNA2\_matrix\_oligo | AACGAGGGCAGCGTTGCACGGGAGGATCCTC |
| SynCrRNA\_coding\_oligo | AACCGCCAAGCTTGCATGCCTGCAGGTCGAG |
| SynCrRNA\_matrix\_oligo | CATCTCGACCTGCAGGCATGCAAGCTTGGCG |

2. The annealing was carried out according to the
   “Oligo Annealing and Ligation Protocol”.

PrymCrRNA1\_coding\_oligo was annealed with
PrymCrRNA1\_matrix\_oligo, PrymCrRNA2\_coding\_oligo with PrymCrRNA2\_matrix\_oligo and
SyncrRNA\_coding\_oligo with SynCrRNA\_matrix\_oligo.

***Note***: After completing step 2 of the protocol, the samples were
stored at -20°C. The plan was to finish the procedure the following week, once
the isolated SVR plasmid backbone with the reporter sequence removed was obtained.
This backbone would then be ready to accept the spacers generated from this
annealing reaction.

### Observations: SVR plasmids

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image25.png)

**Figure
23**: SVR1 and SVR2 clones grown
overnight.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image10.png)

**Figure
24**: Overnight cultures of SVR1 and
SVR2 colonies.

***Conclusions***: The chromoprotein production in SVR vectors is very high
— the colour is easily observed after an overnight incubation on a plate and
in a liquid culture as well, which was not the case in previous cultures. This is
the first time we observed colour in a liquid culture without having to centrifuge
it. The high-copy plasmid backbone we chose for this assembly helped us maximise
colour formation, which was the key goal for our system.

### SVR plasmid isolation

1. The plasmid preparation was carried out from both
   liquid cultures according to “Plasmid Isolation Protocol I”. For
   each sample, 4 ml of the liquid culture were used, and for each culture
   (SVR1 and SVR2) two plasmid preps were carried out, yielding four
   samples.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/zrzut-ekranu-2024-09-22-o-19-45-04.png)

**Figure
25**: SVR pellets formed after
centrifugation during the plasmid isolation protocol.

2. Obtained concentrations were as
   follows:

Table 22. SVR plasmid
concentrations

|  |  |
| --- | --- |
| Plasmid | Concentration [ng/μl] |
| SVR1 sample 1 | 340 |
| SVR1 sample 2 | 346.3 |
| SVR sample 1 | 378.9 |
| SVR sample 2 | 338.7 |

### Sapl digestion of the SVR plasmids

**Purpose**:
To make sure the SapI recognition sites were preserved
within the SVR plasmid sequence, a digestion reaction using this enzyme was
prepared.

1. Digestion reactions were prepared for the
   SVR1 sample 1 and SVR2 sample 2.

Table 23: SapI digestion of SVR
plasmids components.

|  |  |
| --- | --- |
| Component | Volume |
| SVR sample | 1 μl |
| SapI | 0.5 μl |
| Cut Smart Buffer | 0.5 μl |
| H₂O | 5 μl |

2. The digestion was left to incubate at 37°C for
   30 minutes.
3. The digestion results were visualised by gel
   electrophoresis.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image18.png)

**Figure
26**: Gel electrophoresis results of
the digested SVR plasmid samples.

***Conclusion***: Anticipated bands were obtained at 934 and 2115 bp for
both SVR1 and SVR2 samples. A small fraction of the plasmid remained undigested,
marked by the band on 3049 bp. Both SVR plasmids after isolation were correct and
could be used for further experiments.

### PCR adding EcoRI and SpeI restriction sites to the synLOCK Cassette for transfer into a different plasmid backbone

**Purpose**:
After consulting with**Vinoo
Selvarajah**, the Vice President of
Technology at iGEM, we decided to
change the plasmid backbone for our system. This will make it compatible with the
RCF10 standard and allow Cassette transfer between different backbones using the
BioBrick assembly. For that, it is necessary to introduce EcoRI and SpeI sites to
flank our Cassette, which was the aim of this PCR reaction.

To do this, we designed and ordered two primers, which
introduced recognition sites to flank the Cassette fragment.

Table 24: Sequences of
EcoF and SpeR primers

|  |  |
| --- | --- |
| **Primer name** | **Primer Sequence** |
| EcoF | TCTGGAATTCGCGGCCGCTTCTAGAGAATGGAAATTAATACGACTCACTAT |
| SpeR | CGCTACTAGTAAAGCGAAGACATGGAAGAGCCG |

**PCR set up:**

For this PCR the 340 ng/μl sample of SVR1 was chosen. It
was diluted by taking 2 μl of the sample and adding 48 μl of
water.

Two reactions were prepared in final volume of 25
µl

Table 25: PCR Reaction Master
Mix components (for a single reaction):

|  |  |
| --- | --- |
| **Component** | **Volume [µl]** |
| Q5 mix | 12.5 |
| EcoF  primer (10 µM) | 1.25 |
| SpeR primer (10 µM) | 1.25 |
| H₂O | 9.0 |
| **Total** | **24** |

After pipetting the master mix into PCR tubes, 1 µl
of the SVR plasmid dilution was added to all samples except for the negative control
(substituted with H₂O).

**Thermocycler Program:**

1. Initial denaturation: 98°C for 30s
2. Denaturation: 98°C for 10s
3. Annealing: 70°C (for negative control and one
   sample), 65°C (for another sample) for 15s
4. Extension: 72°C for 25s
5. Final extension: 72°C for 2min
6. Hold: 4°C indefinitely

Steps 2, 3, and 4 were looped 30 times.

***Note*****:** The
annealing temperature was calculated using the NEB Tm Calculator tool and was
estimated at 72°C. The negative control was performed at 70°C.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image30.png)

**Figure
27**: Gel electrophoresis of the
SynLOCK Cassette amplified with EcoF and SpeR primers.

**Conclusions**: Expected bands of 1045 bp were obtained.

The PCR products of both reactions were pooled into one
tube and labelled “Cassette\_Reporter”

### DpnI digestion and clean-up

48 µL of the pooled PCR product was digested with 1
µL DpnI to remove any residual template DNA. The reaction was incubated at
37°C for 1 hour. The sample was then cleaned up following the “Post
Enzymatic Reaction Clean-Up” Protocol.

The obtained concentration was 23 ng/μl.

### Overnight digestions of the SVR plasmid

Three overnight digestions were set up according to the
tables below:

Table 26: SapI digestion of the
SVR plasmid setup.

|  |  |
| --- | --- |
| Component | Volume |
| SVR plasmid [340 ng/μl] | 30 μl |
| SapI | 1 μl |
| Cut Smart Buffer | 5 μl |
| H₂O | 14 μl |

Table 27: EcoRI and SpeI
digestion of the A11 plasmid setup.

|  |  |
| --- | --- |
| Component | Volume |
| A11 plasmid [222.6 ng/μl] | 20 μl |
| EcoRI-HF | 1 μl |
| SpeI-HF | 1 μl |
| Cut Smart Buffer | 2.5 μl |
| H₂O | 0.5 μl |

Table 28: EcoRI and SpeI
digestion of the Cassette\_Reporter setup.

|  |  |
| --- | --- |
| Component | Volume |
| Casette\_Reporter [23 ng/μl] | 15 μl |
| EcoRI-HF | 1 μl |
| SpeI-HF | 1 μl |
| Cut Smart Buffer | 2 μl |
| H₂O | 2 μl |

### Gel electrophoresis of the digested plasmid A11 and SVR

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image12.png)

**Figure
28**: Gel electrophoresis of the 5
µl samples of the plasmids digested overnight.

Table 29: Electrophoresis
expectations.

|  |  |  |
| --- | --- | --- |
| **Sample** | **Cutting Enzyme** | **Expected Bands** |
| A11 | EcoRI & SpeI | 2049 bp, 942 bp |
| SVR | SapI | 2115 bp, 934 bp |

***Conclusions***: The plasmids were digested properly, making them suitable
for further experimentation. While a small amount of undigested SVR plasmid remained
in the sample, the majority was successfully digested.

### Gel Extraction and Post-Enzymatic Reaction Clean-Up Testing for the Workflow

***Purpose***: At this stage, we needed to insert the crRNA spacers into
the SVR vector. To streamline and shorten the workflow, we decided
to **compare two DNA purification
techniques**: post-enzymatic
reaction cleanup and the gel extraction method. Our reasoning was that
post-enzymatic cleanup should be sufficient. After ligating the crRNA spacers into
the SVR vector, the purple protein transcription unit would be removed, resulting in
white colonies. This would allow us to easily identify and select the correct
colonies, without needing to perform gel electrophoresis and gel extraction
procedures.

1. The SVR plasmid sample digested with SapI was
   divided into two: 20 µl were used for gel electrophoresis (the 2115 bp
   band was placed in the freezer over the weekend), and 20 µl were
   purified using the “Post Enzymatic Reaction Clean-Up”
   Protocol.
2. The Cassette\_Reporter sample cut with EcoRI and
   SpeI was also purified using the “Post Enzymatic Reaction
   Clean-Up” Protocol, with elution performed using 15 µl of Tris
   pH=8.
3. The resulting concentrations after cleanup
   were:

* SVR cut with SapI:**343.4 ng/µl**

Cassette\_Reporter cut with EcoRI and
SpeI: **15.4
ng/µl**

### 22.07. - 28.07

### Ligation of crRNA spacers into the SVR plasmid backbone

***Purpose***: Inserting the crRNA spacers into the Cassette to create
plasmids ready for linearization and in vitro transcription.

Table 30: Ligation reaction
setup using the SVR plasmid post-enzymatic reaction cleanup.

|  |  |
| --- | --- |
| Component | Volume [µl] |
| SVR cut with SapI post-enzymatic cleanup sample [343.4 ng/µl] | 1.46 |
| T4 buffer | 1 |
| 1:200 dilution of crRNA spacer duplex | 1 |
| H₂O | 5.54 |
| T4 ligase | 1 |

Table 31: Ligation reaction
setup using the SVR plasmid post-DNA gel extraction.

|  |  |
| --- | --- |
| Component | Volume [µl] |
| SVR cut with SapI post-DNA gel extraction sample [37.4 ng/µl] | 1.3 |
| T4 buffer | 1 |
| 1:200 dilution of crRNA spacer duplex | 1 |
| H₂O | 5.7 |
| T4 ligase | 1 |

For the reactions described above, the duplexes used were
PrymCrRNA1, PrymCrRNA2, and SynCrRNA. For each reaction setup, a negative control
was prepared by using water instead of the oligo duplex.

The samples were left at room temperature for 15 minutes.
Afterwards, all of the volume was used for transformation according to the
“Transformation Protocol II”. In the final step, ⅕ of the cells volume
was used for plating.

### Observations: SVR plasmids with crRNA spacers

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image6.png)

**Figure
29:** Transformation results
following the ligation of the crRNA spacers into the SVR plasmid backbone, which was
digested with SapI and purified using post-enzymatic reaction
cleanup. **Row
a):** PrymCrRNA1. **Row b):** PrymCrRNA2. **Row c):** SynCrRNA. The plates were visualised against both
white and purple backgrounds to clearly display the colour differences.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image7.png)

**Figure
30**: Control plate for the
transformation using the SVR plasmid backbone, which was digested with SapI and
purified using post-enzymatic reaction cleanup.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image14.png)

**Figure
31**: Transformation results
following the ligation of crRNA spacers into the SVR plasmid backbone digested with
SapI and obtained through gel extraction. **a):** PrymCrRNA1.**b):** PrymCrRNA2. **c):** SynCrRNA. **d)** negative control

***Conclusions*****:** The
negative controls for both samples were as expected. Because no gel extraction was
done with the post-enzymatic reaction cleanup, some plasmids retained the cut-out
reporter, resulting in the anticipated purple colour. In contrast, the gel
extraction procedure removed any reporter that could potentially relegate into the
vector, leading to no colonies growing on the plates.

The results show that **the ligation efficiency and colour formation is
sufficient to rely solely on visual screening to identify colonies with
correctly ligated crRNA spacers**.
This approach significantly simplifies and accelerates our workflow, confirming that
a gel extraction step is not needed.

### Overnight culture preparation

1. Overnight cultures were prepared for all
   crRNA-carrying plasmids by picking white colonies from plates that had both
   white and purple colonies. Additionally, one false colony was picked to
   demonstrate its appearance the next day, and a negative control was included
   for comparison.
2. For the A11 plasmid cut with EcoRI & SpeI, gel
   electrophoresis was performed, and the 2049 bp band was extracted according
   to the “DNA Gel Extraction Protocol”.

The concentration of the extracted band
was **35.9
ng/µl.**

### The creation of Ultimate System Vector (USV) Plasmid

***Purpose*****:** To
create a plasmid similar to the SVR but withan added BioBrick prefix and suffix, we
chose to transfer the SynLOCK Cassette into the **pSB1C3**plasmid backbone. This modification will ensure
compatibility with both RCF10 and RCF1000 standards in our system.

1. The predigested and extracted A11 plasmid backbone
   was used in the ligation reaction with the Cassette\_Reporter
   fragment.

Table 32: Ligation reaction
components

|  |  |
| --- | --- |
| Component | Volume [µl] |
| A11 digested EcoRI and SpeI | 1.4 |
| T4 buffer | 1 |
| Cassette\_Reporter [15.4 ng/µl] | 5 |
| H₂O | 1,6 |
| T4 ligase | 1 |

For the negative control, water was used in place of the
Cassette\_Reporter. The transformation was carried out according to the
Transformation Protocol II. After transformation, ⅕ of the reaction volume was
plated.

### Observations: USV plasmids

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image21.png)

**Figure 32**: **a)** The
transformation results of the ligation reaction
creating the USV plasmid visualised against white background
and **b)**purple background. **c)** the
negative control

***Conclusions*****:** The
ligation reaction was successful, as indicated by the purple colour of the colonies,
which confirms that the reporter was ligated correctly.

Overnight cultures were prepared from two purple colonies
picked from the plate.

### Plasmid Preparation from Overnight Cultures Carrying SVR + crRNA Plasmids

The plasmid preparation was conducted according to the
“Plasmid Isolation Protocol I”.

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image31.png)

**Figure
33:** Pellets obtained after
centrifuging 4 ml of overnight cultures carrying the respective plasmids. The
results demonstrate that even if the wrong colony is accidentally picked from the
plate, the mistake can be easily identified due to the distinct colour of the
pellet.

Table 33: Concentration of the
plasmids obtained after isolation

|  |  |
| --- | --- |
| Plasmid | Concentration [ng/µl] |
| SVR + SyncrRNA | 84.7 |
| SVR + PrymcrRNA1 | 55.7 |
| SVR + PrymcrRNA2 | 52.6 |

### Linearization of the SVR + crRNAs plasmids with BbsI

All obtained plasmids were digested with BbsI-HF enzyme to
prepare them for in vitro transcription.

Table 34: Digestion reaction
components

|  |  |
| --- | --- |
| Component | Volume |
| Plasmid | 16 μl |
| BbsI-HF | 0.5 μl |
| Cut Smart Buffer | 2.5 μl |
| H₂O | 6 μl |

The digestion reaction was then visualised by gel
electrophoresis:

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image29.png)

**Figure
34**: Gel electrophoresis results of
the digestion of SVR +crRNAs plasmids with BbsI.

***Conclusions***: All plasmids were correctly digested with BbsI, as
indicated by a single band on the gel.

### USV plasmid isolation and digestion

1. USV plasmids were isolated from overnight cultures
   according to the “Plasmid Isolation Protocol I”. The elution
   step was performed in 30 µl 10mM Tris pH=8.

Table 35. Concentrations of
obtained USV plasmid samples.

|  |  |
| --- | --- |
| Sample | Concentration (ng/ul) |
| USV1 | 206 |
| USV2 | 166.2 |

2. The USV1 sample (later referred to as USV) was then
   digested with EcoRI and SpeI to demonstrate that the SynLOCK Cassette unit
   can now be transferred between Biobrick-compatible plasmid backbones.
   Additionally, a SapI digestion was performed to confirm that the reporter
   protein transcription unit could be excised.

Table 36: EcoRI and SpeI
digestion reaction components

|  |  |
| --- | --- |
| Component | Volume |
| USV (206 ng/µl) | 1 μl |
| EcoRI | 0.2 μl |
| SpeI | 0.2 μl |
| Cut Smart Buffer | 1 μl |
| H₂O | 7 μl |

Table 37: SapI digestion
reaction components

|  |  |
| --- | --- |
| Component | Volume |
| USV (206 ng/µl) | 1 μl |
| SapI | 0.2 μl |
| Cut Smart Buffer | 1 μl |
| H₂O | 7 μl |

3. The reactions were incubated at 37°C for 30
   minutes.
4. The results were visualised by gel
   electrophoresis:

**![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image34.png)**

**Figure
35:**Electrophoresis results
displaying the digestion of USV plasmid with the enzymes indicated in the
image.

Table 38: Expected band size
for each digestion of the USV plasmid.

|  |  |
| --- | --- |
| Enzyme/s used | Expected Band Size (bp) |
| EcoRI &SpeI | 2047 & 1031 |
| SapI | 2144 & 934 |

***Conclusion*****:**The
correct bands were obtained, making the experiment successful.

5. Since the results were accurate, the digestion
   reaction was scaled up. A 10 µl sample of the USV plasmid was digested
   for one hour with SapI, followed by clean-up according to the
   “ost-Enzymatic Reaction Clean-Up” Protocol.

The concentration obtained after the clean up
was **30.5
ng/µl**

### Ligation of crRNA spacers into the USV plasmid

Table 39: Ligation reaction
setup using the USV plasmid post-enzymatic reaction clean-up sample.

|  |  |
| --- | --- |
| Component | Volume [µl] |
| USV cut with SapI post-enzymatic cleanup sample [30.5 ng/µl] | 1.65 |
| T4 buffer | 1 |
| 1:200 dilution of crRNA spacer duplex | 1 |
| H₂O | 15.35 |
| T4 ligase | 1 |

For the reactions described above, the duplexes used were
PrymCrRNA1, PrymCrRNA2, and SynCrRNA. For each reaction setup, a negative control
was prepared by using water instead of the oligo duplex.

The samples were left at room temperature for 15 minutes.
Afterwards all of the volume was used for transformation according to the
“Transformation Protocol II”. In the final step 1/7 of the cells’
volume was used for plating.

### IVT on SVR + crRNAs – attempt no. 1

***Purpose*****:** In
vitro transcription (IVT) was conducted on the SVRs containing all crRNAs to
determine if transcripts of the correct length could be produced from the
vector.

A post-enzymatic reaction clean-up was carried out on all
plasmids following linearization with BbsI, according to the “Post Enzymatic
Reaction Clean-Up Protocol”.

Table 40: Concentration of the
plasmids obtained after clean-up:

|  |  |
| --- | --- |
| Plasmid | Concentration [ng/µl] |
| SVR + SyncrRNA | 25.6 |
| SVR + PrymcrRNA1 | 22.7 |
| SVR + PrymcrRNA2 | 12.5 |

IVT was carried out in 20 μl reactions using T7 RNA
polymerase from A&A Biotechnology, following the recommended protocol included
in the reagent manual (<https://www.aabiot.com/en/t7-polimeraza-rna>). The IVT reactions were incubated for 4 hours at 37°C.
A mastermix was prepared (as shown in Table 41) and added to each IVT reaction
(Tables 41A-C).

Table 41: IVT master mix
components.
Volumes were calculated for 3x 20 μl reaction = 60
μl.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| rNTP mix (25 mM) | 4.8 |
| T7 buffer | 12 |
| T7 RNA polymerase | 3 |
| RNase inhibitor (50 U/μl) | 1.2 |

Table 41A: IVT reaction
components for SVR + SyncrRNA.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| Purified and linearized DNA (~200 ng) | 7.8 |
| IVT master mix | 7 |
| RNase-free water | 5.2 |

Table 41B: IVT reaction
components for SVR + PrymcrRNA1.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| Purified and linearized DNA (~200 ng) | 8.8 |
| IVT master mix | 7 |
| RNase-free water | 4.2 |

Table 41C: IVT reaction
components for SVR + PrymcrRNA2.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| Purified and linearized DNA (~200 ng) | 13 |
| IVT master mix | 7 |
| RNase-free water | 0 |

Phenol-chloroform extraction was carried out according to
the protocol described in SHERLOCK Lab documentation.

***Results*****:** No
RNA pellet was recovered during the precipitation and centrifugation steps.

***Conclusions*****:** No
RNA was obtained, possibly due to an experimental error during the phenol-chloroform
extraction or a low starting amount of DNA. The entire procedure should be
repeated.

### Observations: USV plasmids with crRNA spacers

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image19.png)

**Figure
36**: The ligation results of the
crRNA spacers into the USV vector, visualised against both a white (a) and a purple
background (b).

***Conclusions:*** The reaction was successful, as both white and purple
colonies are visible on the plates containing the ligation reaction. In contrast,
only purple colonies appear on the control plate.

### Overnight culture preparation

Overnight cultures were prepared from USV and SVR plasmids
containing three different crRNA spacers. Additionally, a colony from the negative
control and a mistakenly picked purple colony from one of the crRNA plates were
included to show that it's possible to identify a wrongly picked colony based
solely on the colour of the pellet.

### SVR + crRNAs plasmids linearization using BbsI

Samples of SVR plasmids carrying PrymCrRNA1, PrymCrRNA2 and
SynCrRNA were digested with the BbsI-HF enzyme to prepare them
for *in vitro* transcription.

Table 42: Digestion reaction
components

|  |  |
| --- | --- |
| Component | Volume |
| Plasmid [330 ng/μl] | 15 μl |
| BbsI-HF | 0.2 μl |
| Cut Smart Buffer | 5 μl |
| H₂O | 29.8 μl |

The reaction was left to incubate for an hour at
37°C.

The digestion reaction was then visualised by gel
electrophoresis:

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image2.png)

**Figure
37**: Linearization
results. **1**: SVR + PrymCrRNA1,**2**:
PrymCrRNA2, **3**: SynCrRNA

***Conclusion***: All plasmids were successfully linearized, making them
ready for*in vitro* transcription (IVT).

### IVT on SVR + crRNAs – attempt no. 2

IVT was repeated using the same protocol, but this time the
reaction volume was doubled to 40 μl. All calculations remained the same as those
in Table 41, A-C from 25/07/2024, but adjusted for the increased volume. The IVT
reactions were incubated for 2 hours at 37°C. Phenol-chloroform extraction was
performed following the standard protocol.

***Results*****:** No
RNA pellet was recovered during the precipitation and centrifugation steps.

***Conclusions*****:** No
RNA was obtained. After consulting with dr Mateusz Wawro, our instructor, we decided
to repeat the procedure, this time using a larger amount of input DNA and the
HiScribe® T7 High Yield RNA Synthesis Kit from New England Biolabs, in hopes of
achieving a higher RNA yield.

### Observations: Pellets

![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image27.png)

**Figure
38**: Overnight cultures after
centrifugation of 4 mL samples. a) SVR + PrymCrRNA1, b) SVR + PrymCrRNA2, c) SVR +
SynCrRNA, d) USV + PrymCrRNA1, e) USV + PrymCrRNA2, f) USV + SynCrRNA.

### Miniprep repeat on SVR + crRNAs and USV + crRNAs

***Purpose*****:** To
obtain all plasmids carrying crRNAs, including both SVRs and USVs, and to compare
the performance of two available miniprep kits.

Miniprep plasmid isolation was performed using 7 mL of
overnight *E.
coli*culture. Two kits, Syngen
and NEB, were used side by side for comparison, following “Plasmid Isolation
Protocol I” and “Plasmid Isolation Protocol II”,
respectively.

Table 43: Summary of plasmid
concentrations obtained for SVR + crRNAs.

|  |  |  |
| --- | --- | --- |
| Vector | Concentration obtained with Syngen kit [ng/μl] | Concentration obtained with NEB Monarch kit [ng/μl] |
| SVR + PrymcrRNA1 | 284.2 | 139 |
| SVR + PrymcrRNA2 | 224.6 | 125.8 |
| SVR + SyncrRNA | 106.7 | 108.8 |

Table 44: Summary of plasmid
concentrations obtained for USV + crRNAs.

|  |  |  |
| --- | --- | --- |
| Vector | Concentration obtained with Syngen kit [ng/μl] | Concentration obtained with NEB Monarch kit [ng/μl] |
| USV + PrymcrRNA1 | 163.4 | 91.5 |
| USV + PrymcrRNA2 | 174.2 | 82.5 |
| USV + SyncrRNA | 129.6 | 52.4 |

### 29.07. - 4.08.

### Trial IVT on SVR + PrymcrRNA1

**Purpose:** *In
vitro* transcription (IVT)
was conducted on the SVR + PrymcrRNA1 vector to test the procedure using the
HiScribe® T7 High Yield RNA Synthesis Kit and determine
if any RNA could be obtained from the vector.

For this experiment, the HiScribe® T7 High Yield RNA Synthesis Kit from New
England Biolabs was used, following the protocol for short templates (<
0.3 kb). An IVT master mix was prepared according to the protocol, with the
FLuc Control Template from the kit serving as a positive control.

A mastermix was prepared (as shown in Table 45) and
added to each IVT reaction (Tables A-B).

Table 45: IVT master
mix. Volumes were calculated for 3x 20 μl reaction = 60 μl.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| 10X reaction buffer | 4.5 |
| ATP (100 mM) | 4.5 |
| UTP (100 mM) | 4.5 |
| GTP (100 mM) | 4.5 |
| CTP (100 mM) | 4.5 |
| DTT (0.1 M) | 3 |

Table 45A: SVR +
PrymcrRNA1 IVT master mix components.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| IVT master mix | 8.5 |
| RNase-free water | 0 |
| Purified and linearized DNA (~500 ng) | 9.5 |
| T7 RNA Polymerase Mix | 2 |

Table 45B: FLuc control
template IVT master mix components.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| IVT master mix | 8.5 |
| RNase-free water | 5 |
| FLuc Control Template (500 ng/μl) | 1 |
| T7 RNA Polymerase Mix | 2 |

IVT reactions were incubated for 4 h at
37°C.

DNase I treatment was performed to digest any
remaining DNA. The reaction mix was prepared using components from the
Monarch® Total RNA Miniprep Kit and incubated for 15
minutes at 37°C.

Table 46: DNase I
treatment reaction mix.

|  |  |
| --- | --- |
| Component | Volume [μl] |
| IVT reaction | 20 |
| RNase-free water | 70 |
| 10X DNase I Buffer | 10 |
| DNase I | 2 |

Post-enzymatic reaction cleanup was carried out
using the Monarch® Total RNA Miniprep Kit. The RNA Reaction Cleanup
protocol from the Monarch Total RNA Miniprep Kit (NEB #T2010) was
followed.

A protocol for the entire above described procedure
“IVT and RNA Cleanup Protocol” has also been included in the
“Experiments” page.

The concentrations of the obtained RNA samples were
measured using a NanoDrop Microvolume Spectrophotometer.

Table 47:
Concentrations of crRNA samples obtained in the trial IVT.

|  |  |  |  |
| --- | --- | --- | --- |
| crRNA | Concentration [ng/μl] | A260/A280 | A260/A230 |
| SVR PrymcrRNA1 | 610.7 | 2.18 | 2.41 |
| FLuc Control Template | 672.2 | 2.10 | 1.98 |

**Results**: RNA was obtained and purified successfully. The
measured concentration and purity was satisfactory as well.

**Conclusions**: A successful IVT and cleanup procedure workflow
was established, which will later be applied to all vectors.

### Linearization of USV and SVR plasmids carrying crRNA spacers

To carry out the remaining IVT reaction
combinations, we prepared digestions of USV and SVR plasmids containing the
additional crRNA spacers, as outlined in Tables 48 A-E.

Table 48 A: Digestion
reaction components for USV + PrymCrRNA2 plasmid

|  |  |
| --- | --- |
| Component | Volume |
| Plasmid [174 ng/μl] | 28 μl |
| BbsI-HF | 0.2 μl |
| Cut Smart Buffer | 5 μl |
| H₂O | 17 μl |

Table 42 B: Digestion
reaction components for SVR + PrymCrRNA2 plasmid

|  |  |
| --- | --- |
| Component | Volume |
| Plasmid [224.6 ng/μl] | 22 μl |
| BbsI-HF | 0.2 μl |
| Cut Smart Buffer | 5 μl |
| H₂O | 23 μl |

Table 42 C: Digestion
reaction components for USV +SynCrRNA plasmid

|  |  |
| --- | --- |
| Component | Volume |
| Plasmid [129.8 ng/μl] | 39 μl |
| BbsI-HF | 0.2 μl |
| Cut Smart Buffer | 5 μl |
| H₂O | 6 μl |

T

Table 42 D: Digestion
reaction components for SVR + SynCrRNA plasmid

|  |  |
| --- | --- |
| Component | Volume |
| Plasmid [108.8 ng/μl] | 46 μl |
| BbsI-HF | 0.2 μl |
| Cut Smart Buffer | 5 μl |
| H₂O | - |

Table 42 E: Digestion
reaction components for USV + PrymCrRNA 1 plasmid

|  |  |
| --- | --- |
| Component | Volume |
| Plasmid [163.4 ng/μl] | 44 μl |
| BbsI-HF | 0.2 μl |
| Cut Smart Buffer | 5 μl |
| H₂O | 2 μl |

The reactions were left to incubate for an hour at
37°C.

### IVT on SVR + crRNAs and USV + crRNAs

**Purpose:** This experiment aimed to perform IVT on all
vectors following the successful trial IVT conducted the previous day. The
goal was to check if transcripts of the correct length could be obtained
from the vectors.

First, the cleanup of the overnight digest reaction
with BbsI was performed as previously described.

Table 43: Obtained
concentrations of vectors.

|  |  |
| --- | --- |
| Vector | Concentration [ng/μl] |
| SVR + SyncrRNA | 68.9 |
| SVR + PrymcrRNA2 | 65.2 |
| USV + SyncrRNA | 41.7 |
| USV + PrymcrRNA2 | 26.4 |
| USV + PrymcrRNA1 | 106.7 |

IVT was set up following the previously described
procedure and incubated for 4 hours at 37°C. The IVT reactions were then
treated with DNase I, and the cleanup procedure was carried out as
previously described.

Table 44:
Concentrations of obtained crRNA samples.

|  |  |
| --- | --- |
| crRNA | Concentration [ng/μl] |
| SVR SyncrRNA | 1637.9 |
| SVR PrymcrRNA2 | 1665 |
| USV SyncrRNA | 742 |
| USV PrymcrRNA1 | 971.8 |
| USV PrymcrRNA2 | 828.8 |

After obtaining all RNAs, gel electrophoresis was
performed using the previously established protocol described in the
SHERLOCK Lab documentation, with one modification: a loading buffer without
added EtBr was used to prepare the samples. Following separation at 90V for
approximately 40 minutes, the gel was first washed in the TBE buffer for 10
minutes. It was then washed in a TBE + EtBr solution for 10 minutes, and
finally washed again in the TBE buffer for 10 minutes.

The Thermo Scientific RiboRuler High Range RNA
Ladder (200 to 6000 bases) was used. All transcripts expected length was 64
bp, so bands below 200 bp would be assumed to be the correct
transcripts.

*![](https://static.igem.wiki/teams/5087/pics/syn-lab-not/image32.png)*

**Figure
39**: RNA electrophoresis
– IVT results.

**Results:** Transcripts of the correct length were
successfully obtained, but some bands appeared faint. This could indicate
that the vectors may not have been fully digested with BbsI, or that the
DNase I treatment was only partially effective.

**Conclusions:** The correct transcript length confirms that
the system is functioning as expected. However, further technical
optimization is needed to improve the efficiency of RNA synthesis. Despite
this, we have established a proof of concept demonstrating that both SVR and
USV crRNA synthesis systems work correctly.

## Protein Lab

---

### Protein Lab intro

#### **Protein Lab Goals**

**Primary Objective:**

**1. Production and Purification of huLwCas13a:**

* The initial goal was to produce and purify the huLwCas13a protein for use in our
  SHERLOCK assay.

**Expanded Objectives (Post Consultation):**

**2. Production and Purification of CcaCas13b:**

* After consulting with Prof. Andrzej
  Górecki, we decided to produce and purify an additional protein, CcaCas13b, as a
  backup in case huLwCas13a faced issues such as inefficient purification or
  inactivity. This strategy ensured that the project's workflow would continue
  smoothly without interruption.

**3. Comparative Analysis of Proteins with and without Tags:**

* We aimed to compare the structure, stability, and activity of huLwCas13a and
  CcaCas13b both with and without the 6xHis-TwinStrep-SUMO tag. This comparison would
  help determine whether the expensive process of cleaving off the tag using SUMO
  protease is necessary.

**Final Goals:**

* **Production and Purification of Both Proteins:** Successfully produce
  and purify both
  huLwCas13a and CcaCas13b.
* **Assessment of 6xHis-TwinStrep-SUMO Tag Impact:** Evaluate how
  the presence of the 6xHis-TwinStrep-SUMO tag influences the proteins' structure,
  thermal
  stability, and activity.
* **Optimization of the Purification Protocol:** Refine the
  purification process to
  maximize the yield and activity of the proteins, whether tagged or untagged.

#### **How Cas13 Proteins Work in the SHERLOCK System**

One of the essential components for detecting DNA/RNA using the SHERLOCK system is a
protein from the **Cas (CRISPR-associated) family**. Cas proteins generally
target and
cleave invading DNA (types I, II, IV, V) or RNA (types III and VI). **Cas13a and
Cas13b**, specifically, belong to type VI, which targets **RNA**
[1].

In addition to the *cis*-cleavage activity common among Cas proteins (such as
Cas9),
Cas13 proteins possess a unique ***trans*-cleavage** or
**collateral
activity**. This
collateral activity enables Cas13 to cleave **any single-stranded RNA (ssRNA) in
the
vicinity of the protein**, regardless of its sequence. When the CRISPR RNA
(crRNA)
binds to its specific target RNA, it induces a conformational change in the Cas13
protein, activating its nucleolytic domain [2, 3].

![](https://static.igem.wiki/teams/5087/pics/homepage/protein.webp)

Once activated, Cas13 indiscriminately cuts any nearby ssRNA, a feature that SHERLOCK
exploits to generate a **detectable signal**. The SHERLOCK system detects
the cleavage
of ssRNA reporters tagged with a fluorophore and a quencher. When the reporter is
cleaved, fluorescence is emitted, indicating the presence of the target RNA. The
Lateral Flow Assay (LFA) test also relies on this collateral activity, but uses
biotin and fluorescein tags for detection.

#### **Background**

**Numerous procedures** have been described in the literature for purifying
Cas13 proteins,
with huLwCas13a being the most commonly studied. These procedures often involve
**single-step** purification using Immobilized Metal Affinity
Chromatography (**IMAC**) [4-6],
and sometimes include an additional Size Exclusion Chromatography (**SEC**)
step [7].
Another approach involves a more complex **three-step** purification
process: starting with
affinity chromatography using a **streptavidin** [8] or
**histidine** [9] tag (**IMAC**), followed
by Ion Exchange Chromatography (**IEC**), and finishing with
**SEC**.

In many protocols, the **6xHis-TwinStrep-SUMO tag located at the N-terminus of the
protein
is cleaved off** after purification [5][6][8]. However, no specific data
justifying the
removal of the tag, which requires the use of the relatively expensive SUMO protease,
has been found in the literature.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/bia-ko-tag.png)

This study focuses on **optimizing the purification protocol to obtain the purest,
most
active form of huLwCas13a protein with minimal labor and cost**. Based on our
literature
research, particularly Kellner et al. [8], and the experience of our instructors, we
developed our own protocols for the purification of huLwCas13a and CcaCas13b.

#### **Key Steps in Protein Production and Purification Process**

The primary goal was to obtain pure and active Cas13a and Cas13b proteins for use in
SHERLOCK detection. Below are the key steps:

1. **Protein Expression:** Proteins were expressed in *E. coli*
   strain Rosetta 2(DE3)pLysS using the pET system.
2. **Bacterial Cell Disruption:** Sonication was used to lyse the
   bacterial cells.
3. **Protein Purification:**
   * **IMAC:** Evaluated HisTag versus StrepTag purification
     efficiency (Figures 11-14).
   * **IEC:** Removed remaining contaminants and concentrated
     the sample before SEC (Figures 15-18).
   * **SEC:** Achieved highly pure Cas13a and Cas13b proteins
     (Figures 21-22).
4. **Comparative Analysis:** The tag’s effect on structure, thermal
   stability, and activity was evaluated via Circular Dichroism (Figures 27-28),
   NanoDSF (Figures 29-30), and SHERLOCK activity tests (Figure 26).

#### **Conclusion:**

The optimized protocol enabled the production of highly pure and active Cas13a and
Cas13b proteins suitable for SHERLOCK detection assays, **eliminating the need for
the
tag cleavage step** as it does not significantly affect protein structure,
stability,
or activity.

The optimised protocol can be found here:
[Click for protocol.](https://static.igem.wiki/teams/5087/pics/protocols/cas13-purification.pdf)

### 13.11. - 19.11

### Plasmids

Plasmids:
1. pC013 (huLwCas13a) (pC013 - Twinstrep-SUMO-huLwCas13a was a gift from Feng
Zhang (Addgene plasmid # 90097 ;
http://n2t.net/addgene:90097 ; RRID:Addgene\_90097)) [3].

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image14.png)

**Figure 1.** pC013 plasmid coding 6xHis-TwinStrep-SUMO-huLwCas13a.

2. pR0306 (CcaCas13b) (pR0306 CcaCas13b His6-TwinStrep-SUMO was a gift from Feng
Zhang
(Addgene plasmid # 182687 ; http://n2t.net/addgene:182687 ;
RRID:Addgene\_182687)) [10].

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image15.png)

**Figure 2.** pR0306 plasmid coding 6xHis-TwinStrep-SUMO-CcaCas13b.

were bought in Addgene.

### Plasmid isolation and electrophoresis

**GOAL:** Isolate purchased plasmids and prepare samples for
sequencing to confirm the correct nucleotide sequence.

**METHODS:** Bacteria were streaked on agar plates
(ampicillin 100 μg/ml for pR0306, ampicillin 100 μg/ml and chloramphenicol 34
μg/ml for pC013). Bacteria were incubated overnight at 37°C.

The next day 15 ml TB liquid cultures were inoculated (antibiotics in
concentrations as in agar plates). Cultures grew overnight in a shaker – 300
r.p.m., 37°C. The next day plasmids were isolated using NZYMiniprep (NZYtech) –
protocol is available in the protocols section.

0.8% agarose gel was prepared by dissolving 0.8 g of agarose in 100 ml of
TAE buffer in a microwave. The mixture was cooled to a temperature of about
50°C, 3 μl of Midori Green DNA Stain (Nippon Genetics) was added. Agarose was
poured into a gel tray with the well comb in place. When the gel was solid 1 kb
GeneRuler marker and plasmid samples were loaded into the wells. Gel ran for 30
min, 99 V.

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image1.jpg)

**Figure 3.** Agarose gel after pC013 (Cas13a) and pR0306 (Cad13b)
plasmid isolation. Key: d = plasmid digested using enzymes BamHI and XbaI, nd =
plasmid non-digested.

Table 1. Plasmid pC013 and pR0306 concentration measurement

|  |  |
| --- | --- |
| Sample | DNA concentration [ng/μl] |
| pC013 (1) | 561.4 |
| pC013 (2) | 596.6 |
| pR0306 | 138.8 |

In the digested sample, a single band was observed for the pR0306 plasmid (Figure
3), indicating that the sample was ready for sequencing (Figure 1).
However, the pC013 plasmid showed more than two bands, which we suspected was
due to the presence of the pRARE plasmid in the **Rosetta
2(DE3)pLysS** cells.
This plasmid codes for rare tRNAs and has a selectable marker for
chloramphenicol.
To obtain clear sequencing results, we decided to remove the
**pRARE** plasmid from the bacteria.

To achieve this, **DH5α** cells were transformed with varying
amounts of the pC013 plasmid – 100 ng, 10 ng, and 0.1 ng – added to
approximately 1 ml of competent bacteria.
The bacteria were then streaked onto **agar plates containing ampicillin
but no chloramphenicol**.
After incubation overnight at 37°C, growth was observed only on the plates with
10 ng and 100 ng of plasmid DNA.
Notably, the number of colonies on both plates was similar.

### 20.11. - 26.11

### DH5α colonies liquid cultures inoculation, selection, plasmid isolation

**GOAL:** Selection of colonies that lost the pRARE plasmid.

**METHODS:** To select colonies that had lost the pRARE plasmid but
retained pC013, colonies were picked from the plate with bacteria transformed
with 10 ng of plasmid DNA. These colonies were resuspended in 200 μl LB, and 100
μl was transferred to glass tubes containing **LB with ampicillin**, while
another
100 μl was transferred to **LB with ampicillin and chloramphenicol**. A
total of 19
such samples were prepared and incubated overnight in a shaker at 37°C, 300
r.p.m.

The following day, bacterial growth was observed in 18 out of 19 tubes containing
LB with ampicillin. **No growth was seen in any tube containing
chloramphenicol**,
indicating successful **removal of the pRARE** plasmid from the cells.
Plasmid
isolation was performed using the NZYMiniprep kit on six randomly selected
tubes.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image17.png)

**Figure 4.** Agarose gel after pC013 plasmid isolation. Numbers
correspond to the following samples of grown bacteria. d = plasmid digested
using enzymes BamHI and XbaI, nd = plasmid non-digested.

Figure 4 confirms successful removal of the pRARE plasmid, as only one band is
observed in every digested (d) sample.

Table 2. Plasmid pC013 concentration measurement

|  |  |  |
| --- | --- | --- |
| Sample | DNA concentration [ng/μl] | A260/A280 |
| 1 | 48.3 | 2.15 |
| 2 | 42.6 | 2.1 |
| 3 | 38.3 | 2.31 |
| 4 | 34.9 | 2.05 |
| 5 | 39.6 | 2.2 |
| 6 | 36.1 | 2.19 |

### Plasmids sequencing

**GOAL:** Confirming that pC013 and pR0306 plasmids have the
proper sequence coding for Cas proteins.

**METHODS:** For pC013 sequencing, we selected sample 1 from
plasmid isolation (22.11). For pR0306 sequencing, we chose a sample from
plasmid isolation performed on 16.11.

Sequencing was ordered using both T7
promoter and T7 terminator primers, ensuring thorough verification of the
sequence. Comparing the 5' and 3' ends of the sequence with the one
deposited by the vendor allows for greater confidence in the accuracy of the
CDS sequence for the Cas13 protein.

**Results for pC013:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image16.png)

**Figure 5.** Alignment of sequenced fragment and pC013 plasmid
sequence (T7 promoter primer).

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image19.png)

**Figure 6.** Alignment of sequenced fragment and pC013 plasmid
sequence (T7 terminator primer).

**Results for pR0306:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image18.png)

**Figure 7.** Alignment of sequenced fragment and pR0306
plasmid sequence (T7 promoter primer).

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image21.png)

**Figure 8.** Alignment of sequenced fragment and pR0306
plasmid sequence (T7 terminator primer).

**Conclusions:**

The obtained sequencing reads demonstrate **high quality**, with
all nucleotides in the sequence successfully determined. The substitutions
observed in the initial segments of the sequence are attributed to the high
abundance of these DNA fragments post-amplification, which caused
fluorescence reading distortions due to signal saturation. This phenomenon
is characteristic of Sanger sequencing methodology, as are the mismatches
observed closer to the end of sequenced fragments. Consequently, it can be
concluded that the **sequencing results align with the plasmid
sequence deposited on the vendor's (Addgene) website.**

### 04.12 - 10.12

### Bacteria transformation

**GOALS:** Transforming Rosetta 2(DE3)pLysS cells with sequenced
plasmids (pC013, pR0306).

**METHODS:** Transformation was performed using a **heat
shock** method
(20 min on ice, 45 s in a thermoblock at 42°C, 2 min on ice) into the competent
Rosetta 2(DE3) pLysS cells. 1 µl of plasmid was used per 1 ml of competent
bacteria. The bacteria were streaked on LB-agar plates containing ampicillin
(100 μg/ml) and chloramphenicol (34 μg/ml) and incubated overnight at 37°C.

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image20.jpg)

**Figure 9.** Single colony grown after transformation of Rosetta
2(DE3)pLysS cells with sequenced pC013 sample.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image24.jpg)

**Figure 10.** Colonies grown after transformation of Rosetta
2(DE3)pLysS cells with sequenced pR0306 sample.

**CONCLUSIONS:** For the colony transformed with pC013 (Figure 9),
precultures will be prepared before inoculating mother cultures. For pR0306
(Figure 10), there are plenty of colonies that will be used for mother culture
inoculation.

### Preculture for huLwCas13a production

**GOAL:** Multiplying the number of bacterial cells containing
pC013.

**METHODS:** 5 ml of TB media supplemented with ampicillin (100
μg/ml) and chloramphenicol (34 μg/ml) was inoculated using the colony observed
in **Figure 9**. The bacteria were grown overnight at 37°C in a
shaker at 300 r.p.m.

**RESULTS:** Bacterial growth was observed in the glass tube.

**CONCLUSIONS:** The preculture will be used the next day for mother
culture inoculation.

### Mother cultures for protein expression and purification (Cas13a)

**GOAL:** Preparing mother cultures (50 ml) for protein expression.

**METHODS:** Single colonies for Cas13b (see Figure 10) were used to prepare
six mother cultures. These cultures were incubated overnight with shaking at 37°C in 50
ml of TB medium supplemented with antibiotics (ampicillin 100 μg/ml and chloramphenicol
34 μg/ml).

**RESULTS:** Bacterial growth was observed in all six flasks.

**CONCLUSIONS:** The mother cultures will be used the next day for
inoculating large-scale cultures for protein expression.

### Large scale cultures (Cas13a)

**GOAL:** Producing Cas proteins in large-scale cultures (9 L).

**METHODS:** Six flasks, each containing 1.5 L of TB medium with the
same composition as the mother cultures, were inoculated with 30 ml of the
mother cultures. OD600 was monitored, and when it reached between
0.4-0.6, the flasks were cooled to 4°C for 30 minutes in a cold room. Then, 1.5
ml of 0.5 M IPTG (isopropyl-β-D-thiogalactopyranoside) inducer was added, and
the cultures were shaken for 18 hours at 21°C. After incubation, the cultures
were centrifuged (7 min, 5,000 g, 4°C), yielding six Falcon tubes of bacterial
pellet, with masses ranging from 13-18 g per tube. The pellets were stored at
-20°C until further use.

**RESULTS:**

* For Cas13a, 95.18 g of bacterial pellet was obtained, of which 15.12 g was
  used for further purification.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image22.jpg)

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image23.png)

### 11.12 - 17.12

### Large scale cultures (Cas13b)

**GOAL:** Producing Cas proteins in large-scale cultures (9 L).

**METHODS:** Six flasks, each containing 1.5 L of TB medium with the
same composition as the mother cultures, were inoculated with 30 ml of the
mother cultures. OD600 was monitored, and when it reached between
0.4-0.6, the flasks were cooled to 4°C for 30 minutes in a cold room. Then, 1.5
ml of 0.5 M IPTG (isopropyl-β-D-thiogalactopyranoside) inducer was added, and
the cultures were shaken for 18 hours at 21°C. After incubation, the cultures
were centrifuged (7 min, 5,000 g, 4°C), yielding six Falcon tubes of bacterial
pellet, with masses ranging from 13-18 g per tube. The pellets were stored at
-20°C until further use.

**RESULTS:**

* For Cas13b, 96.7 g of bacterial pellet was obtained, of which 18.82 g was
  used for further purification.

### 08.01 - 14.01

### Cell disruption - sonication

**GOAL:** Disrupting bacteria and releasing accumulated Cas
proteins.

**METHODS:** The bacterial pellets were resuspended in double the
volume of NI buffer with the following additions:

* 1 cOmplete Ultra tablet
* 30 mg of lysozyme
* 10 μl benzonase nuclease (activity ≥ 250 U/μl)
* 45 μl DTT (1 M)

The tube was placed on ice, and 20 sonication cycles were performed. Each cycle
consisted of 30 seconds of sonication followed by a 30-second pause. The
sonicator was set to not exceed a temperature of 30°C, with an amplitude of 40%.
After sonication, the sample was centrifuged for 30 minutes at 30,000 g at 4°C.

**Buffer without imidazole (NI):** 0.5 M NaCl, 20 mM Na₃PO₄, 5%
glycerol, 1 mM DTT, protease inhibitors, pH = 8.0.

### Protein purification - IMAC

**GOALS:** Reduce contamination and concentrate Cas proteins in
samples.

**METHODS:** The first stage of protein purification was carried out
due to the presence of an N-terminal **histidine tag, which binds to the nickel
resin column**. The process was conducted at 4°C.

The column with packed resin was
washed with NI buffer (flow rate: 3 ml/min, 5 column volumes (cv)), the sample
(supernatant from centrifugation in the previous step) was loaded (flow rate: 1.5
ml/min), and the column was washed with 5 cv of NI buffer (flow rate: 3 ml/min)
to remove unbound residues. A two-step elution was performed: first with a
buffer mixing NI and I buffers in a 1:1 ratio (final imidazole concentration of
**150 mM**), followed by buffer I with **300 mM imidazole**.
Fifteen 2-ml fractions were
collected for each elution step.

**Buffers:**

* **NI buffer (without imidazole):** 0.5 M NaCl, 20 mM Na₃PO₄, 5%
  glycerol, 1 mM DTT, protease inhibitors, pH 8.0.
* **I buffer (with 300 mM imidazole):** 0.5 M NaCl, 20 mM Na₃PO₄,
  5% glycerol, 1 mM DTT, protease inhibitors, 300 mM imidazole, pH 8.0.

*Note:* Before use, add 1 cOmplete Ultra tablet per 30 ml of buffer.

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image26.jpg)

**Figure 11.** SDS-Page gel for huLwCas13a - samples from
expression and IMAC purification. Cas13a is indicated by a violet arrow.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image27.jpg)

**Figure 12.** SDS-Page gel for CcaCas13b - samples from
expression and IMAC purification. Cas13b is indicated by a yellow arrow.

**Wells key:** unind. – uninduced bacterial culture, ind. – culture
after 18h IPTG induction, sonic. – supernatant after sonication and
centrifugation, prec. – sediment after sonication and centrifugation, FT –
flow-through from sample application on His-resin column, wash – flow-through
from column washing before elution; E1, E2, E3… - fractions eluted with 150 mM
imidazole buffer, \*E1, \*E2, \*E3 - fractions eluted with 300 mM imidazole buffer.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image28.png)

**Figure 13.** Total eluted protein concentrations in fractions
after IMAC (Cas13a).

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image29.png)

**Figure 14.** Total eluted protein concentrations in fractions
after IMAC (Cas13b).

**CONCLUSIONS:** **IMAC was effective in partially purifying both
proteins**, reducing contaminant levels. Although contaminants were still
present,
the fractions were enriched with the produced proteins, as indicated by thicker
bands on the SDS-PAGE gel (marked by arrows).

* For Cas13a, 150 mM imidazole elution was efficient, but a small portion of
  the protein was eluted with 300 mM imidazole. In future purifications, 300
  mM elution is recommended (see Figures 11 and 13).
* For Cas13b, a significant amount of protein remained bound to the column
  after 150 mM elution. The 300 mM elution was more effective (see Figures 12
  and 14).

### 15.01-21.01 (Cas13a), 11.03-17.03 (Cas13b)

### SUMO protease digestion (Cas13a)

**GOALS:** Divide Cas13a and Cas13b into two pools – SUMO protease digested
and undigested (with and without the 6xHis-TwinStrep-SUMO tag), in order to compare
their activity, structure, and stability after final purification.

**METHODS:** 10 ml of selected samples for Cas13a (samples 1, 3, 5, 7, and
9) and 14 ml of Cas13b (samples 1, 3, 5, 7, 9, 11, and 13) were diluted to 30 ml with
buffer A. To this, 10 μl of DTT (1 M), 60 μl of NP-40 detergent, and 50 μl of SUMO
protease (1 U/μl) were added. The mixtures were digested overnight on a rocker at 4°C.

**Buffer A:** 20 mM Na₃PO₄, 5% glycerol, 1 mM DTT, pH 7.5

### 22.01-28.01 (Cas13a), 11.03-17.03 (Cas13b)

### Protein purification – IEC

**GOALS:** Further removal of protein contaminants and concentrating
the Cas protein sample before final purification using SEC.

**METHODS:** This purification stage was conducted separately for
two samples: the digested protein (with a theoretical pI of 9.62 for Cas13b and
9.72 for Cas13a) and the undigested protein (pI 9.5 for Cas13b and 9.62 for
Cas13a).

The separation was performed using an **ӒKTA Pure FPLC** (Fast Protein
Liquid Chromatography) system and a cation exchange column **Mono S
4.6/100PE**
(Cytiva) with a volume of 1.7 ml.

The device was programmed based on the protocol [8], starting with
pre-equilibrating the column with 12.5% buffer B (5 column volumes (cv)). The
undigested protein sample (Cas13a) was diluted with buffer A (pH 7.5) to 30 ml
and loaded onto the column. The column was washed with 12.5% buffer B (5 cv) and
the bound protein was eluted with a gradient of 12.5% to 100% buffer B (10 cv),
collecting 2 ml fractions. The column was washed with 100% buffer B (10 cv) and
equilibrated with 12.5% buffer B (5 cv). The flow-rate for all steps was 1
ml/min.

The same procedure was repeated for the digested Cas13a sample and both Cas13b
samples with the following changes: for elution, a gradient of 12.5% to 80%
buffer B was used, and the volume of collected fractions was 0.5 ml.

**Buffers:**

* **Buffer A:** 20 mM Na₃PO₄, 5% glycerol, 1 mM DTT, pH 7.5
* **Buffer B:** 2 M NaCl, 20 mM Na₃PO₄, 5% glycerol, 1 mM DTT, pH
  7.5

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image30.png)

**Figure 15.** SDS-Page gel for huLwCas13a - samples after IEC.
Cas13a with the tag is indicated by the violet arrow, Cas13a without the tag by
the blue arrow. The purple rectangle marks two bands corresponding to the
cleaved 6xHis-TwinStrep-SUMO tag.

**Wells key:**

* Entry – pooled fractions after IMAC
* Sample appl. – flow-through from sample application on the column
* Wash – flow-through from column washing
* E1, E2, E3… - eluted fractions, numbers correspond to the numbers of fractions on
  chromatograms (Figure 17, Figure 18). Samples with \* correspond to samples after
  SUMO protease digestion

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image31.png)

**Figure 16.** SDS-Page gel for CcaCas13b - samples after IEC.
Cas13b with the tag is indicated by the yellow arrow, Cas13b without by the
orange arrow. The black circle marks the cleaved tag.

**Wells key:**

* Eluted fractions of Cas13b (left picture for SUMO protease undigested, right for
  digested) E6, E8, E10... -  numbers correspond to the numbers of fractions on
  chromatograms (Figure 19,  Figure 20).
* Flow-through - from sample application on the column
* Wash – flow-through from column washing. The area where a band for the digested tag
  was expected is marked with black circles. For the undigested protein there is one
  band in this area, while for the digested protein, there are two. Therefore, the
  additional band is likely the tag.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image32.png)

**Figure 17.** Chromatogram. Cas13a IEC for SUMO protease
undigested sample.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image4.png)

**Figure 18.** Chromatogram. Cas13a IEC for SUMO protease
digested sample.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image5.png)

**Figure 19.** Chromatogram. Cas13b IEC for undigested sample
of Cas13b.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image6.png)

**Figure 20.** Chromatogram. Cas13b IEC for SUMO protease
digested sample.

**CONCLUSIONS:** For both Cas13a and Cas13b (SUMO protease digested
and undigested samples), a 2-fold reduction in protein contaminants was
observed, though **contaminants still remain in significant amounts**. The
concentration of Cas13a and Cas13b in the collected fractions was visible, with
the elution maximum for both proteins occurring at approximately 35% buffer B,
which corresponds to 0.7 M NaCl.

Using a cation-exchanger was effective in separating the cleaved tag (marked by
the purple rectangle in Figure 15 for huLwCas13a and the black circle in Figure
16 for CcaCas13b). The pI of the 6xHis-TwinStrep-SUMO tag is 6.57, which
prevents the tag from binding to the column bedding.

### 04.03-10.03 (Cas13a), 11.03-17.03 (Cas13b)

### Protein concentration

**Protein samples concentration**

**GOALS:** Reducing the volume of the sample to inject it into the
SEC column.

**METHODS:** Chosen samples (4-6 and 9-20 for Cas13a, 9-14 for
digested and undigested Cas13b) were concentrated in a centrifugal concentrator
to a volume of approximately 0.5 ml (centrifugation parameters: 15 min, 4,000 g,
4°C).

**RESULTS:** The sample volume for both proteins was reduced to
approximately 0.5 ml.

### SEC

**GOALS:** Obtaining pure huLwCas13a and CcaCas13b.

**METHODS:** Size-exclusion chromatography (SEC) was performed on
the same device as IEC, with a flow rate of 0.5 ml/min. **The Superdex 200 10/300
GL** (Merck) column was equilibrated with buffer S (1 column volume (cv)),
and the
concentrated sample of undigested protein was loaded. The sample was eluted,
collecting 0.5 ml fractions (1 cv), followed by washing and re-equilibrating the
column (1 cv). The SUMO-digested sample was then loaded.

**Buffer S:** 50 mM Tris-HCl (pH 7.5), 0.6 M NaCl, 5% glycerol. pH =
7.5.

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image7.png)

**Figure 21.** SDS-Page gel for huLwCas13a - samples after SEC.
Cas13a with tag is indicated by the violet arrow, without tag by the blue arrow.
Samples with \* correspond to samples after SUMO protease digestion.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image8.png)

**Figure 22.** SDS-Page gel for Cas13b - samples after SEC.
Cas13b with tag is indicated by the yellow arrow, without by the orange arrow.
Samples with \* correspond to samples after SUMO protease digestion.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image9.png)

**Figure 24.** Chromatogram. Cas13a SEC. First sample is SUMO
protease undigested, second is digested.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image10.png)

**Figure 25.** Chromatogram. Cas13b SEC. First sample is SUMO
protease undigested, second is digested.

**CONCLUSIONS:** **We successfully completed the protein production
and purification process for both Cas proteins**. However, two bands are
present
for SUMO protease-digested samples (Figures 21 and 22). The higher band
corresponds to the protein with the 6xHis-TwinStrep-SUMO tag (violet arrow for
Cas13a, yellow for Cas13b), and the lower band corresponds to the protein
without the tag (blue for Cas13a, orange for Cas13b).

**Pooled fractions for huLwCas13a:**

* **Undigested:** Fractions 17-20 (0.5 ml each, total 2 ml).
  Protein concentration: 0.73 mg/ml (measured using the Bradford method).
  Total huLwCas13a mass: 1.46 mg.
* **Digested:** Fractions 40-44 (0.5 ml each, total 2.5 ml).
  Protein concentration: 0.87 mg/ml. Total huLwCas13a mass: 2.175 mg.

**Pooled fractions for CcaCas13b:**

* **Undigested:** Fractions 44-48 (0.5 ml each, total 2.5 ml).
  Protein concentration: 0.52 mg/ml. Total CcaCas13b mass: 1.3 mg.
* **Digested:** Fractions 75-79 (0.5 ml each, total 2.5 ml).
  Protein concentration: 0.35 mg/ml. Total CcaCas13b mass: 0.875 mg.

### 15.04 - 21.04

### SHERLOCK activity test

**GOAL:** Comparing the activity of huLwCas13a with and without
the 6xHis-TwinStrep-SUMO tag.

**METHODS:** The SHERLOCK test was conducted by Kasia and Nina
in the SHERLOCK Lab. The procedure followed the protocol described by
Kellner [1].

The test focused on huLwCas13a, and we decided not to order specific
synthetic DNA for CcaCas13b activity testing because redesigning the synDNA
provided by Kellner by changing the DR sequence specific to CcaCas13b would
be required. Initially, we aimed to test huLwCas13a activity, and since it
worked, we used it for further SHERLOCK reactions.

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image11.jpg)

**Figure 26.** Comparing activity of huLwCas13a with and
without the 6xHis-TwinStrep-SUMO tag.

**Figure key:**

* 1 – SUMO protease digested huLwCas13a added to SHERLOCK mix
* 2 – SUMO protease undigested huLwCas13a added to SHERLOCK mix
* 3 – Water added instead of Cas13a (negative control)
* 4 – RNase A added instead of Cas13a (positive control)
* 5 – Target RNA and crRNA replaced with water in SHERLOCK mix, for SUMO
  digested huLwCas13a
* 6 – Target RNA and crRNA replaced with water in SHERLOCK mix, for SUMO
  undigested huLwCas13a

**CONCLUSIONS:** The produced huLwCas13a is active. There are **no
statistically significant differences** in activity between the SUMO protease
digested (1) and undigested (2) samples. The protein does not exhibit
constitutive collateral activity, as indicated by the significantly lower
signal observed in samples without target RNA and crRNA (5-6). **The produced
huLwCas13a can be used in further SHERLOCK tests**.

### 22.04 - 28.04

### Circular Dichroism

**GOAL:** Comparing the structure of both proteins (huLwCas13a
and CcaCas13b) with and without the 6xHis-TwinStrep-SUMO tag.

**METHODS:** Measurements were conducted using a Jasco J-710
spectropolarimeter with the following parameters:

* Wavelength range: 190-250 nm
* Resolution: 1 nm
* Scanning mode: continuous
* Scanning speed: 20 nm/min
* Response time: 4 s
* Bandwidth: 2.0 nm

Five data series were collected using a quartz cuvette with an optical path
length of 0.1 mm.

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/cd-cas13a-fix.png)

**Figure 27.** Dependence of mean residue molar ellipticity
for huLwCas13a with and without the tag in the far-UV range.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/cd-cas13b-fix.png)

**Figure 28.** Dependence of mean residue molar ellipticity
for CcaCas13b with and without the tag in the far-UV range.

**Table 2.** Proportions of various secondary structure types
determined from Circular Dichroism spectra for huLwCas13a.

| huLwCas13a | α-helix | β-sheet | β-turn | unordered |
| --- | --- | --- | --- | --- |
| Undigested | 64.5 ± 2.1 | 3.8 ± 0.9 | 10.2 ± 0.7 | 20.9 ± 2.5 |
| Digested | 58.1 ± 10.2 | 4.7 ± 6.3 | 18.2 ± 9.9 | 19.5 ± 7.2 |

**Table 3.** Proportions of various secondary structure types
determined from Circular Dichroism spectra for CcaCas13b.

| CcaCas13b | α-helix | β-sheet | β-turn | unordered |
| --- | --- | --- | --- | --- |
| Undigested | 76.8 ± 11.1 | 1.5 ± 2 | 6.9 ± 4.3 | 15.2 ± 10.5 |
| Digested | 80.4 ± 7.4 | 0.5 ± 2 | 4.4 ± 3.3 | 14.7 ± 7.8 |

**CONCLUSIONS:** For both proteins, the mean residue ellipticity
spectra (Figures 27, 28) overlap, and the proportions of the individual
secondary structures (Tables 2 and 3) do not differ significantly. This
indicates that the **tag’s presence does not significantly impact the protein
structure**, and therefore, it should not affect the protein's function, as
confirmed in earlier activity tests.

### NanoDSF

**GOALS:** Comparing the thermal stability of both proteins
(huLwCas13a and CcaCas13b) with and without the 6xHis-TwinStrep-SUMO tag.

**METHODS:** Measurements were conducted using the Prometheus
NT.48 device (NanoTemper Technologies). The heating range was from 20°C to
95°C, at a rate of 1°C/min. Measurements were performed in triplicates.

**RESULTS:**

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image2.png)

**Figure 29.** Dependence of the first derivative of the
fluorescence intensity change from temperature for huLwCas13a.

![](https://static.igem.wiki/teams/5087/pics/prot-lab-not/image3.png)

**Figure 30.** Dependence of the first derivative of the
fluorescence intensity change from temperature for CcaCas13b.

**Key:**

* D – SUMO protease digested sample
* UD – SUMO protease undigested sample

**CONCLUSIONS:** The curves corresponding to the digested (D)
and undigested (UD) proteins overlap, indicating that the **tag’s presence
does not significantly impact the proteins’ thermal stability**. This
justifies not cleaving off the 6xHis-TwinStrep-SUMO tag, as the tag does not
alter protein function, which has also been confirmed in earlier tests.

### Final Conclusions from Our Tests

**FINAL CONCLUSIONS FROM OUR TESTS:**

We recommend purifying huLwCas13a and CcaCas13b according to the following
protocol: [Click
for protocol](https://static.igem.wiki/teams/5087/pics/protocols/cas13-purification.pdf).

From the sediments used for protein purification, we obtained:

* 1.46 mg tagged huLwCas13a – enough for 11,500 reactions according to
  Kellner’s protocol [8]
* 2.175 mg untagged huLwCas13a – enough for 17,000 reactions
* 1.3 mg tagged CcaCas13b – enough for 10,300 reactions
* 0.875 mg untagged CcaCas13b – enough for 6,900 reactions

This supports the use of a longer and more costly purification protocol
(compared to 1-step purification protocols suggested in some publications
[4], [5], [6]) to obtain the purest possible Cas protein preparation,
**ensuring greater reliability of SHERLOCK test results.**

We also observed that IMAC can effectively replace StrepTag affinity
chromatography as suggested by Kellner [8]. In fact, IMAC may be more
effective, as we obtained 3.635 mg of pure Cas13a from 1.5 L of culture and
2.175 mg of Cas13b, while Kellner reports only 0.5-1 mg from a 4 L culture.

**Not cleaving off the 6xHis-TwinStrep-SUMO tag** is justified by
**activity tests**
(Figure 26), **CD** (Figure 27, Figure 28), and **NanoDSF**
(Figure 29, Figure 30)
results, making the purification protocol faster and cheaper.

**Additional recommendations (not tested):**

* Use more protease inhibitors or change the product to reduce proteolysis
  observed on the SDS-Page gel after IMAC (Figure 11, Figure 12).
* Use a different cell disruption method (e.g., French press) – during
  sonication, the mixture heated up quickly, extending the process to more
  than 30 minutes instead of the predicted 20 minutes.
* Prewash the His-NiNTA column with 30 mM imidazole buffer before elution
  with 300 mM (or higher) imidazole – this could result in purer eluted
  fractions.

### Bibliography

1. Y. Xu and Z. Li, “CRISPR-Cas systems: Overview, innovations and
   applications in human disease research and gene therapy,” *Comput
   Struct Biotechnol J*, vol. 18, pp. 2401–2415, 2020, doi:
   10.1016/j.csbj.2020.08.031.
2. O. O. Abudayyeh et al., “C2c2 is a single-component programmable
   RNA-guided RNA-targeting CRISPR effector.,” *Science*, vol. 353,
   no. 6299, p. aaf5573, Aug. 2016, doi: 10.1126/science.aaf5573.
3. J. S. Gootenberg et al., “Nucleic acid detection with
   CRISPR-Cas13a/C2c2,” *Science (1979)*, vol. 356, no. 6336, pp.
   438–442, Apr. 2017, doi: 10.1126/science.aam9321.
4. A. S. Savinova, E. Yu. Koptev, E. V. Usachev, A. P. Tkachuk, and V. A.
   Guschin, “Cas13a: purification and use for detection of viral RNA,”
   *Bulletin of Russian State Medical University*, no. 2, pp. 21–25,
   2018, doi: 10.24075/brsmu.2018.021.
5. B. An et al., “Rapid and Sensitive Detection of Salmonella spp. Using
   CRISPR-Cas13a Combined With Recombinase Polymerase Amplification,”
   *Front Microbiol*, vol. 12, Oct. 2021, doi:
   10.3389/fmicb.2021.732426.
6. H. Khan et al., “CRISPR-Cas13a mediated nanosystem for attomolar
   detection of canine parvovirus type 2,” *Chinese Chemical
   Letters*, vol. 30, no. 12, pp. 2201–2204, Dec. 2019, doi:
   10.1016/j.cclet.2019.10.032.
7. M. 2022 iGEM Team, “Cas13a Purification Protocol.” Accessed: Jun. 05,
   2024. [Online]. Available: https://2022.igem.wiki/montpellier/results
8. M. J. Kellner, J. G. Koob, J. S. Gootenberg, O. O. Abudayyeh, and F.
   Zhang, “SHERLOCK: nucleic acid detection with CRISPR nucleases.,”
   *Nat Protoc*, vol. 14, no. 10, pp. 2986–3012, Oct. 2019, doi:
   10.1038/s41596-019-0210-2.
9. A. East-Seletsky et al., “Two distinct RNase activities of CRISPR-C2c2
   enable guide-RNA processing and RNA detection,” *Nature*, vol.
   538, no. 7624, pp. 270–273, Oct. 2016, doi: 10.1038/nature19802.
10. J. S. Gootenberg, O. O. Abudayyeh, M. J. Kellner, J. Joung, J. J.
    Collins, and F. Zhang, “Multiplexed and portable nucleic acid detection
    platform with Cas13, Cas12a, and Csm6.,” *Science*, vol. 360, no.
    6387, pp. 439–444, Apr. 2018, doi: 10.1126/science.aaq0179.

## PrymFlow Lab

---

### Background information

### Background

The **SHERLOCK** method, like other CRISPR/Cas-based detection
techniques, is compatible with a **Lateral Flow readout format**. The
Lateral Flow Test is known for its speed, simplicity, and ease of interpretation.
This study aimed to adapt the SHERLOCK components, previously studied and optimized
by the SHERLOCK Lab, for use with Lateral Flow dipsticks, resulting in the
development of the **PrymFlow** test for detecting the presence of
***Prymnesium parvum***.

The **HybriDetect - Universal Lateral Flow Assay Kit** (Milena Biotec)
is the most commonly used dipstick for SHERLOCK assays and was also utilized in this
study. This test strip, based on lateral flow technology with Gold Nanoparticles
(GNPs), offers a simple and rapid method for developing custom rapid tests [1]. The
HybriDetect strip allows users to assess the status of the RNA reporter used in the
SHERLOCK method (for a detailed overview of the SHERLOCK method principle, see
Project Description page). In the Lateral Flow Assay procedure, a single-stranded
RNA reporter labeled with 6-Carboxyfluorescein (**6-FAM**) at the
**5′** end and **biotin** at the
**3′** end is used. Cas13-dependent cleavage of the reporter
results in the separation of these terminal labels, enabling the HybriDetect
dipstick to distinguish between intact and cleaved reporters, providing a
semi-quantitative readout [2].

### LFA result interpretation

If the target is present, the **T-line** becomes visible, indicating a
positive result. Meanwhile, the **C-line** serves as a control that is
less visible when greater amounts of the target sequence are present. Gold
nanoparticles (GNPs) with anti-FAM antibodies provide the visual indication of the
test result (**Figure 1**).

#### **WHAT HAPPENS WHEN *PRYMNESIUM PARVUM* IS NOT PRESENT IN THE WATER SAMPLE?**

Streptavidin, immobilized on the C-line, captures the **biotin-labeled**
ends of the intact reporters. The reporters are captured on the C-line, and the
binding of gold nanoparticles (GNPs) conjugated with anti-FAM antibodies makes only
the C (control) line visible.

#### **AND WHEN IT IS PRESENT?**

The reporters are cleaved by the activated Cas protein. Consequently, gold
nanoparticles (GNPs) with anti-FAM antibodies capture the FAM-labeled fragments,
which then bind to the anti-anti-FAM antibody immobilized on the test line (T-line),
producing a strong signal. This strong T-line indicates the presence of Prymnesium
parvum. Some intact reporters might remain in the mix (the amount depends on how
many target DNA molecules were present in the sample, influencing the ratio of
activated to non-activated Cas13 protein and the number of cleaved reporter
molecules). As a result, a weak control line (C-line) is visible.

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image12.png)

***Figure 1****: Explanation of
the lines visible on the Lateral Flow test during a negative and positive
result.*

[1] Milenia GenLine HybriDetect, Instructions for Use <https://www.milenia-biotec.com/uploads/2024/08/MGHD_1_IFU_D.pdf>
(Accessed 21.08.2024)

[2] Milenia GenLine HybriDetect, A Short Guide for Improved Readout-Performance <https://www.milenia-biotec.com/uploads/2019/07/improved-CRISPR-readout_final-1.pdf>
(Accessed 21.08.2024)

### 24.06. - 30.06

### First LFA test on Prymnesium genomic DNA

**Purpose:**The purpose of this experiment was to attempt the LFA
detection procedure for the first time and test whether all reagents work
correctly. Secondly, it aimed to see whether *Prymnesium*DNA
isolated from cultures can be directly detected
using the LFA.

The dual-labelled RNA reporter was resuspended in 697
μl of RNase-free water, as per the manufacturer’s instructions.

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image20.png)

***Figure
2****:
Manufacturer’s manual for the RNA reporter for use in the LFA
procedure.*

This experiment utilized
the GalF and GalR primers as well as PrymcrRNA1 and PrymcrRNA2.
The sequences as well as descriptions of these parts can be found in the iGEM
Registry. RPA was performed according to the “RPA Protocol” included
in the “Experiments” page, which was adapted from [3].

Samples labelled
“*Prymnesium* gDNA” indicate *Prymnesium parvum* genomic DNA isolated from our culture. For this
experiment, the sample labelled “DNA P 27/05” was used (see AlgaLab
documentation for a description of the culture’s origin).

In this experiment, 7 RPA
reactions were assembled:

***Table
1****: RPA samples
summarized.*

| **Sample number** | **Sample name\*** | **Template DNA used for RPA** | **RPA primers used** |
| --- | --- | --- | --- |
| 1 | RPA (-) | – | GalF + GalR |
| 2 | synDNA | synDNA | SynF + SynR |
| 3-4 | *Prymnesium* gDNA | *Prymnesium* gDNA | GalF + GalR |
| 5 | SHERLOCK (-) | – | GalF + GalR |
| 6 | RNase A (+) | – | – |
| 7 | RNase A (+) no inhibitor | – | – |

**\*Note**:
Sample names in Table 1 indicate the following:

* RPA (-): negative
  control for RPA – UltraPure water was  added instead of DNA
  to RPA reaction
* SHERLOCK (-):
  negative control for LFA detection – water added instead of RPA
  mixture for detection
* RNase A (+): RNAse
  A added instead of Cas13a to mixture for detection
* RNase A (+) no
  inhibitor: RNAse A added instead of Cas13a to mixture for detection,
  with no RNase inhibitor added to reaction mix.
* synDNA and
  syncrRNA. These samples serve as a positive control for the whole
  procedure since they utilize example sequences for testing SHERLOCK
  (Table 2 in [3]). These parts have also been included in the iGEM
  Repository by our team.

The same nomenclature has
been adapted throughout this notebook.

Next, SHERLOCK LFA
detection was carried out according to the “SHERLOCK LFA Protocol”
included in the “Experiments” page, which was adapted from
[3].

**SHERLOCK** **LFA
samples**:

1. RPA (-) +
   PrymcrRNA1/2\* (RPA negative control)
2. synDNA + syncrRNA
   (SHERLOCK positive control)
3. *Prymnesium* gDNA + PrymcrRNA1
4. *Prymnesium* gDNA + PrymcrRNA2
5. SHERLOCK (-) +
   PrymcrRNA1/2 (SHERLOCK negative control)
6. RNase (+)
7. RNase (+) no
   inhibitor\*\*

**\*Note**: *PrymcrRNA1/2* refers to an equimolar mixture of the two
PrymcrRNAs, with a 1:1 ratio of PrymcrRNA1 to PrymcrRNA2. This mixture was used
for the negative control to minimize the number of LFA strips needed. Since
neither of the individual crRNAs should yield a positive result, a positive
outcome for this negative control would indicate a need for further
investigation.

**\*\*Note**:
Reaction no. 7 was prepared after it was realized that
an RNase inhibitor had been mistakenly included in reaction no. 6, which was
intended to be the positive control. The negative result observed led to the
hypothesis that the RNase inhibitor might have caused the negative outcome. To
test this, reaction 7 consisted of 100 μl assay buffer, 2 μl RNase, and
1.2 μl LFA probe, with the dipstick being directly placed in the solution for
detection.

**Results**

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image14.jpg)

***Figure
3****: LFA test
result 28/06/2024.*

***Table
2****: SHERLOCK LFA
samples and results summarized.*

| **Strip number** | **Input sample name** | **crRNA added** | **Result** | **Comment(s)** |
| --- | --- | --- | --- | --- |
| 1 | RPA (-) | PrymcrRNA1/2 | Positive | Unexpected positive result for negative control |
| 2 | synDNA | syncrRNA | Fully positive, only T-band visible | Expected positive result for positive control |
| 3 | *Prymnesium* gDNA | PrymcrRNA1 | Positive |  |
| 4 | *Prymnesium* gDNA | PrymcrRNA2 | Negative |  |
| 5 | SHERLOCK (-) | PrymcrRNA1/2 | Negative | As expected |
| 6 | RNase (+) | – | Negative | Unexpected negative result for positive control |
| 7 | RNase (+) no inhibitor | – | Slightly positive |  |

**Conclusions**:

* The results validate that
  the **procedure and all
  test components function as intended**, as evidenced by a strong positive result for
  the synDNA + syncrRNA positive control and a negative result for the
  SHERLOCK (-) + PrymcrRNA1/2 negative control.
* However, an **unexpected positive result was observed for
  the RPA (-) control**,
  an issue previously noted in the fluorescent readout tests (as
  documented in the SHERLOCK Lab records on 24/06/2024). This suggests
  a **possible interaction
  between PrymcrRNA1 and one of the RPA primers: GalF or
  GalR**. The negative
  outcome for the SHERLOCK (-) control further supports the hypothesis
  that one of the RPA components is responsible for this issue. In future
  experiments, separate RPA (-) controls should be conducted for
  PrymcrRNA1 and PrymcrRNA2 to verify preliminary fluorescent readout
  results indicating that PrymcrRNA1, and not PrymcrRNA2, is causing this
  problem.
* It's unusual that reaction 7 did not yield
  a fully positive result, which could be attributed to its preparation
  differing from the other reactions (as noted earlier). This reaction
  should be repeated with identical conditions and incubation as the other
  samples to ensure consistent results.

[3] Kellner, Max J., Jeremy G. Koob, Jonathan S.
Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid
Detection with CRISPR Nucleases.” *Nature
Protocols* 14, no. 10 (October
2019):
2986–3012. <https://doi.org/10.1038/s41596-019-0210-2>.

### 01.07. - 07.07

### RPA on Alt and Mod primers

**Purpose**: The
purpose of this experiment was to test the new
ModF & ModF and AltF & AltR primers.

We performed RPA using
primers that were designed based on those described in the paper [4]. These
primer sequences were obtained by the GenomeLab, as detailed in their
documentation. To facilitate transcription, the primers were modified by adding
a T7 promoter to the 5' end of the forward primer.

The sequences of the
primers used were as follows:

**Primers
"mod"/"M"**

PR-RPA-4-F-mod+T7:
GAAATTAATACGACTCACTATAGGGAATCATCGAACTTTTGAACGCAACTGG

PR-RPA-4-R-mod:
GTAGGCGGCCTACTAGCACGTCGGCACA

**Primers
"alt"/"A"**

RPA-alt-F+T7:
GAAATTAATACGACTCACTATAGGCTTCCAGGTTCCGCCTGGGAGCATGTTTCTTC

RPA-alt-R:
GTACCGGTGGGCGGAAACGCGACGACCTA

These primers have also
been added to the iGEM Repository by our team.

Primers were dissolved in
water to make a 100 μM stock solution according to the manufacturer's
instructions.

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/futur.png)

***Figure
4****:
Manufacturer’s manual for the Alt and Mod primers.*

Samples labeled
“*Prymnesium* gDNA” indicate *Prymnesium parvum* genomic DNA isolated from our culture. In this
experiment, the gDNA sample "NOW5 DNA" (concentration of 164 ng/μl,
sample origin is described in AlgaLab documentation). RPA was performed
according to previously described procedure.

***Table
3****: RPA samples
summarized.*

| **Sample number** | **Sample name** | **Template DNA used for RPA** | **RPA primers used** |
| --- | --- | --- | --- |
| 1-3 | *Prymnesium* gDNA (Mod) | *Prymnesium* gDNA | ModF + ModF |
| 4 | RPA (-) (Mod) | – | ModF + ModF |
| 5-7 | *Prymnesium* gDNA (Alt) | *Prymnesium* gDNA | AltF + AltR |
| 8 | RPA (-) (Alt) | *–* | AltF + AltR |

The RPA samples were stored at -20°C for use the
following day.

### LFA on Alt and Mod primers

**Purpose:**The
purpose of this experiment was to obtain a readout
from the RPA reactions conducted on 03/07/2024.

SHERLOCK LFA detection was
performed according to the established protocol. However, we did not follow the
correct order of adding components as outlined in the Kellner paper [3]. This
was an oversight, and care should be taken in future experiments to ensure that
components are added in the correct sequence.

**SHERLOCK** **LFA
samples**:

1. RPA (-) (Alt) +
   PrymcrRNA1
2. RPA (-) (Alt) +
   PrymcrRNA2
3. *Prymnesium* gDNA (Alt) + PrymcrRNA1
4. *Prymnesium* gDNA (Alt) + PrymcrRNA2
5. RPA (-) (Mod) +
   PrymcrRNA1
6. RPA (-) (Mod) +
   PrymcrRNA2
7. *Prymnesium* gDNA (Mod) + PrymcrRNA1
8. *Prymnesium* gDNA (Mod) + PrymcrRNA2
9. RNase (+)
10. RNase (+) no
    inhibitor

**Results**

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image1.jpg)

***Figure
5****: LFA test
result 05/07/2024.*

***Table
4****: SHERLOCK LFA
samples and results summarized.*

| **Strip number** | **Input sample name** | **crRNA added** | **Result** | **Comment(s)** |
| --- | --- | --- | --- | --- |
| 1 | RPA (-) (Alt) | PrymcrRNA1 | Negative |  |
| 2 | RPA (-) (Alt) | PrymcrRNA2 | Negative |  |
| 3 | *Prymnesium* gDNA (Alt) | PrymcrRNA1 | Negative |  |
| 4 | *Prymnesium* gDNA (Alt) | PrymcrRNA2 | Negative |  |
| 5 | RPA (-) (Mod) | PrymcrRNA1 | Negative |  |
| 6 | RPA (-) (Mod) | PrymcrRNA2 | Negative |  |
| 7 | *Prymnesium* gDNA (Mod) | PrymcrRNA1 | Negative |  |
| 8 | *Prymnesium* gDNA (Mod) | PrymcrRNA2 | Negative |  |
| 9 | RNase (+) | – | Negative |  |
| 10 | RNase (+) no inhibitor | – | Negative |  |

**Conclusions:**

* **All tests
  returned negative results**,
  indicating that *Prymnesium* DNA was not detected. This outcome
  contrasts with previous experiments where negative controls showed
  unexpected positive results, suggesting that the issue
  of **false positives has
  been resolved by using different primer pairs**.
* However, *Prymnesium* gDNA has also not been detected
  This **might be due to a
  low concentration of the target DNA** – further testing is
  required.
* The positive
  control with RNase did not yield a positive result, regardless of the
  presence of an inhibitor. This could suggest that
  the **RNase used does
  not cleave the reporter as expected.**
* For future
  experiments, the synDNA samples will always be included as a positive
  control since the RNase positive control has proven unreliable.

[4] Luo, Huang, and Jiang,
“Establishment of Methods for Rapid Detection of Prymnesium Parvum by
Recombinase Polymerase Amplification Combined with a Lateral Flow
Dipstick.”

### 08.07. - 14.07

### PCR with KAC39, ITS and Gal primers

**Purpose**:

The purpose of this
experiment was to evaluate the usefulness of three sets of primers for PCR
amplification of *Prymnesium* gDNA, intended for use as input in future LFA
(Lateral Flow Assay) tests.

This was done to address
previously encountered issues with the low concentration
of *Prymnesium* gDNA. **By
PCR-amplifying the detected fragment, we aimed to
obtain a higher concentration of the target DNA for more reliable testing
and to enable precise quantification of its
concentration**.

Direct measurement of gDNA
concentration is unreliable because our algal cultures were established from
an **environmental
sample** taken from a water
body affected by *Prymnesium
parvum* blooms, meaning they
are not pure cultures and may contain DNA from various other organisms.
Consequently, the gDNA isolated from these cultures likely includes DNA from
these other organisms in addition to *Prymnesium parvum*.
When DNA concentration is measured using a NanoDrop
Microspectrophotometer, it quantifies all DNA present in the sample, of
which *Prymnesium
parvum*DNA might constitute
only a small percentage.

PCR was carried out using
3 sets of primers:

* KAC39
  primers:

  + Forward:
    ATCATTACCGGTCTTTCCACCCAC
  + Reverse: GAGTCCAATGGTGCGCGC
* ITS
  primers:

  + ItsF:
    TCCGTAGGTGAACCTGCGG
  + ItsR:
    TCCTCCGCTTATTGATATGC
* Gal
  primers:

  + Gal\_T7:
    GAAATTAATACGACTCACTATAGGGTGTCTGCCGTGGACTTAGTGCT
  + GalR:
    ATGGCACAACGACTTGGTAGG

The origins of these
sequences have been described in the GenomeLab documentation. The sequences
themselves have been added to the iGEM Repository by our team.

PCR and post-PCR clean-up
were carried out according to the “PCR on *Prymnesium parvum* genome Protocol” included in the
“Experiments” page, with the exception that
a **gradient of primer annealing
temperatures was used: 55°C, 60°C, 65°C and
70°C**to assess
temperature where we get the most pure amplicon.

A mix of the “NOW
5.1” and “Maj1” samples of *Prymnesium* gDNA
were used (sample origin is described in
AlgaLab documentation).

Anticipated PCR product
lengths were:

* 157 bp for
  Galluzzi RPA primers
* 672 bp for KAC39
  primers
* 760 bp for ITS
  primers

**Results**

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image3.png)

***Figure
6****: PCR result
from 08/07/2024.*

**Note:** The gel did not run evenly, which caused the bands
for the KAC39 primers to appear higher than their actual position. For future
experiments, ensure that a ladder is included on both sides of the gel to avoid
this issue.

**Observations:**

* The **Galluzzi**RPA primers **failed**to amplify any product.
* The **ITS** primers did amplify a product,
  but **multiple
  bands** were
  observed, indicating non-specific amplification.
* The **KAC39** primers, at annealing temperatures of
  65°C and 70°C, produced a **single product of the correct
  length**. This product
  was successfully purified and will be used in future experiments.

The measured concentration of the obtained KAC39 PCR
product (pooled samples from 65°C and 70°C annealing temperatures) was
98 ng/μl.

**Conclusions**:

* The KAC39 primers are the most effective for
  this procedure and will be used for all subsequent experiments at
  70°C annealing temperature.

### LFA test on Gal PCR product

**Purpose**:
The purpose of this experiment was to evaluate the LFA
procedure using PCR-amplified *Prymnesium* gDNA
as the input.

PCR using the Gal primers
was carried out by the AlgaLab and its results are included in their
documentation.

This experiment utilized
the RPA reactions performed by the SHERLOCK Lab on 28/06/2024. Table 5
summarizes the components of these samples, however the numbers and formatting
has been changed in relation to the original in the SHERLOCK Lab
documentation.

***Table
5****: RPA samples
summarized.*

| **Sample number** | **Sample name** | **Template DNA used for RPA** | **RPA primers used** |
| --- | --- | --- | --- |
| 1 | Gal PCR 200 nM | DNA PCR P+Maj2\* (200 nM) | GalF + GalR |
| 2 | Gal PCR 200 pM | DNA PCR P+Maj2\* (200 pM) | GalF + GalR |
| 3 | Gal PCR 200 fM | DNA PCR P+Maj2\* (200 fM) | GalF + GalR |
| 4 | RPA (-) | – | GalF + GalR |
| 5 | synDNA | synDNA | SynF + SynR |

\*DNA PCR P+Maj2 refers to
a sample of the PCR-amplified Prymnesium gDNA using Gal primers. Since the
reaction was performed by the AlgaeLab, this is their naming convention.
Concentrations of this PCR product after clean-up are given in brackets.

SHERLOCK LFA detection was
carried out according to the established protocol.

**SHERLOCK LFA
samples**:

1. RPA (-) +
   PrymcrRNA1
2. RPA (-) +
   PrymcrRNA2
3. Gal PCR 200 nM +
   PrymcrRNA1
4. Gal PCR 200 pM +
   PrymcrRNA1
5. Gal PCR 200 fM +
   PrymcrRNA1
6. Gal PCR 200 nM +
   PrymcrRNA2
7. Gal PCR 200 pM +
   PrymcrRNA2
8. Gal PCR 200 fM +
   PrymcrRNA2
9. synDNA +
   syncrRNA

**Results**

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image5.jpg "LFA 08.07.2024")

***Figure
7****: LFA test
result 08/07/2024.*

***Table
6****: SHERLOCK LFA
samples and results summarized.*

| **Strip number** | **Input sample name** | **crRNA added** | **Result** | **Comment(s)** |
| --- | --- | --- | --- | --- |
| 1 | RPA (-) | PrymcrRNA1 | Positive | Unexpected positive result for negative control |
| 2 | RPA (-) | PrymcrRNA2 | Negative | Expected negative result for negative control |
| 3 | Gal PCR 200 nM | PrymcrRNA1 | Positive | Result cannot be reliably interpreted |
| 4 | Gal PCR 200 pM | PrymcrRNA1 | Positive | Result cannot be reliably interpreted |
| 5 | Gal PCR 200 fM | PrymcrRNA1 | Positive | Result cannot be reliably interpreted |
| 6 | Gal PCR 200 nM | PrymcrRNA2 | Positive |  |
| 7 | Gal PCR 200 pM | PrymcrRNA2 | Positive | Primary LOD is between 200 pM and 200 fM |
| 8 | Gal PCR 200 fM | PrymcrRNA2 | Negative |
| 9 | synDNA | syncrRNA | Fully positive, only T-band visible |  |

**Conclusions**:

The results of this experiment align with the
observations made in the fluorescent readout tests by the SHERLOCK Lab (24,
28/06/2024:

* The combination of PrymcrRNA1 and Gal primers
  causes false positives, confirming previous concerns about its
  specificity. Therefore, results of **PCR-amplified*****Prymnesium*****DNA for strips 3-5 cannot reliably be
  interpreted**. As a
  result, **PrymcrRNA1
  will not be used in combination with Gal primers in future
  experiments**.

Positive results for PrymcrRNA2 with Gal primers were
observed for both 200 nM and 200 pM concentrations, but not for the 200 fM
concentration. This suggests that the Limit of Detection (**LOD**)
for this primer and crRNA combination **likely falls between 200 pM and 200
fM**.

### 29.07. - 04.08

### LFA test on Prymnesium genomic DNA – repeat

**Purpose**: The purpose of this experiment was to repeat the
SHERLOCK reaction conducted on 19/07/2024 to confirm the previously obtained
results.

**SHERLOCK samples**:

1. L1 Mod-Gal + PrymcrRNA1
2. Szczecin  Mod-Gal + PrymcrRNA1
3. RPA (-) Mod-Gal + PrymcrRNA1
4. KAC39 PCR (480 nM) Mod-Gal + PrymcrRNA1
5. synDNA + syncrRNA

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image4.jpg)

***Figure
12****: LFA test
results 29/07/2024.*

***Table
13****: SHERLOCK LFA
samples and results summarized.*

| **Strip number** | **Input sample name** | **crRNA added** | **Result** | **Comment(s)** |
| --- | --- | --- | --- | --- |
| 1 | L1 Mod-Gal | PrymcrRNA1 | Positive | T-line less intense than C-line |
| 2 | Szczecin Mod-Gal | PrymcrRNA1 | Positive | T-line more intense than C-line |
| 3 | RPA (-) Mod-Gal | PrymcrRNA1 | Negative |  |
| 4 | KAC39 PCR (480 nM) Mod-Gal | PrymcrRNA1 | Positive | T-line more intense than C-line |
| 5 | synDNA | syncrRNA | Fully positive | Only T-line visible |

**Conclusions**:

Results remained **consistent with the results from
22/07/2024**. The test
successfully detected *Prymnesium* gDNA in samples isolated from our algal culture
("Szczecin" samples) as well as in samples directly obtained from a
water body affected by Prymnesium parvum blooms ("L1" samples).
Indicating that our **LFA test
can detect the*****Prymnesium
parvum’s*****genomic DNA obtained directly from water
samples.**

### Hand incubation vs thermocycler incubation

**Purpose**: The purpose of this experiment was to compare the
effectiveness of incubating the RPA and SHERLOCK reactions using body heat
versus thermocycler incubation. This was done to
assess **whether the reaction
can be effectively carried out by the user holding it in their
hand.**

RPA and SHERLOCK reactions were carried out according
to the established protocol. Each of the five RPA reactions was prepared in
duplicate: one set was incubated in a thermocycler, and the other set was
incubated in the palm of a hand. Post-RPA samples that were hand-incubated were
also hand-incubated for the SHERLOCK reaction, while those incubated in the
thermocycler continued to be incubated in the thermocycler for the SHERLOCK
reaction.

***Table
14****: RPA samples
summarized.*

| **Sample number** | **Sample name** | **Template DNA used for RPA** | **RPA primers used** |
| --- | --- | --- | --- |
| 1, 6 | KAC39 PCR (480 nM) | KAC39 PCR (480 nM) | ModF + GalR |
| 2, 7 | KAC39 PCR (200 fM) | KAC39 PCR (200 fM) | ModF + GalR |
| 3, 8 | Szczecin | Szczecin | ModF + GalR |
| 4, 9 | L1 | L1 | ModF + GalR |
| 5, 10 | RPA (-) | – | ModF + GalR |

**SHERLOCK samples:**

1. KAC39 PCR (480 nM) + PrymcrRNA1
2. KAC39 PCR (200 fM) + PrymcrRNA1
3. Szczecin + PrymcrRNA1
4. L1 + PrymcrRNA1
5. RPA (-) + PrymcrRNA1

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image18.jpg)

***Figure
13****: LFA test
results from 29/07/2024. Samples incubated using the thermocycler are
labeled in red, while those incubated by hand (palm of the hand) are denoted
in black.*

***Table
15****: SHERLOCK LFA
samples and results summarized.*

| **Strip number** | **Input sample name** | **crRNA added** | **Result** | **Comment(s)** |
| --- | --- | --- | --- | --- |
| 1 (black) | KAC39 PCR (480 nM) | PrymcrRNA1 | Positive | T-line more intense than C-line |
| 2 (black) | KAC39 PCR (200 fM) | PrymcrRNA1 | Negative |  |
| 3 (black) | Szczecin | PrymcrRNA1 | Negative | Extremely faint T-line visible, but despite this interpreted as negative result |
| 4 (black) | L1 | PrymcrRNA1 | Negative |  |
| 5 (black) | RPA (-) | PrymcrRNA1 | Negative | T-line faint |
| 1 (red) | KAC39 PCR (480 nM) | PrymcrRNA1 | Positive | T-line more intense than C-line |
| 2 (red) | KAC39 PCR (200 fM) | PrymcrRNA1 | Negative | Extremely faint T-line visible, but despite this interpreted as negative result |
| 3 (red) | Szczecin | PrymcrRNA1 | Negative | Extremely faint T-line visible, but despite this interpreted as negative result |
| 4 (red) | L1 | PrymcrRNA1 | Negative |  |
| 5 (red) | RPA (-) | PrymcrRNA1 | Negative |  |

**Conclusions**:

The test results for both thermocycler and hand
incubation methods **do not show
significant differences**,
suggesting that hand incubation is as effective as thermocycler incubation for
this protocol. However, only test 1 one the tested conditions yielded a positive
result, indicating that there may be issues with either the RPA reaction
reagents or the DNA templates used in the other tests, since other conditions
have been positive in previous tests. **This inconsistency in results points to the need for
further optimization or troubleshooting of the reaction components to ensure
reliable outcomes across all tests.**

### Limit of detection (LOD) determination

**Purpose**: The purpose of this experiment was to determine the
final Limit of Detection (LOD) for our LFA test.

The Limit of Detection (LOD) was determined as the
lowest concentration of PCR-amplified Prymnesium gDNA for which the test result
remained positive, indicated by the presence of a visible T-line. For this
experiment, samples were incubated in a PCR thermocycler.

***Table
16****: RPA samples
summarized.*

| **Sample number** | **Sample name** | **Template DNA used for RPA** | **RPA primers used** |
| --- | --- | --- | --- |
| 1 | 200 nM | KAC39 PCR (200 nM) | ModF + GalR |
| 2 | 20 nM | KAC39 PCR (20 nM) | ModF + GalR |
| 3 | 2 nM | KAC39 PCR (2 nM) | ModF + GalR |
| 4 | 200 pM | KAC39 PCR (200 pM) | ModF + GalR |
| 5 | 40 pM | KAC39 PCR (40 pM) | ModF + GalR |
| 6 | 20 pM | KAC39 PCR (20 pM) | ModF + GalR |
| 7 | 10 pM | KAC39 PCR (10 pM) | ModF + GalR |
| 8 | 5 pM | KAC39 PCR (5 pM) | ModF + GalR |
| 9 | 2 pM | KAC39 PCR (2 pM) | ModF + GalR |
| 10 | 1 pM | KAC39 PCR (1 pM) | ModF + GalR |
| 11 | 200 fM | KAC39 PCR (200 fM) | ModF + GalR |
| 12 | 20 fM | KAC39 PCR (20 fM) | ModF + GalR |
| 13 | 2 fM | KAC39 PCR (2 fM) | ModF + GalR |
| 14 | L1 | L1 | ModF + GalR |
| 15 | Szczecin | Szczecin | ModF + GalR |
| 16 | RPA (-) | – | ModF + GalR |
| 17 | synDNA | synDNA | SynF + SynR |

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image16.jpg)

***Figure
14****: LFA test
results from 30/07/2024.*

Conclusions:

* Tests 1-13 were conducted using progressively
  decreasing amounts of *Prymnesium parvum* DNA, as reflected by the corresponding
  decrease in test band intensity. By comparing these with test 16, a
  negative control, it can be estimated that the **limit of detection (LOD) for our Sherlock
  assay is approximately 10 pM**, as indicated by test 8. However, this
  conclusion is somewhat subjective, given that band intensity was not
  quantified. To obtain a more precise and reliable LOD determination,
  this test should be repeated.
* The synDNA positive control (stripe 17) yielded
  a positive result. However, the test performed on older RPA samples
  previously provided a more definitive positive result (C-line was not
  visible). This suggests there may be inefficiencies in the RPA procedure
  or potential contamination of its reagents, which could be affecting the
  consistency of the results.

The tests for both the L1 (water DNA isolation) and
Szczecin samples returned negative results, indicating
that *Prymnesium
parvum* DNA was not detected
in these samples. This is likely due to unidentified issues within the
procedure, possibly linked to the RPA step or sample handling.

### One-pot SHERLOCK LFA attempt no. 2

**Purpose:** The purpose of this experiment was to test out the
one-pot Sherlock procedure using **proportions and concentrations similar to those in
the two-pot** procedure
instead of ones suggested in Kellner et al.

Half of the tested samples were incubated in the palm
of a hand and half in a thermocycler set to 37°C. Different incubation times
were also tested, some reactions were incubated for 30 minutes, some for 60
min.

***Table
23****: 1-pot
Sherlock mix components..*

| **Component** | **Volume x1 [μl]** | **One-pot mix (volume x4.5) [μl]** |
| --- | --- | --- |
| **RPA primer F** | 2.24 | 10.08 |
| **RPA primer R** | 2.24 | 10.08 |
| **RPA rehydration buffer** | 7.34 | 33.03 |
| **LwaCas13a in SB DTT** | 2 | 9 |
| **LFA sensor** | 0.2 | 0.9 |
| **RNAse inhibitor** | 1 | 4.5 |
| **rNTP mix** | 0.8 | 3.6 |
| **T7 polymerase (5U/μl)** | 0.5 | 2.25 |
| **MgCl****2** | 0.18 | 0.81 |
| **MgAc** | 1.25 | 5.625 |
| **Sum** | 17.75 | 79.875 |

**Procedure:**

* Two one-pot mixes were prepared, one with ModF
  + GalR RPA primers, one with syn RPA primers.
* 4.5 μl of crRNA (PrymcrRNA 1 or syn) was
  added.
* 75 μl of a mix was added per RPA reaction
  aliquot. Such reaction mix was pipetted up and down until the
  lyophilizate was dissolved.
* 18.75 μl of reaction mix was transferred per
  PCR tube.
* 1.25 μl of DNA (PrymcrRNA 1 or syn) was
  added per PCR tube

***Table
24****: 1-pot
Sherlock samples summarized.*

| **Sample number** | **Sample name** | **Template DNA used for RPA** | **RPA primers used** |
| --- | --- | --- | --- |
| 1 | KAC PCR thermocycler 60 min | KAC PCR | ModF + GalR |
| 2 | KAC PCR thermocycler 30 min | KAC PCR | ModF + GalR |
| 3 | KAC PCR hand 60 min | KAC PCR | ModF + GalR |
| 4 | KAC PCR hand 30 min | KAC PCR | ModF + GalR |
| 5 | synDNA thermocycler 60 min | synDNA | SynF + SynR |
| 6 | synDNA thermocycler 30 min | synDNA | SynF + SynR |
| 7 | synDNA hand 60 min | synDNA | SynF + SynR |
| 8 | synDNA hand 30 min | synDNA | SynF + SynR |

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image17.jpg "453868356_1008523960961457_5470321161705542432_n.jpg")

***Figure
17****: LFA test
results from 1/08/2024.*

***Table
25****: SHERLOCK LFA
samples and results summarized.*

| **Strip number** | **Input sample name** | **crRNA added** | **Result** | **Comment(s)** |
| --- | --- | --- | --- | --- |
| 1 | KAC PCR thermocycler 60 min | PrymcrRNA1 | Negative |  |
| 2 | KAC PCR thermocycler 30 min | PrymcrRNA1 | Negative |  |
| 3 | KAC PCR hand 60 min | PrymcrRNA1 | Negative |  |
| 4 | KAC PCR hand 30 min | PrymcrRNA1 | Negative |  |
| 5 | synDNA thermocycler 60 min | syncrRNA | Negative |  |
| 6 | synDNA thermocycler 30 min | syncrRNA | Negative |  |
| 7 | synDNA hand 60 min | syncrRNA | Negative |  |
| 8 | synDNA hand 30 min | syncrRNA | Negative |  |

**Conclusions**:

Since all of the tests gave a negative
result,**there is a chance that
the 1-pot procedure described in Kellner et al. was inadequate or that the
alterations made for this experiment were not suitable.**

### Reduced incubation time attempt

**Purpose:** The purpose of this experiment was to determine if
performing the LFA detection of*Prymnesium parvum* in half of the standard time would produce
significant results.

Labeling of the samples refers to the RPA incubation
times. When RPA time was reduced, the Sherlock reaction time was also half the
standard protocol time.

***Table
26****: RPA samples
summarized.*

| **Sample number** | **Sample name** | **Template DNA used for RPA** | **RPA primers used** |
| --- | --- | --- | --- |
| 1 | KAC PCR thermocycler 30 min | KAC PCR | ModF + GalR |
| 2 | KAC PCR thermocycler 15 min | KAC PCR | ModF + GalR |
| 3 | KAC PCR hand 30 min | KAC PCR | ModF + GalR |
| 4 | KAC PCR hand 15 min | KAC PCR | ModF + GalR |
| 5 | synDNA thermocycler 30 min | synDNA | SynF + SynR |
| 6 | synDNA thermocycler 15 min | synDNA | SynF + SynR |
| 7 | synDNA hand 30 min | synDNA | SynF + SynR |
| 8 | synDNA hand 15 min | synDNA | SynF + SynR |

**Sherlock samples:**

1. KAC PCR thermocycler 30 min + PrymcrRNA1
2. KAC PCR thermocycler 15 min + PrymcrRNA1
3. KAC PCR hand 30 min + PrymcrRNA1
4. KAC PCR hand 15 min + PrymcrRNA1
5. synDNA thermocycler 30 min + syncrRNA
6. synDNA thermocycler 15 min + syncrRNA
7. synDNA hand 30 min + syncrRNA
8. synDNA hand 15 min + syncrRNA

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image2.jpg "453791794_3759303107643436_6887006336096453930_n.jpg")

***Figure
18****: LFA test
results from 1/08/2024.*

***Table
27****: SHERLOCK LFA
samples and results summarized.*

| **Strip number** | **Input sample name** | **crRNA added** | **Result** | **Comment(s)** |
| --- | --- | --- | --- | --- |
| 1 | KAC PCR thermocycler 30 min | PrymcrRNA1 | Negative | Extremely faint T-line visible, but despite this interpreted as negative result |
| 2 | KAC PCR thermocycler 15 min | PrymcrRNA1 | Negative | Extremely faint T-line visible, but despite this interpreted as negative result |
| 3 | KAC PCR hand 30 min | PrymcrRNA1 | Positive |  |
| 4 | KAC PCR hand 15 min | PrymcrRNA1 | Negative | Extremely faint T-line visible, but despite this interpreted as negative result |
| 5 | synDNA thermocycler 30 min | syncrRNA | Positive |  |
| 6 | synDNA thermocycler 15 min | syncrRNA | Positive |  |
| 7 | synDNA hand 30 min | syncrRNA | Positive |  |
| 8 | synDNA hand 15 min | syncrRNA | Positive |  |

**Conclusions**:

A positive result was
obtained after a shortened incubation time for the synDNA samples, but this was
not observed in all samples containing Prymnesium parvum DNA. This inconsistency
might be due to the poor quality of the original DNA, as the test was conducted
on KAC39 PCR products, which showed blurred bands on the PCR gel. However, based
on preliminary observations, the **T-band intensity in the synDNA samples did not
significantly decrease despite the shortened incubation
time**. This suggests that the
incubation period could potentially be reduced, allowing
for **quicker test
results**.

That said, **further experiments are needed** to confirm whether the incubation time can indeed
be shortened to 15 minutes. Additionally, a detailed investigation should be
conducted to determine how the reduced incubation time affects the limit of
detection (LOD).

## **Algae Lab**

### Research Purpose

The main purpose of the Algae lab was to create and maintain *Prymnesium parvum*
laboratory culture in order to isolate DNA of the golden algae, which was necessary for
further experiments with the SHERLOCK system. Furthermore, the lab aimed to optimise
culture conditions by testing different sea salt concentrations and lighting conditions.

### 06-12.05

### Introduction to the Lab

Reviewing the Occupational Safety and Health (BHP) Principles and On-the-Job Training.

### F/2 Medium Preparation

The medium preparation protocol is also available on the Experiments page under the F/2
medium preparation protocol.

F/2 is a widely utilised medium specifically designed for cultivating coastal marine
algae, although it also works well for freshwater species.

Prior to preparing the medium, 100x concentrated stock solutions of each medium
ingredient were made. Table 1 shows the final concentrations.

Table 1: F/2 medium
ingredients.

|  |  |
| --- | --- |
| **Ingredient name** | **Final concentration [mg/L]** |
| ZnSO4 · 7 H2O | 22 |
| CoCl2 · 6 H2O | 10 |
| MnCl­2 · 4 H2O | 180 |
| Na2Mo4·2 H2O | 6 |
| NaNO3 | 75 |
| NaH2PO4 · 2 H2O | 56.5 |
| Na2EDTA | 4160 |
| FeCl­3 · 6 H2O | 3150 |
| CuSO4 · 5 H2O | 10 |

F/2 medium also contains sea salt, which is added after mixing all the other ingredients.
The default concentration of sea salt is 16 g/L.

Total volume of 800 ml was prepared, which contains:

* 8 ml of each of 9 stocks concentrated 100x (72 ml in total)
* 728 ml of distilled water
* 12.8 g of sea salt (16 g/L)

The medium was divided into two bottles: one containing 300 ml and the other 400 ml.
Since the total volume was too large for sterilisation, 100 ml was discarded. To prepare
F/2 agar medium for future cultures on plates, 3 g of agar was added to the 300 ml
portion. Both portions were then sterilised using a microwave autoclave.

##### Vitamin stock solutions

1000x concentrated stock solutions of vitamins B1, B7, B12 were prepared, each with a
volume of 6 ml. Table 2 shows the final concentration of each vitamin solution:

Table 2: Final concentration of
vitamin solutions.

|  |  |
| --- | --- |
| **Vitamin** | **Concentration [mg/L]** |
| B1 | 100 |
| B7 | 0.5 |
| B12 | 0.5 |

The stocks were sterilised by filtration with a membrane filter.

**Sterilisation procedure:**

* Previously prepared vitamin solution was taken in sterile conditions with a syringe
  and a needle.
* The needle was removed and a membrane filter was attached to the syringe.
* The solution was filtered to a new sterile tube and secured.

The vitamins cannot be added directly to the F/2 medium, as they degrade under the
extreme conditions of the sterilisation process. They are added separately to every new
liquid culture. In this notebook, the combination of vitamins B1, B7, B12 will be
referred to as “the vitamins”.

### 13-19.05

### Environmental Samples from Oder Water

Water samples were collected from the Czernica reservoir in Lower Silesia, Poland. This
water body is directly connected to the main channel of the Oder River and has
experienced recurring *Prymnesium parvum* blooms since the Oder River disaster in
2022.

Three samples of 1.5 L were collected from three distinct places:

1. Middle of the lake

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image13.png)

**Figure 1.** Environmental sample collection site (middle of the
lake).

2. Bay area

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image31.png)

**Figure 2.** Environmental sample collection site (bay area).

3. The outlet to the Oder River

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image7.png)

**Figure 3.** Environmental sample collection site (the outlet to the
Oder River).

A sample of river mud was also collected from the same place as sample no. 2. For each
place, there is a sample containing vitamins (W) and a sample with no vitamins added (BW).
Therefore, the names of the samples are: 1W, 1BW, 2W, 2BW, 3W, 3BW.

1L of each water sample was transferred to a separate water bottle, and vitamins B1, B7,
and B12 were added to the cultures. Vitamins were purchased from the pharmacy and were
in pill form that contained only the given vitamin (not multivitamins). Before adding to
the water sample, they were ground in a mortar. Table 4 shows the vitamin content in the
water samples.

Table 4: Vitamin content [mg]
in Oder water samples.

|  |  |  |  |
| --- | --- | --- | --- |
| **Water sample** | **B1** | **B7** | **B12** |
| 1W | 33 | 10 | 0 |
| 2W | 33 | 10 | 0 |
| 3W | 100 | 10 | 0.5 |

### Prymnesium parvum Samples from Gdańsk

Two centrifuged samples of *Prymnesium parvum* were obtained from Gdańsk. The
samples were obtained in the form of sediments. One of the samples was fully utilised
for DNA isolation, while the other was used to establish liquid cultures.

### Microscopic Imaging of Water and Culture Samples

**Purpose:** Observation of Oder water samples and received *Prymnesium
parvum* samples and identification of *Prymnesium parvum* based on
morphological features.

This protocol is also available on the Experiments page under the Preparing liquid algal
cultures samples for imaging protocol.

##### Preparation of a Sample for Imaging

* Transfer 1-1.5 mL of water/culture sample into Eppendorf tube.
* Centrifuge using table-top centrifuge at 2000 rpm for 1 minute.
* Discard supernatants.
* Resuspend the pellet in the small volume left in the tube after discarding the
  supernatant.
* Apply sample on glass slide, cover with coverslip.
* Image sample using the 40x air objective.
* Use the TopView Software.
* Use fluorescence to image chloroplasts.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image12.jpg)

**Figure 4.** 2W Oder water sample – microscopic image.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image32.jpg)

**Figure 5.** 1W Oder water sample – microscopic image.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image19.jpg)

**Figure 6.** 1BW Oder water sample – microscopic image.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image27.jpg)

**Figure 7.** 2BW Oder water sample – microscopic image.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image18.jpg)

**Figure 8.** 3BW Oder water sample – fluorescent microscopic image.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image11.png)

**Figure 9.** *Prymnesium parvum* Gdańsk sample – microscopic
image.

##### Notes

* We can add some glycerol to a sample (1:1 with water, for example) to slow down
  algae movements to make imaging easier.
* We could also glue the coverslip to the slide next time.
* When saving images, save them to a folder on the computer and only later transfer
  everything to a disk/pendrive.

### Preparation of Liquid Algal Cultures

This protocol is also available on the Experiments page under the **Setting up
liquid algal cultures protocol**.

All liquid cultures were prepared under sterile conditions using sterilised tools and
culture media. After preparation, the cultures were sealed and placed in the growth
chamber. This protocol applies to all future liquid cultures as well.

##### Oder Water Liquid Cultures

Six liquid algal cultures were prepared from the Oder water samples in previously
sterilised glass bottles. The volumes of the cultures were 80 ml, 40 ml, and 10 ml. Each
culture contained Oder water (or river mud) and the F/2 medium in a 1:1 ratio.

Table 4: Characteristics of the
liquid cultures from Oder water.

|  |  |  |  |
| --- | --- | --- | --- |
| **Culture volume** | **80 ml** | **40 ml** | **10 ml** |
| **Type of inoculated sample** | 1W | 2W | River mud |
|  | 1BW | 2BW | 3BW |

The Oder water samples already contain vitamins, therefore there was no need for extra
enrichment with the vitamins.

##### Gdańsk Liquid Cultures

20 µl of *Prymnesium parvum* sample was transferred into a 12-well plate
containing 3 ml of F/2 medium in 4 wells. Serial dilutions were made.

The rest of the sample was split and transferred into two flasks that contain 20 ml of
F/2 medium with the addition of the vitamins. Culture names: Gdańsk 1, Gdańsk 2.

### Preparation of Algal Cultures on Plates

This protocol is also available on the Experiments page under the **Establishing
algae cultures on solid medium protocol**.

All plate cultures were prepared under sterile conditions using sterilised tools and
media. After preparation, the cultures were secured and placed in the growth chamber.
This protocol applied to all future plate cultures as well.

Six culture plates with sterilised and cooled F/2 agar medium were prepared. Three of
them were inoculated with 100 µl of water samples 1BW, 2BW, and 3BW, and the remaining
three were inoculated with 100 µl of water samples 1W, 2W, and 3W.

### Algal DNA Isolation

**Purpose:** Collecting DNA of *Prymnesium parvum* in the largest
possible quantity.

The isolation protocol is available on the Experiments page under the **Algal DNA
isolation with QIAwave DNA blood and tissue kit protocol**.

DNA was isolated from 2W and Gdańsk 1 liquid cultures. Total culture volumes used for the
isolation:

* **Gdańsk 1** – 6 ml
* **2W** – 66 ml

The isolation was performed with the QIAwave DNA Blood & Tissue Kit (Qiagen), with some
modifications to the manufacturer's protocol: Purification of Total DNA from Animal
Tissues.

##### Steps:

* The cultures were split into 2 ml samples and centrifuged (5 min, 14,000 rpm).
  Supernatants were discarded, and pellets were collected.
* 20 µl of proteinase K and 180 µl of ATL buffer were added to each sample. The
  samples were mixed thoroughly by vortexing.
* The samples were incubated at 56°C for 7 hours with shaking.
* A pre-mix of 96% ethanol and AL buffer was prepared.
* After incubation, 400 µl of the ethanol-AL buffer pre-mix was added to each sample
  and mixed thoroughly by vortexing.
* For each sample, a DNeasy Mini Spin Column was put in a 2 ml Waste Tube provided in
  the isolation kit.
* The mixtures from step 5 were transferred into the DNeasy Spin Columns. The samples
  were centrifuged at 8000 rpm for 1 min. The flow-through was discarded.
* 500 µl of AW1 buffer was added to the columns, followed by centrifugation at 8000
  rpm for 1 min. The flow-through was discarded.
* 500 µl of AW2 buffer was added to the columns, followed by centrifugation at 14,000
  rpm for 3 min. The flow-through and Waste Tubes were discarded.
* The columns were put in 2 ml tubes, and 200 µl of AE buffer was pipetted directly
  onto the membrane of the column.
* The samples were incubated at room temperature for 1 min and centrifuged at 8000 rpm
  for 1 min.

As a result, 6 DNA solutions of 200 µl were obtained. Half of them come from our
*Prymnesium parvum* cultures (K samples – from “Kraków”), and the other half come
from Gdańsk (G samples – from “Gdańsk”). DNA concentration was measured with a NanoDrop
spectrophotometer.

Table 5: Concentration of the
isolated DNA.

|  |  |  |
| --- | --- | --- |
| **Sample name** | **DNA concentration [ng/µl]** | **A260/A280** |
| K1 | 6.5 | 1.47 |
| K2 | 4.8 | 1.50 |
| K3 | 5.8 | 1.34 |
| G1 | 8.2 | 1.64 |
| G2 | 14.9 | 1.46 |
| G3 | 19.5 | 1.09 |

**Legend:**

* **K1, K2, K3** – DNA isolated from Oder water liquid cultures
* **G1, G2, G3** – DNA isolated from Gdańsk liquid cultures

### PCR on the Isolated Algal DNA

##### Purposes

* Confirming that the isolated DNA contains the ITS2 sequence, which is specific to
  *Prymnesium parvum*. The sequence is 132 bp in length.
* Determining the optimal primer concentration for the reaction.

The PCR protocol is also available on the Experiments page under the **PCR reaction
on DNA isolated from algae cultures protocol**.

##### Primers Sequences

* **GalF** 5' TGTCTGCCGTGGACTTAGTGCT 3'
* **GalR** 5' ATGGCACAACGACTTGGTAGG 3'

The PCR reaction was performed on two DNA samples named Kz (DNA isolated from Oder liquid
cultures) and Gz (DNA isolated from Gdańsk liquid cultures), which were previously
concentrated in the SHERLOCK lab. DNA concentration:

* **Kz** – 170 ng/µl
* **Gz** – 32.5 ng/µl

Reaction volume was 20 µl. The reaction was performed using two different primer
concentrations.

##### Preparation of Primer Solutions

* **Concentration 200 nM:** Primers FOR and REV were pre-diluted 10x in
  one tube. 1 µl of each primer was added to 8 µl of pure water. Then 0.8 µl of the
  primers mixture was added to the reaction mixture.
* **Concentration 400 nM:** Primers FOR and REV were separately
  pre-diluted. 1 µl of each primer was added to 9 µl of pure water in two separate
  tubes. Then 0.8 µl of each primer was added to the reaction mixture.

Table 6: PCR samples
composition. Primers concentration 200 nM. Reaction volume = 20 µl

|  |  |  |
| --- | --- | --- |
| **PCR reagent** | **Volume [µl]** | |
|  | **Sample Gz** | **Sample Kz** |
| DNA template | 2 | 0,5 |
| PCR Mix (A&A Biotechnology) | 10 | 10 |
| Primers mix | 0,8 | 0,8 |
| Water | 7,2 | 8,7 |

Table 7: PCR samples
composition. Primers concentration 400 nM. Reaction volume = 20 µl

|  |  |  |
| --- | --- | --- |
| **PCR reagent** | **Volume [µl]** | |
|  | **Sample Gz** | **Sample Kz** |
| DNA template | 2 | 0,5 |
| PCR Mix (A&A Biotechnology) | 10 | 10 |
| FOR primer | 0,8 | 0,8 |
| REV primer | 0,8 | 0,8 |
| Water | 6,4 | 7,9 |

Table 8: PCR Reaction
Conditions.

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final elongation | 72 | 300 |

**Number of cycles:** 40

##### DNA Electrophoresis of the PCR Product

Electrophoresis was conducted on a 2% agarose gel at 85 V for 45 minutes. A total of 5 µl
of each PCR product was loaded into the gel wells. Two distinct DNA standards were used
for reference: the GeneRuler 1kb DNA Ladder and the O'GeneRuler DNA Ladder Mix.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image36.png)

**Figure 10.** Gel electrophoresis of the PCR product – result.

##### Legend:

* **O'GeneRuler DNA Ladder Mix**
* **Gz** primers 200 nM
* **Gz** primers 400 nM
* **Kz** primers 200 nM
* **Kz** primers 400 nM
* **GeneRuler 1kb DNA Ladder Mix**

##### Conclusions

Samples Gz and Kz contain the ITS2 sequence of 132 bp, with clear bands visible between
the 200 bp and 100 bp markers. No specific product was observed in row 2, likely due to
an error during the preparation of the reaction mix. Based on these results, it can be
concluded that the algal DNA isolated on 21/05/2024 contains *Prymnesium parvum*
genomic DNA.

There were no significant differences in product visibility across the different primer
concentrations tested, so a concentration of 200 nM will be used for future reactions.

### Preparation of Isolated DNA for Sequencing

##### Preparation of Isolated DNA for Sequencing

* DNA isolated from the 2W culture was concentrated.
* 250 µl of the precipitation buffer was added to 250 µl of DNA.
* The sample was centrifuged at 14,000 rpm for 10 minutes.
* The supernatant was discarded.
* The pellet was dried in an open tube for 20 minutes.
* The DNA pellet was suspended in 30 µl of ultrapure water.
* **DNA concentration was measured:** 33.9 ng/µl.
* The primers used in the previous PCR reaction were diluted to a concentration of 5
  µM in 20 µl of ultrapure water, following the manufacturer’s guidelines.
* The samples containing the PCR products and primers were then secured and sent to
  Genomed S.A. for sequencing.

### PCR with KAC 39 Primers

##### PCR with KAC 39 Primers

**Purpose:** Amplification of a specific fragment of *Prymnesium
parvum* genome that contains the ITS2 sequence. The fragment length is about 700
bp. To achieve this, KAC 39 primers, complementary to the desired DNA fragment, were
designed.

**KAC 39 Primers Sequences:**

* **FOR:** 5' ATCATTACCGGTCTTTCCACCCAC 3'
* **REV:** 5' GAGTCCAATGGTGCGCGC 3'

The PCR reaction was performed on two DNA samples named Kz (DNA isolated from Oder liquid
cultures) and Gz (DNA isolated from Gdańsk liquid cultures), which were previously
concentrated in the SHERLOCK lab. DNA concentrations were as follows:

* **Kz:** 170 ng/µl (11 samples)
* **Gz:** 32.5 ng/µl (2 samples)

The primers’ initial solution (100 µM) was diluted twice: 10x and 20x to the final
concentration of 500 nM:

* First dilution (10x): 2 µl + 2 µl + 16 µl (pure water)
* Second dilution (20x): 1 µl + 19 µl (reaction mix)

Table 9. PCR samples
composition. Reaction volume = 20 ul

|  |  |  |
| --- | --- | --- |
| **PCR reagent** | **Volume [µl]** | |
|  | **Sample Gz** | **Sample Kz** |
| DNA template | 2 | 0,5 |
| PCR Mix (A&A Biotechnology) | 10 | 10 |
| Primers mix | 1 | 1 |
| Water | 7 | 8,5 |

Table 10: PCR Reaction
Conditions

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 65 | 30 |
| Elongation | 72 | 60 |
| Final Elongation | 72 | 120 |

**Number of Cycles:** 40

##### Gel Electrophoresis of the PCR Product

Electrophoresis was conducted using a 2% agarose gel, run at 85 V for 45 minutes. 8 µl of
each PCR product was loaded into the gel wells. Two DNA standards were used: GeneRuler 1
kb DNA Ladder and O'GeneRuler DNA Ladder Mix.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image2.jpg)
**Figure 11.** Gel Electrophoresis of the PCR Products –
Results.

* **1** – O'GeneRuler DNA Ladder Mix
* **2 - 3** – DNA template Kz
* **4 - 14** – DNA template Gz
* **15** – GeneRuler 1 kb DNA Ladder

##### Conclusions

No specific product was obtained. Blurred stripes visible at the bottom of the gel are
probably primer dimers. Optimization of PCR conditions is necessary.

### 20-26.05

### F/2 Medium Preparation

##### F/2 Medium Preparation

The medium preparation protocol is also available on the Experiments page under the
**F/2 Medium Preparation Protocol**.

The F/2 medium was prepared by using previously made 100x concentrated stocks. The total
medium
volume: 400 ml, which contains:

* 4 ml of each of 9 stocks, 36 ml in total
* 364 ml of distilled water
* 6.4 g of sea salt (16 g/L)

After mixing all the ingredients, the medium was sterilised with a microwave autoclave.

### Plate Cultures – Microscopic Screening

##### Plate Cultures – Microscopic Screening

Algal plate cultures made 13/05 were observed under the microscope.

###### Notes

* The brown areas are mostly diatoms, the green spots contain organisms we are more
  interested in.
* Morphology of the organisms within the green spots varies between the plates and
  often also within one plate. For example, some are more round, and some more
  longitudinal.

###### Imaging Difficulties:

* The microscope is not fully tailored toward the shape of our plates. Hence the
  plates sometimes keep moving as they are being scanned, requiring frequent image
  sharpening.
* The agar is quite thick in certain places.
* There is a need to readjust the image sharpness when switching from microscope view
  to the computer screen.
* At times, it was nearly impossible to clearly see the large colonies of organisms
  within the green areas.

Our instructor, Dr Paweł Jedynak, suggests creating a large-volume liquid culture to
reduce the need for frequent algal culture passages and to have a backup culture in case
issues arise with the others. The algae are expected to grow very slowly, which is why a
high volume is recommended, likely in a dimly lit environment.

Another suggestion is that we create a culture in a bottle and place it in a dimly lit
place, similar to the Chlamydomonas cultures, to create a sustainable culture.

Most of the plate cultures did not contain *Prymnesium parvum* or other organisms
with similar morphological characteristics. The 1W plate was the only one on which a
colony potentially identifiable as *Prymnesium parvum* was observed.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image4.jpg)

**Figure 12.** 1W plate culture – microscopic image.

### New Oder Water Samples

##### New Oder Water Samples

Eight water samples of 0.5 L were taken from different places in the Oder river. The
samples were
observed under the microscope. *Prymnesium parvum* was present in sample number
5. Samples 3,
4 and 8 also looked promising.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image37.jpg)

**Figure 13.** Environmental sample collection site of NOW1 and NOW2.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image34.jpg)

**Figure 14.** Environmental sample collection site of NOW3.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image1.jpg)

**Figure 15.** Environmental sample collection site of NOW4 and NOW5.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image23.jpg)

**Figure 16.** Environmental sample collection site of NOW6.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image26.jpg)

**Figure 17.** Environmental sample collection site of NOW7.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image25.jpg)

**Figure 18.** Environmental sample collection site of NOW8.

### New Oder Water (NOW) Liquid Cultures

##### New Oder Water (NOW) Liquid Cultures

**Purpose:** New liquid cultures were set up in order to increase the amount
of biological
material for future DNA isolations.

This protocol is also available on the Experiments page under the **Setting up
liquid algal cultures
protocol**.

Five liquid cultures were made: NOW 3, NOW 4, NOW 5, NOW 5.1, NOW 8 (NOW – New Oder
Water). Based on
microscopic imaging, water sample number 5 contained the highest concentration of
*Prymnesium parvum*
cells, so an additional liquid culture, NOW 5.1, was prepared.

The cultures were made in glass flasks that contained portions of previously prepared F/2
medium
(salt 16 g/L). Oder water was transferred into the flask with F/2 medium. The Oder water
to F/2 medium
volume ratio was either 1:3 or 1:2, depending on the capacity of the flask. Stocks of
vitamins were
added to each flask.

Independently from the flask cultures, 8 mini-liquid cultures with a volume of 3 ml each
were set up in a
12-well plate. The cultures contained the same mix of F/2 medium and vitamins.

### New Oder Water 5 – Serial Dilutions

##### New Oder Water 5 – Serial Dilutions

Serial dilutions of the sample 5 from the New Oder Water were made on a 12-well plate.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image22.jpg)

**Figure 19.** Scheme of the serial dilutions performed.

All the wells of the plate were filled with 3 ml of F/2 medium. 1 ml of sample 5 was
added to wells
1A, 2A, 3A, and mixed by pipetting. Then, in each row, 1 ml from the A well was
transferred into the
B well and mixed. The procedure was repeated for the C and D wells. The vitamins were
added into each well.

### PCR Reactions with Galluzzi, KAC 39, and White Primers

##### PCR Reactions with Galluzzi, KAC 39, and White Primers

**Purposes:**

* Amplification of the ITS2 sequence from *Prymnesium parvum* genome with
  Galluzzi primers.
* Optimization of PCR reaction conditions for KAC 39 and White primers by performing a
  gradient PCR.

The PCR protocol is also available on the Experiments page under the **PCR reaction
on DNA isolated from algae cultures protocol**.

White primers were designed by White et. al. [2]. They amplify the same DNA sequence as
KAC 39 primers designed by our team members.

Table 11. Sequences of the
Primers Used in PCR.

|  |  |
| --- | --- |
| **PCR Primer Name** | **Sequence** |
| GalF | 5' TGTCTGCCGTGGACTTAGTGCT 3' |
| GalR | 5' ATGGCACAACGACTTGGTAGG 3' |
| KacF | 5' ATCATTACCGGTCTTTCCACCCAC 3' |
| KacR | 5' GAGTCCAATGGTGCGCGC 3' |
| ItsF\*\* | 5' TCCGTAGGTGAACCTGCGG 3' |
| ItsR\*\* | 5' TCCTCCGCTTATTGATATGC 3' |

\*Primers designed by Galluzzi et. al. [1], that were used in previous PCR reactions.
\*\* Primers designed by White et. al. [2].

The PCR reaction was performed on two DNA samples named K (DNA isolated from Oder liquid
cultures) and G (DNA isolated from Gdańsk liquid cultures), which were previously
concentrated in SHERLOCK lab. DNA concentration:

* **K:** 118.6 ng/µl
* **G:** 147.6 ng/µl

**Galluzzi PCR**

The primers’ initial solution 100 µM was diluted twice: 10x and 50x to the final
concentration of 200 nM.

* First dilution (10x): 1 µl + 1 µl + 8 µl (pure water)
* Second dilution (50x): 1 µl + 49 µl (reaction mix)

Table 12. PCR Samples
Composition. Reaction Volume = 50 µl

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA Template | 1 |
| PCR Mix (A&A Biotechnology) | 25 |
| Primer Mix | 1 |
| Water | 23 |

Table 13. PCR Reaction
Conditions (16/05 Source Description)

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final Elongation | 72 | 300 |

Table 14. PCR Samples
Composition – KAC 39 and White. Reaction Volume = 20 ul

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA Template | 0.5 |
| PCR Mix (A&A Biotechnology) | 10 |
| Primer Mix | 0.4 |
| Water | 9.1 |

Table 15. PCR Reaction
Conditions

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 95 | 180 |
| Denaturation | 95 | 30 |
| Annealing | 50/55/60/65/70 | 30 |
| Elongation | 72 | 30 |
| Final Elongation | 72 | 600 |

##### Gel Electrophoresis of the PCR Product

Electrophoresis was carried out on a 2% agarose gel at 85 V for 45 minutes. 8 µl of each
PCR product were loaded into the gel wells. Two distinct DNA standards were used:
GeneRuler 1 kb DNA Ladder and O'GeneRuler DNA Ladder Mix.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image9.jpg)

**Figure 20.** DNA Electrophoresis of the PCR Products – Results.

* **1** – O'GeneRuler DNA Ladder Mix
* **2 – 5** – Galluzzi, DNA sample: K
* **6 – 7** – Galluzzi, DNA sample: G
* **8 – 12** – Gradient PCR, KAC 39; 50/55/60/65/70°C respectively,
  (sample K)
* **13 – 17** – Gradient PCR, White; 50/55/60/65/70°C respectively
  (sample K)
* **18** – O'GeneRuler 1 kb DNA Ladder Mix

##### Conclusions

In the PCR reaction with Galluzzi primers, the ITS2 DNA fragment was successfully
amplified using DNA sample K as a template. However, DNA sample G, derived from liquid
cultures from Gdańsk, did not show a specific and clear band on the gel, indicating that
these PCR reactions were not successful.

For the PCR with KAC39 primers, no specific product was visible on the gel, suggesting
that further optimization is needed.

In the PCR with White primers, two distinct bands were observed in lanes 13 and 14,
corresponding to annealing temperatures of 50°C and 55°C, respectively. One of the bands
in each lane had the expected length of approximately 700 bp, which may indicate the
desired product. The reaction should be repeated at these two annealing temperatures for
confirmation.

### Another Water Samples – Brand New Oder Water (BOW)

##### Another Water Samples – Brand New Oder Water (BOW)

Two water samples of 0.5 L were collected from another area of Czernica lake.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image28.jpg)

**Figure 21.** Environmental sample collection site of BOW1 and BOW2

### Brand New Oder Water Liquid Cultures

##### Brand New Oder Water Liquid Cultures

**Purpose:** New liquid cultures were set up in order to increase the amount
and variety of biological material for future DNA isolations.

This protocol is also available on the Experiments page under the **Setting up
liquid algal cultures protocol**.

Two liquid cultures were made, one for each water sample: 5 ml of each Oder water sample
was added to 20 ml of F/2 medium (sea salt 16 g/L) with the addition of vitamins in
glass flasks. The cultures were named BOW 1 and BOW 2.

Mini-cultures on a 12-well plate were also prepared. 1 ml of each Oder water sample was
transferred into two wells of a 12-well plate containing 3 ml of F/2 medium with the
addition of vitamins.

### PCR with Galluzzi Primers

##### PCR with Galluzzi Primers

**Purpose:** The aim of the experiment was to evaluate whether the ITS2
sequence is present in the DNA samples isolated on 16/05 and concentrated in the
SHERLOCK lab.

This protocol is also available on the Experiments page under the **PCR reaction on
DNA isolated from algae cultures protocol**.

**P. parvum** genomic DNA isolated from Krakow liquid cultures was used from
the following samples:

* **G1:** 95.1 ng/µl
* **K1:** 125.2 ng/µl
* **K2:** 188.3 ng/µl

Primers complementary for the ITS2 region were designed by Galluzzi et al. [1].

* **KacF** 5' TGTCTGCCGTGGACTTAGTGCT 3'
* **KacR** 5' ATGGCACAACGACTTGGTAGG 3'

Primers stock solutions (100 µM) were diluted 500x to the final concentration of 200 nM:

* First dilution (25x): 1 µl + 1 µl (primers) + 23 µl (water)
* Second dilution (20x): 2.5 µl (primer mix) + 47.5 µl (PCR reaction mix)

Table 16. PCR Samples
Composition. Reaction Volume = 50 µl

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA Template | 2.5 |
| PCR Mix (A&A Biotechnology) | 25 |
| Primers Mix | 2.5 |
| Water | 20 |

Table 17. PCR Reaction
Conditions (Source Description - 16/05)

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final Elongation | 72 | 300 |

**Number of Cycles:** 40

##### DNA Electrophoresis of the PCR Product

Electrophoresis was performed in a 2% agar gel at 85 V for 45 minutes. 10 µl of each PCR
product was added to the gel wells. Two distinct DNA standards were used: GeneRuler 1kB
DNA Ladder and O'GeneRuler DNA Ladder Mix (SM033).

**Results:** No specific product was observed. The photo could not be taken
due to the device failure.

##### Conclusions

None of the tested samples yielded a PCR product. The experiment is not reproducible with
the DNA samples isolated on 16/05. Newly isolated DNA samples are needed.

### 27.05 - 02.06

### Algal DNA Isolation

##### Algal DNA Isolation

**Purpose:** DNA isolation from liquid algal cultures containing
*Prymnesium parvum*'s cells and verification of the presence of the
*Prymnesium parvum* genome at the request of the SHERLOCK lab team.

The isolation protocol is available on the Experiments page under the **Algal DNA
isolation with QIAwave DNA blood and tissue kit protocol**.

Before isolation, all liquid cultures were examined macroscopically, and the best-looking
ones were selected:

* **NOW 5**
* **NOW 5.1**
* **P** (samples from two 12-well plates mixed together)
* **BOW 1**
* **BOW 2**
* **2W**
* **Gdańsk 2**

DNA isolation was performed in the same way as described on 16/05/2024.

Elution was performed twice, with 200 µl of AE buffer each time. As a result, 7 DNA
samples of 400 µl were received. The concentration of the isolated DNA was measured with
a NanoDrop spectrophotometer.

Table 18. DNA Concentrations
After the Isolation

|  |  |  |
| --- | --- | --- |
| **Sample** | **Concentration [ng/µl]** | **A260/A280** |
| NOW 5 | 5.8 | 1.66 |
| NOW 5.1 | 4.9 | 1.65 |
| P | 12.7 | 1.67 |
| BOW 1 | 5.6 | 1.53 |
| BOW 2 | 6.4 | 1.55 |
| 2W | 11.2 | 1.28 |
| Gdańsk 2 | 5.2 | 1.8 |

### Increasing the Concentration of Isolated DNA

##### Increasing the Concentration of Isolated DNA

This protocol is also available on the Experiments page under the **increasing the
DNA concentration protocol**.

The concentration of samples NOW 5, P, BOW 2, Gdańsk 2 was increased according to the
following protocol:

1. 1/10 volume of sodium acetate (pH=5,5) was added to the samples.
2. Isopropanol equivalent to the volume of the sample was added and the samples were
   mixed by gentle rotating.
3. The samples were centrifuged at maximum speed for 10 min.
4. Samples were then washed twice with ethanol (with 5 min centrifugation in between
   and gentle rotation to mix them).
5. Pellets were suspended in 15 μl of water and the concentration was measured. The
   concentrations were as follows:

Table 19. DNA Concentrations
After the Increase

|  |  |  |  |
| --- | --- | --- | --- |
| **Sample Name** | **Concentration [ng/µl]** | **A260/A280** | **A260/A230** |
| NOW 5 | 164.9 | 1.63 | 0.93 |
| P | 280.6 | 1.60 | 0.93 |
| BOW 2 | 154.3 | 1.63 | 0.94 |
| Gdańsk 2 | 127.2 | 1.78 | 1.19 |

Samples BOW 2 and NOW 5.1 were left and secured in case the concentration process fails.

### PCR with Galluzzi Primers on DNA Isolated 27/05

##### PCR with Galluzzi Primers on DNA Isolated 27/05

**Purposes:**

* To determine whether the *Prymnesium parvum* ITS2 sequence is present in the
  DNA samples isolated from liquid cultures and concentrated the previous day.
* To optimize PCR conditions by testing those recommended by the PCR Mix manufacturer.

*Prymnesium parvum* genomic DNA was used from the following samples:

* **NOW 5:** 164.9 ng/µl
* **P:** 280.6 ng/µl
* **BOW 2:** 154.3 ng/µl
* **Gdańsk 2:** 127.2 ng/µl

Primers complementary for the ITS2 region were designed by Galluzzi et. al. [1]

* **KacF:** 5' TGTCTGCCGTGGACTTAGTGCT 3'
* **KacR:** 5' ATGGCACAACGACTTGGTAGG 3'

Primers stock solutions (100 µM) were diluted 500x to the final concentration of 200 nM.

* **First dilution (25x):** 1 µl + 1 µl (primers) + 23 µl (water)
* **Second dilution (20x):** 1 µl (primer mix) + 19 µl (PCR reaction mix)

Table 20. PCR Samples
Composition. Reaction Volume = 20 ul

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA Template | 2 |
| PCR Mix (A&A Biotechnology) | 10 |
| Primers Mix | 0.8 |
| Water | 7.2 |

**Number of Cycles:** 40

The PCR was performed in two different conditions. A – conditions used routinely in our
lab for Galluzzi primers (source description - 16/05). B – conditions adjusted for the
Taq polymerase that was used, according to the manufacturer's instruction.

Table 21. PCR Conditions Tested
in the Experiment

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Reaction step** | **Conditions A** | | **Conditions B** | |
| **Temperature [°C]** | **Time [s]** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 | 95 | 60 |
| Denaturation | 98 | 10 | 95 | 15 |
| Annealing | 67 | 60 | 67 | 60 |
| Elongation | 72 | 5 | 72 | 15 |
| Final elongation | 72 | 300 | 72 | 300 |

##### DNA Electrophoresis of the PCR Product

Electrophoresis was performed in 2% agar gel, at 85 V for 45 min. 5 µl of each PCR
product were added to wells of the gel. Two distinct DNA standards were used: GeneRuler
1kb DNA Ladder and O'GeneRuler DNA Ladder Mix (SM033).

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image35.png)

**Figure 22.** Gel Electrophoresis of PCR Products – Results.

* **1** – O'GeneRuler DNA Ladder Mix
* **2** – negative control (water)
* **3** – P
* **4** – BOW 2
* **5** – NOW 5
* **6** – Gdańsk 2
* **7** – negative control (water)
* **8** – P
* **9** – BOW 2
* **10** – NOW 5
* **11** – Gdańsk 2
* **12** – GeneRuler 1kb DNA Ladder Mix

##### Conclusions

A product of the correct length was observed in rows 8 and 10 (conditions B, samples P
and NOW 5, respectively). The negative control sample showed no visible band. This
indicates that samples P and NOW 5 contain the *Prymnesium parvum* genome and are
suitable for further experiments in the SHERLOCK lab.

Additionally, a band of the proper length was visible in the 3rd row for conditions A.
However, since the negative control sample also displayed a product of the same length,
conditions A will be discontinued. Conditions B will be used for subsequent PCR
reactions.

### PCR with White Primers

##### PCR with White Primers

**Purposes:**

* To amplify the ribosomal cistron sequence of *Prymnesium parvum* using PCR
  with White primers.
* To optimize reaction conditions.

**Primers used in the reaction were designed by White et. al. [2]:**

* **ItsF:** 5' TCCGTAGGTGAACCTGCGG 3'
* **ItsR:** 5' TCCTCCGCTTATTGATATGC 3'

**DNA template –** coming from liquid cultures set up 13/05, concentrated in
the SHERLOCK lab:

* **K:** 118.6 ng/µl

The primers’ initial solution (100 µM) was diluted twice: 10x and 50x to the final
concentration of 200 nM:

* **First dilution (10x):** 1 µl + 1 µl + 8 µl (pure water)
* **Second dilution (50x):** 1 µl + 49 µl (reaction mix)

Table 22. PCR Samples
Composition. Reaction Volume = 50 ul

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA Template | 1 |
| PCR Mix (A&A Biotechnology) | 25 |
| Primers Mix | 1 |
| Water | 23 |

**PCR was performed under two different conditions (A and B).** Condition A
followed the protocol described by Binzer et al. [3], who used these primers. Due to the
use of a different polymerase in our reaction compared to Binzer et al., the conditions
were adjusted for our polymerase, resulting in Condition B. Each set of conditions was
tested in duplicate, with the samples labeled as A1, A2, B1, and B2.

Table 23. PCR Reaction
Conditions

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Reaction step** | **Conditions A** | | **Conditions B** | |
| **Temperature [°C]** | **Time [s]** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 95 | 720 | 95 | 180 |
| Denaturation | 95 | 30 | 95 | 30 |
| Annealing | 54 | 30 | 50 | 30 |
| Elongation | 72 | 40 | 72 | 120 |
| Final elongation | 72 | 300 | 72 | 600 |

##### Gel Electrophoresis of the PCR Product

Electrophoresis was performed in 2% agar gel, at 85 V for 45 min. 20 µl of each PCR
product were added to wells of the gel (10 µl per well). Standard: GeneRuler 1kB DNA
Ladder (Thermo Fisher).

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image21.jpg)

**Figure 23.** DNA Electrophoresis of PCR Products – Results.

* **1, 10** – GeneRuler 1 kB DNA Ladder
* **2, 3** – A1
* **4, 5** – A2
* **6, 7** – B1
* **8, 9** – B2

##### Conclusions

The only DNA products visible on the gel were in rows 6 and 7. Both rows displayed two
bands: a longer one, approximately 700 bp, and a shorter one, around 600 bp. All stripes
were cut out from the gel before photographing to prevent DNA degradation from prolonged
UV exposure.

The DNA was isolated from the gel with the Syngen kit, following the manufacturer's
instructions. DNA concentration was measured:

* **Shorter product:** 0.3 ng/µl
* **Longer product:** 0.1 ng/µl

Concentration of both PCR products isolated from the gel was too low for Sanger
sequencing procedure.

##### Conclusions

The stripes that were cut out of the gel were poorly visible, likely due to low
amplification efficiency. Reaction conditions require further optimization.

### 03.06 - 09.06

### F/2 Medium Preparation

##### F/2 Medium Preparation

The medium preparation protocol is also available on the Experiments page under the
**F/2
Medium Preparation Protocol**.

The F/2 medium was prepared by using previously made 100x concentrated stocks. Total
medium volume: 700 ml, which contains:

* 7 ml of each of 9 stocks (63 ml in total)
* 637 ml of distilled water
* 11,2 g of sea salt (16 g/L)

After mixing all the ingredients, the medium was sterilised with a microwave autoclave.

### Algal Cell Passage

##### Algal Cell Passage

This protocol is also available on the Experiments page under the **Passaging
Liquid Algal
Cultures Protocol**.

Some of the liquid cultures made on 13/05 were already dark yellow, therefore the first
passage of
algal liquid culture was performed, following these steps:

* A portion of F/2 medium was transferred in sterile conditions into a flask. The
  medium amount
  should be sufficient to form a layer approximately 1 cm thick at the bottom.
* 1 ml of the 1W liquid culture was added to the flask.
* A proper amount of the vitamins stocks was added to the flask.
* The new culture was gently mixed by rotating, secured, and left in the growth
  chamber.

As a result, new liquid cultures were made: 1W1, 2W1, 1BW1, 2BW1, 3BW1, Gdańsk 11, Gdańsk
21.

##### Notes

* **Culture Classification System:** Each successive "1" in the culture
  name
  indicates an additional passage.
* **Biohazard Waste:** All waste, including pipette tips and liquid waste
  that
  have come into contact with *Prymnesium*, should be treated as a biohazard.
  Store this
  waste in a separate beaker and dispose of it according to Dr Jedynak's
  instructions.
* **Sterilisation Warning:** When sterilising the F/2 medium with agar
  for plate
  cultures, do not tighten the cap of the bottle. Avoid cooling the capped bottle
  immediately
  after removing it from the water bath or autoclave, as this can cause the bottle to
  explode
  or implode.

Information about growth life cycle of *Prymnesium*:
[Prymnesium Life Cycle Information](https://tpwd.texas.gov/landwater/water/environconcerns/hab/ga/workshop/media/edvardsen.pdf?fbclid=IwAR0EhX46378b5XavsDxhzCr5aEU5PLTOCK0MmjSbbR9P6q-B-eZ-6MNTN8)

### Testing Various Lighting Conditions

##### Testing Various Lighting Conditions

Cultures **2W1** and **1BW1** were left in a place with lower
light intensity in the growth chamber. They will serve as a test to determine whether
*Prymnesium parvum* can thrive under such conditions.

### High Volume Liquid Culture

##### High Volume Liquid Culture

1 ml of **2W** liquid culture was added in sterile conditions to a glass
bottle with 700 ml F/2 medium. The cap of the bottle was left untightened and the bottle
was left in the growth chamber.

### Prymnesium parvum Sample from Szczecin

##### Prymnesium parvum Sample from Szczecin

A 30 ml sample of **Prymnesium parvum** liquid sample was obtained from
Szczecin. Figure 24 shows a microscopic image of the sample.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image29.jpg)

**Figure 24.** *Prymnesium parvum* liquid sample from Szczecin –
microscopic image.

Liquid stocks of the F/2 medium ingredients, concentrated 1000x, were received along with
the *Prymnesium parvum* sample. The ingredients were as follows:

* NaNO3
* H2PO4
* NaSiO2
* Trace metal solution
* Vitamin mix solution

The sea salt was received as a solid.

### F/2 Medium Preparation from the Received Ingredients Stocks

##### F/2 Medium Preparation from the Received Ingredients Stocks

The F/2 medium was prepared using the stocks received from Szczecin. The stocks are 1000x
concentrated. Total medium volume: 200 ml, which contains:

* 200 µl of each of the stocks (1 ml in total)
* 199 ml of distilled water
* 0,6 g of sea salt (3 g/L)

According to the instructions from the team in Szczecin, a sea salt concentration of 3
g/L was used.

After mixing all the ingredients, the medium was sterilised in a microwave autoclave. The
vitamin solution was added after the sterilisation in order to avoid the vitamin
degradation.

### Liquid Cultures from the Szczecin Sample

##### Liquid Cultures from the Szczecin Sample

**Purpose:** Propagation of a *Prymnesium parvum* laboratory liquid
culture so as to compare it with liquid algal cultures made from environmental samples.

This protocol is also available on the Experiments page under the **Setting up
liquid algal cultures protocol**.

The sample from Szczecin was split and two liquid cultures were prepared, in different
medium versions:

* 7 ml of the sample was added to 23 ml of F/2 medium (salt 16 g/L). Sample name:
  **Szczecin**
* 15 ml of the sample was added to 15 ml of F/2 medium (salt 3 g/L). Sample name:
  **Szczecin F/2**

New liquid cultures were left in a growth chamber, where the conditions described below
prevailed, following the recommendations of the team from Szczecin.

* **Temperature:** 22°C
* **Lighting:** 100 photon fluence rate
* **Light/dark cycle:** 14 h/10 h

**Note:** The culture density was too low for DNA isolation, therefore the
liquid cultures were set up to increase the amount of *Prymnesium parvum* cells.

### 10.06 - 16.06

### PCR with Galluzzi Primers on Selected DNA Samples Isolated on 27/05

##### PCR with Galluzzi Primers on Selected DNA Samples Isolated on 27/05

**Purpose:** The goal was to provide DNA templates for sensitivity tests of
the SHERLOCK assay. This involved using two samples that had been successfully amplified
in the previous PCR with Galluzzi primers (P and NOW 5), along with two samples that had
not been successfully amplified (BOW 2 and Gdańsk 2).

**P. parvum** genomic DNA isolated from Krakow liquid cultures was used from
the following samples:

* **NOW 5** – 164.9 ng/µl (2 PCR reactions)
* **P** – 280.6 ng/µl (2 PCR reactions)
* **BOW 2** – 154.3 ng/µl (1 PCR reaction)
* **Gdańsk 2** – 127.2 ng/µl (1 PCR reaction)

**Primers Sequences:**

* **KacF:** 5' TGTCTGCCGTGGACTTAGTGCT 3'
* **KacR:** 5' ATGGCACAACGACTTGGTAGG 3'

Primers stock solutions (100 uM) were diluted 500x to the final concentration of 200 nM:

* First dilution (25x) – 1 µl + 1 µl (primers) + 23 µl (water)
* Second dilution (20x) – 2.5 µl (primer mix) + 47.5 µl (PCR reaction mix)

Table 24. PCR Samples
Composition. Reaction Volume = 50 µl

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA Template | 1 |
| PCR Mix (A&A Biotechnology) | 25 |
| Primers Mix | 2.5 |
| Water | 21.5 |

Table 25: PCR Reaction
Conditions Based on the Manufacturer’s Instruction (A&A Biotechnology)

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 95 | 60 |
| Denaturation | 95 | 15 |
| Annealing | 67 | 60 |
| Elongation | 72 | 15 |
| Final Elongation | 72 | 300 |

**Number of Cycles:** 30

##### DNA Electrophoresis of the PCR Product

Electrophoresis was performed in 2% agar gel, at the 85 V voltage for 45 min. 5 µl of
each PCR product were added to wells of the gel. Two distinct DNA standards were used:
GeneRuler 1kB DNA Ladder and O'GeneRuler DNA Ladder Mix (SM033).

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image10.png)
**Figure 25.** DNA Electrophoresis of the PCR Product –
Results.

* **1** – GeneRuler 1kB DNA Ladder
* **2** – Negative control (water)
* **3, 4** – P
* **5, 6** – NOW 5
* **7** – Gdańsk 2
* **8** – BOW 2
* **9** – O'GeneRuler DNA Ladder Mix

##### Conclusion

Specific products of the correct length were obtained for DNA samples P, NOW5, and BOW 2,
indicating successful amplification, unlike the results from the PCR conducted on 28/05.
PCR with Gdańsk 2 as the DNA template did not yield any specific product.

The PCR products were purified after the enzymatic reaction to remove any potential PCR
inhibitors.

### Macroscopic Observations of Liquid Cultures

##### Macroscopic Observations of Liquid Cultures

**Observation:** Cultures growing in standard light conditions exhibit
slightly faster growth compared to those left in a darker environment.

### 17.06 - 23.06

### Changes in Algal Morphology

##### Changes in Algal Morphology

The sample preparation for imaging protocol is available on the Experiments page under
the **Preparing liquid algal cultures samples for imaging protocol**.

We observed a significant change in algal morphology in liquid cultures made 13/05 from
environmental samples. In every observed culture, the algal cells became oval, small,
and very similar to each other. Two chloroplasts, which are a characteristic feature of
*Prymnesium parvum*, are no longer visible in the fluorescent light of the
microscope. We hypothesise that under stressful conditions, *Prymnesium parvum*
transitioned to the haploid cyst stage, which accounts for the observed morphological
changes. By stressful conditions, we mean laboratory conditions, which differ from the
natural habitat of the algae.

Figure 26 and 27, respectively, show a sample view of the cultures before and after the
morphological change.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image20.jpg)

**Figure 26.** Algae liquid culture before the morphological change.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image6.jpg)

**Figure 27.** Algae liquid culture after the morphological change.

**High Volume Liquid Culture:** We observed slight growth of algae as a
golden-brown precipitate at the bottom of a flask.

### F/2 Medium – Testing Various Sea Salt Concentrations

##### F/2 Medium – Testing Various Sea Salt Concentrations

**Purpose:** Determining which sea salt concentration will be optimal for
algae growth.

The medium preparation protocol is also available on the Experiments page under the
**F/2 medium preparation protocol**.

Three portions of F/2 medium with varying concentrations of sea salt were prepared. The
medium was made following the same procedure as outlined on 10/05, with a volume of 200
ml for each salt concentration. The sea salt concentrations tested were: **2.5
g/L**, **5 g/L**, and **10 g/L**.

3 ml of each of the medium variants was put into two wells of a 12-well plate, and the
vitamin mix was added. The liquid culture chosen for the test was 2W1, as it was in the
best condition. Then, 100 µl of 2W1 liquid culture was transferred from the flask to
each well. The plate was secured and left in the growth chamber.

### 24.06 - 30.06

### F/2 Medium Preparation

##### F/2 Medium Preparation

The medium preparation protocol is also available on the Experiments page under the
**F/2 medium preparation protocol**.

The F/2 medium was prepared by using previously made 100x concentrated stocks. Total
medium volume: 800 ml, which contains:

* 8 ml of each of 9 stocks (72 ml in total)
* 728 ml of distilled water
* 12,8 g of sea salt (16 g/L)

After mixing all the ingredients, the medium was sterilised in an autoclave.

### Algal Cell Passage

##### Algal Cell Passage

This protocol is also available on the Experiments page under the **Passaging
liquid algal cultures protocol**.

The passage was carried out in the same manner as described on 03/06. Cultures 2BW1,
3BW1, Gdańsk 11, and Gdańsk 21 looked badly – they were fully coloured green, which is
probably because of green algae growth that had replaced *Prymnesium parvum*.
Therefore, the listed cultures were not passaged and 2BW1 and 3BW1 were disposed of.

As a result, new liquid cultures were made:

* 1W11
* 2W11
* 1BW11
* NOW 31
* NOW 41
* NOW 51
* NOW 5.11
* NOW 81
* BOW 11
* BOW 21

### Disposal of Old Cultures

##### Disposal of Old Cultures

This protocol is also available on the Experiments page under the **Algae liquid
culture disposal protocol**.

Liquid cultures 2BW1 and 3BW1 were disposed of following these steps:

* Medicarine solution was prepared – 1 pill of Medicarine was dissolved in 1 L of tap
  water.
* The Medicarine solution was added to the cultures until the culture flask was nearly
  full.
* The cultures were left for 15 minutes so that all the microorganisms were
  neutralised.
* After that time, the flasks were emptied and washed.

### Gradient PCR with Galluzzi Primers Containing T7 Promoter Sequence

##### Gradient PCR with Galluzzi Primers Containing T7 Promoter Sequence

**Purpose:** Introduction of the T7 promoter sequence into the ITS2 sequence
from *Prymnesium parvum* genome by PCR amplification. To achieve this, the
forward primer designed by Galluzzi et.al [1] was modified by adding the T7 promoter
sequence.

**Primers Sequences:**

* **ModF:** 5' GAAATTAATACGACTCACTATAGGGTGTCTGCCGTGGACTTAGTGCT 3'
* **ModR:** 5' ATGGCACAACGACTTGGTAGG 3'

To optimize the annealing temperature, five different temperatures were investigated in a
gradient: 55/59,2/65,5/67,6/70°C.

Primers stock solutions (100 µM) were diluted 500x to a final concentration of 200 nM:

* First dilution (25x): 1 µl + 1 µl (primers) + 23 µl (water)
* Second dilution (20x): 1 µl (primer mix) + 19 µl (PCR reaction mix)

**DNA Template:** DNA isolated from Krakow liquid cultures – sample K3.2,
with a concentration of 62,4 ng/µl (concentrated in the SHERLOCK lab).

Table 26. PCR Samples
Composition. Reaction Volume = 20 µl

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA template | 1 |
| PCR Mix A&A Biotechnology | 10 |
| Primer mix | 1 |
| Water | 8 |

Table 27. PCR Reaction
Conditions

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 55/59,2/65,5/67,6/70 | 60 |
| Elongation | 72 | 5 |
| Final Elongation | 72 | 300 |

**Number of cycles:** 32

##### Gel Electrophoresis of the PCR Product

Electrophoresis was performed in 2% agar gel, at 85 V for 45 minutes. 10 µl of each PCR
product were added to the wells. Two distinct DNA standards were used: GeneRuler 1 kb
DNA Ladder and O'GeneRuler DNA Ladder Mix.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image15.jpg)
**Figure 28.** DNA Electrophoresis – Results.

* **1** – O'GeneRuler DNA Ladder Mix
* **2** – 55°C
* **3** – 59,2°C
* **4** – 65,5°C
* **5** – 67,6°C
* **6** – 70°C
* **7** – GeneRuler 1 kb DNA Ladder Mix

##### Conclusions

No specific product was obtained in the performed PCR. The shadows visible below 100 bp
are primer dimers. The reaction conditions require optimization.

### Sea Salt Experiment – Observations

##### Sea Salt Experiment – Observations

The cultures on a 12-well plate with three different sea salt concentrations were
examined under the microscope. No significant changes were observed at concentrations of
2.5 g/L and 5 g/L. However, at a concentration of 10 g/L, morphological changes were
noted—small, oval cysts transformed into larger, more rounded cells. Movement of some of
the cells was also observed.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image24.png)

**Figure 29.** 12-well Plate. Sea Salt Concentration – 10 g/L.
Microscopic Image.

### Gradient PCR with Galluzzi Primers Containing T7 Promoter Sequence – Modified Reaction Conditions

##### Gradient PCR with Galluzzi Primers Containing T7 Promoter Sequence – Modified Reaction Conditions

**Purpose:** Second attempt to introduce the T7 promoter sequence into the
ITS2 sequence from the *Prymnesium parvum* genome. The PCR conditions were
modified and adjusted to the Taq polymerase that was used.

**Primers sequences:**

* **ModF:** 5' GAAATTAATACGACTCACTATAGGGTGTCTGCCGTGGACTTAGTGCT 3'
* **ModR:** 5' ATGGCACAACGACTTGGTAGG 3'

Primers stock solutions (100 uM) were diluted 500x to the final concentration of 200 nM.
First dilution (25x): 1 µl + 1 µl (primers) + 23 µl (water).
Second dilution (20x): 1 µl (primer mix) + 19 µl (PCR reaction mix).

**DNA template:** coming from liquid cultures set up 13/05, concentrated in
the SHERLOCK lab: sample K3.2 (62.4 ng/µl).

In order to save the DNA template, reaction volume was reduced to 10 µl.

Table 28. PCR Samples
Composition. Reaction Volume = 10 µl

|  |  |
| --- | --- |
| **PCR reagent** | **Volume [µl]** |
| DNA template | 0.5 |
| PCR Mix (A&A Biotechnology) | 5 |
| Primer mix | 0.5 |
| Water | 4 |

Table 29. PCR Reaction
Conditions

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 95 | 120 |
| Denaturation | 95 | 20 |
| Annealing | 55/59.2/65.5/67.6/70 | 60 |
| Elongation | 72 | 30 |
| Final Elongation | 72 | 300 |

**Number of cycles:** 30

##### Gel Electrophoresis of the PCR Product

Electrophoresis was performed in 2% agar gel, at the 85 V voltage for 45 min. 5 µl of
each PCR product were added to wells of the gel. Two distinct DNA standards were used:
GeneRuler 1kB DNA Ladder and O'GeneRuler DNA Ladder Mix.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image14.jpg)
**Figure 30.** DNA Electrophoresis of the PCR Product –
Results.

* **1** – O'GeneRuler DNA Ladder mix
* **2** – 55°C
* **3** – 59.2°C
* **4** – 65.5°C
* **5** – 67.6°C
* **6** – 70°C
* **7** – GeneRuler 1kB DNA Ladder Mix

##### Conclusion

Rows 2, 3, 4 contain a slight trace of the specific product. There is no sign of the
product in rows 5 and 6. The signal may be weak because of the small amount of DNA
template and fewer PCR cycles.

### DNA Isolation – New Method

##### DNA Isolation – New Method

**Purpose:** Checking the efficiency of a new DNA isolation method that
utilises the Monarch Genomic DNA Purification Kit (NEB).

The isolation protocol is available on the Experiments page under the **Algal DNA
isolation with NEB isolation kit protocol**.

1.5 ml of liquid cultures NOW 5 and BOW 2 (cultures that had previously given specific
products in PCR with Galluzzi primers) were centrifuged (13,500 rpm, 10 min). The
supernatants were disposed of and the pellets were utilised for DNA isolation, following
the manufacturer's protocol.

The steps followed were:

1. 10 µl of proteinase K and 200 µl of Lysis Buffer were added to each sample and mixed
   by vortexing.
2. The samples were incubated at 56°C with shaking (1400 rpm) for 1 hour.
3. 400 µl of Binding Buffer was added to each sample and mixed by vortexing for 10
   seconds.
4. The mix from step 3 was transferred to a Purification Column, previously inserted
   into a collection tube.
5. The samples were centrifuged twice: first for 3 minutes at 1000 g so that DNA binds
   to the columns, and second for 1 minute at 13,500 g to clear the membrane. The
   flow-through was discarded.
6. 500 µl of Wash Buffer was added to the columns. Caps were closed and the samples
   were inverted a few times so that the wash buffer reached the cap. The samples were
   centrifuged for 1 minute at 13,500 g. The flow-through was discarded.
7. Step 6 was repeated.
8. The columns were placed in 1.5 ml collection tubes. 50 µl of preheated water was
   added, and the samples were incubated at room temperature for 1 minute.
9. The samples were centrifuged for 1 minute at 13,500 g in order to elute the isolated
   DNA.

**Note:** Steps including RNase addition, described in the manual, were
skipped, as they are not necessary for this experiment.

Table 30. Concentration of the
DNA Isolated with a Monarch Kit

|  |  |  |  |
| --- | --- | --- | --- |
| **Sample Name** | **DNA Concentration [ng/µl]** | **A260/A280** | **A260/A230** |
| NOW 5 | 16 | 1.68 | 1.34 |
| BOW 2 | 24.1 | 1.79 | 1.56 |

### 01.07 - 07.07

### Increasing the Concentration of DNA Isolated 27/05

##### Increasing the Concentration of DNA Isolated 27/05

After the DNA isolation with the QIAwave Blood and Tissue Kit (27/05), three DNA samples
of 400 µl remained that required concentration increase: NOW 5.1, 2W, and BOW 1.

This protocol is also available on the Experiments page under the **Increasing the
DNA concentration protocol**.

The concentration of isolated DNA was increased according to the following protocol:

1. 1/10 volume of sodium acetate (pH=5.5) was added to the samples.
2. Isopropanol equivalent to the volume of the sample was added, and the samples were
   mixed by gentle rotating.
3. The samples were centrifuged at maximum speed for 10 minutes.
4. The samples were washed twice with ethanol (with 5 minutes centrifugation in between
   and gentle rotation to mix them).
5. Pellets were suspended in 15 µl of water, and the concentration was measured.

Table 31. Concentration of the
Isolated DNA

|  |  |  |  |
| --- | --- | --- | --- |
| **Sample Name** | **Concentration [ng/µl]** | **A260/A280** | **A260/A230** |
| NOW 5.1 | 31.5 | 1.57 | 0.88 |
| BOW 1 | 39.3 | 1.62 | 0.88 |
| 2W | 16.6 | 1.88 | 0.01 |

**Note:** Significant contamination of the samples with organic compounds.

### PCR with Galluzzi Primers on DNA Isolated on 27/05 and 27/06

##### PCR with Galluzzi Primers on DNA Isolated on 27/05 and 27/06

**Purpose:** Verification of the presence of *Prymnesium parvum*'s
ITS2 sequence in DNA samples isolated with the Monarch isolation kit (NOW 5 and BOW 2)
and the newly concentrated DNA samples (NOW 5.1, BOW 1, 2W). Galluzzi primers were used
for this purpose.

The PCR protocol is also available on the Experiments page under the **PCR reaction
on DNA isolated from algae cultures protocol**.

**DNA Samples Used as Template:**

* **NOW 5.1:** 31.5 ng/µl
* **2W:** 16.6 ng/µl
* **BOW 1:** 39.3 ng/µl
* **NOW 5:** 16 ng/µl
* **BOW 2:** 24.1 ng/µl

**Primers Sequences:**

* **GalF:** 5' TGTCTGCCGTGGACTTAGTGCT 3'
* **GalR:** 5' ATGGCACAACGACTTGGTAGG 3'

Primers stock solutions (100 uM) were diluted 500x to the final concentration of 200 nM.

* First dilution (25x): 1 µl + 1 µl (primers) + 23 µl (water)
* Second dilution (20x): 2.5 µl (primer mix) + 47.5 µl (PCR reaction mix)

Table 32. PCR Samples
Composition. Reaction Volume = 50 µl

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA Template | 3 |
| PCR Mix (A&A Biotechnology) | 25 |
| Primers Mix | 2.5 |
| Water | 19.5 |

Table 33. PCR Reaction
Conditions Based on the PCR Mix Manufacturer’s Instruction (A&A
Biotechnology)

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 95 | 60 |
| Denaturation | 95 | 15 |
| Annealing | 67 | 60 |
| Elongation | 72 | 15 |
| Final Elongation | 72 | 300 |

**Number of Cycles:** 30

##### DNA Electrophoresis of the PCR Product

Electrophoresis was performed in 2% agar gel, at the 85 V voltage for 45 minutes. 5 µl of
each PCR product were added to wells of the gel. Two distinct DNA standards were used:
GeneRuler 1kB DNA Ladder and O'GeneRuler DNA Ladder Mix (SM033).

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image30.jpg)
**Figure 31.** Gel Electrophoresis of the PCR Products –
Results.

* **1** – GeneRuler 1kB DNA Ladder
* **2** – Negative control (water)
* **3** – BOW 2
* **4** – NOW 5
* **5** – NOW 5.1
* **6** – 2W
* **7** – BOW 1
* **8** – O'GeneRuler DNA Ladder Mix (SM033)

##### Conclusion

Three specific products of the right length were received for the samples BOW 2, NOW 5.1,
and BOW 1. Samples NOW 5.1 and BOW 1 were tested with Galluzzi primers for the first
time, and the presence of the ITS2 sequence was confirmed.

### 08.07 - 14.07

### Microscopic Observation – Liquid and Plate Cultures

##### Microscopic Observation – Liquid and Plate Cultures

###### Plate Cultures

Except for the 1W plate culture, no changes in algae growth were observed on the plates.
None of the microorganisms observed were morphologically similar to *Prymnesium
parvum*.

###### Liquid Cultures

Hardly any differences in the growth and quantity of algae have been observed since the
last microscopic evaluation. Very few cells morphologically resembled *Prymnesium
parvum*. However, another change in general morphology of the culture occurred –
algal cells became round, and small, oval cells were no longer visible. **Figure
32** shows the microscopic image of 1BW11 liquid culture, where the changes
are most visible.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image17.png)
**Figure 32.** 1BW11 Liquid Culture – Microscopic Image.

### Algal DNA Isolation

##### Algal DNA Isolation

**Purpose:** Verification of *Prymnesium parvum* genome presence in
newly created liquid cultures after the passage.

The isolation protocol is also available on the Experiments page under the **Algal
DNA Isolation with QIAwave DNA Blood and Tissue Kit protocol**.

Samples of 2 ml were taken from the liquid cultures 2W11, 1BW11, 2W1D (culture from
Darkness), and the high-volume culture. The samples were centrifuged (5 min, 14 000
rpm). DNA was isolated in the same manner as described 16/05. Elution was performed
twice, with 200 µl of AE buffer each time. As a result, 4 DNA samples of 400 µl were
received. The concentration of the isolated DNA was measured with a NanoDrop
spectrophotometer.

Table 34. Concentration of the
isolated DNA.

|  |  |  |
| --- | --- | --- |
| **Sample name** | **DNA concentration [ng/µl]** | **A260/A280** |
| High-volume culture | 11,3 | 1,41 |
| 2W1D | 9,9 | 1,26 |
| 2W11 | 9,4 | 1,15 |
| 1BW11 | 11,2 | 1,12 |

### PCR of the Isolated DNA and Colony PCR

##### PCR of the Isolated DNA and Colony PCR

**Purpose:** Identification of the ITS2 sequence that is placed in the
*Prymnesium parvum* genome and is specific to this species. The sequence length
is 132 bp.

The PCR protocol is also available on the Experiments page under the **PCR Reaction
on DNA Isolated from Algae Cultures Protocol**.

**Colony PCR** of the sample taken from the high-volume culture was
performed simultaneously. Sample preparation for the colony PCR included:

* Taking 2 ml sample from the high-volume culture
* Centrifugation of the sample (2 min, 14 000 rpm)
* Washing the sample with ultra-pure water
* Adding the sample directly to the PCR reaction mixture

Primers for the PCR reaction were the same as used by Galluzzi et al. [1].

* **GalF:** 5' TGTCTGCCGTGGACTTAGTGCT 3'
* **GalR** 5' ATGGCACAACGACTTGGTAGG 3'

Initial concentration of the primers was 100 µM. They were diluted twice, first by 10x
and then by 50x, to the final concentration of 200 nM.

Table 35. PCR Samples
Composition. Reaction volume = 20 µl

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| DNA template | 9,2 |
| PCR Mix (A&A Biotechnology) | 10 |
| Primer FOR | 0,4 |
| Primer REV | 0,4 |

Table 36. PCR Reaction
Conditions (source description - 16/05)

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final Elongation | 72 | 300 |

**Number of cycles:** 40

##### DNA Electrophoresis of the PCR Product

Electrophoresis was performed in 2% agar gel at 85 V for 45 min. 10 µl of each PCR
product were added to the wells of the gel. Two distinct DNA standards were used:
GeneRuler 1kB DNA Ladder and O'GeneRuler DNA Ladder Mix.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image3.jpg)
**Figure 33.** Gel Electrophoresis of the PCR Product –
Results.

* **1** – O'GeneRuler DNA Ladder Mix
* **2** – high-volume culture
* **3** – 2W1D
* **4** – 1BW11
* **5** – 2W11
* **6** – colony PCR
* **7** – O'GeneRuler 1 kB DNA Ladder Mix

##### Conclusion

The specific PCR product of the correct length was observed only in row 3, which refers
to the 2W1D culture. The other rows either contain unspecific products or no products at
all. Based on these results, it can be concluded that the 2W1D culture (the one growing
in darkness) contains the ITS2 sequence from the *Prymnesium parvum* genome.

### Oder Water Environmental Samples Collected in July

##### Oder Water Environmental Samples Collected in July

Six environmental samples of Oder water were collected from the Czernica lake.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image8.jpg)
**Figure 34.** Environmental sample collection site of
L1, L2, and L3

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image16.jpg)
**Figure 35.** Environmental sample collection site of L4

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image23.jpg)
**Figure 36.** Environmental sample collection site of L5

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image38.jpg)
**Figure 37.** Environmental sample collection site of L6

### Liquid Cultures from the July Oder Water Samples

##### Liquid Cultures from the July Oder Water Samples

This protocol is also available on the Experiments page under the **Setting up
liquid algal
cultures protocol**.

Six liquid algae cultures were prepared from the collected Oder water samples. The
samples were
centrifuged for 5 minutes at 2000 rpm and the sediment was suspended in 10 ml of F/2
medium prepared
from Szczecin ingredients.

The cultures were named L1, L2, L3, L4, L5, L6 from the Polish word for “July” –
“lipiec”.

### Algal DNA Isolation

##### Algal DNA Isolation

**Purposes:**

* DNA template supply for the SHERLOCK lab experiments
* Obtaining algal DNA directly from Oder water samples

The isolation protocol is available on the Experiments page under the **Algal DNA
isolation with NEB isolation kit protocol**.

DNA was isolated from the liquid cultures NOW 5.1, BOW 2, Szczecin/F2 Szczecin, and from
Oder water samples L1, L4, L6.

Liquid cultures were centrifuged (13 500 rpm, 5 min). The supernatants were disposed of
and the pellets were utilized for DNA isolation with the Monarch Genomic DNA
Purification Kit (NEB) following the modified manufacturer's protocol, which was
described in detail on 27/06. DNA was eluted with 50 µl of pure water.

Table 37. Concentration of the
isolated DNA

|  |  |  |  |
| --- | --- | --- | --- |
| **Sample name** | **DNA concentration [ng/µl]** | **A260/A280** | **A260/A230** |
| Szczecin/F2 Szczecin | 7,3 | 1,71 | 1,08 |
| BOW 2 | 71,4 | 1,51 | 0,66 |
| NOW 5.1 | 19,0 | 1,69 | 1,20 |
| L1 | 80,6 | 1,52 | 0,62 |
| L4 | 27,5 | 1,56 | 0,65 |
| L6 | 6,6 | 1,63 | 0,76 |

### PCR on the isolated DNA

##### PCR on the isolated DNA

**Purpose:** Verification of the *Prymnesium parvum* ITS2 sequence in
newly isolated DNA samples. Galluzi primers were used.

The PCR protocol is also available on the Experiments page under the **PCR reaction
on DNA isolated from algae cultures protocol**.

Primers for the PCR reaction were the same as used by Galluzzi et. al. [1]:

* **FOR primer:** 5' TGTCTGCCGTGGACTTAGTGCT 3'
* **REV primer:** 5' ATGGCACAACGACTTGGTAGG 3'

Initial concentration of the primers was 100 µM. They were diluted twice, firstly 10x and
then 50x, to the proper final concentration of 200 nM.

**Newly isolated DNA samples used as the template:**

* Szczecin/F2 Szczecin
* BOW 2
* NOW 5.1
* L1
* L4
* L6

Table 38. PCR samples
composition. Reaction volume = 10 µl

|  |  |
| --- | --- |
| **PCR reagent** | **Volume [µl]** |
| DNA template | 2 |
| PCR Mix (A&A Biotechnology) | 5 |
| Primer mix | 0,5 |
| Water | 2,5 |

Table 39. PCR reaction
conditions (source description - 16/05)

|  |  |  |
| --- | --- | --- |
| **Reaction step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final elongation | 72 | 300 |

**Number of cycles:** 40

**DNA electrophoresis** of the PCR product will be performed in a few days
(described 17/07 in this notebook).

### 15.07 - 21.07

### Algae culture – consultation

##### Algae culture – consultation

After consulting with **Paweł Brzezowski**, a staff member from the
Department of Biochemistry and Plant Physiology JU, attention was drawn to two aspects:

* **Concentrations of the stock solutions** used for F/2 medium
  preparation. The concentration of each stock was ten times lower than intended
  because of a calculation error during the initial medium preparation.
* **Appropriate pH** of F/2 medium. So far, the pH of the newly prepared
  portions of F/2 medium has not been monitored. This oversight is due to lack of
  expertise in algae culture.

##### Conclusions after the consultation

Taking these two factors into account, algae growth in all cultures prepared so far was
not optimal due to disturbances in the F/2 medium composition and its pH. A hypothesis
was made that these stressful conditions could trigger the observed algal morphological
change and contribute to growth slowdown.

### New stock solutions for F/2 medium

##### New stock solutions for F/2 medium

The concentrations of stock solutions were recalculated and corrected. New stock
solutions were prepared in the same final quantities as described on 10/05 (Table 1).

### F/2 Medium Preparation (Corrected Version)

##### F/2 Medium Preparation (Corrected Version)

The medium preparation protocol is also available on the Experiments page under the
**F/2
medium preparation protocol**.

The F/2 medium was prepared by using newly made 100x concentrated stocks. Total medium
volume: 400 ml, which contains:

* 4 ml of each of 9 stocks (36 ml in total)
* 364 ml of distilled water
* 6.4 g of sea salt (16 g/L)
* 1320 µl of NaOH to optimise pH of the medium (from pH=5.12 to pH=7.9)

According to the literature data, the pH of the F/2 medium should be approximately 8. The
pH of the medium was measured with a pH metre.

After mixing all the ingredients, the medium was sterilised in a microwave autoclave.

### Algal Cell Passage

##### Algal Cell Passage

Newly prepared F/2 medium was used to perform this passage. The passage was carried out
in the same
manner as described on 03/06. Cultures **NOW 31** and **NOW
81** were
coloured green and were not passaged. As a result, new liquid cultures were made:

* **1W111**
* **2W111**
* **1BW111**
* **NOW 411**
* **NOW 511**
* **NOW 5.111**
* **BOW 111**
* **BOW 211**

### Attempt of Setting up a Pure Prymnesium parvum Culture

##### Attempt of Setting up a Pure Prymnesium parvum Culture

**Purpose:** Isolating single *Prymnesium parvum* colonies from algae
liquid
cultures. Such colonies can give a start to a pure *Prymnesium parvum* laboratory
culture.

5 ml of **NOW 5** liquid culture was collected to a 15 ml Falcon tube and a
series of
centrifugation steps were performed. **Table 40** shows the centrifugation
conditions that
were utilised. After each centrifugation, the supernatants were transferred into new
tubes for
another centrifugation. The pellets were suspended in 500 µl of F/2 medium,
appropriately diluted, and
then transferred into previously prepared 1% agar plates containing the F/2 medium. The
plates were left
in the culture room with a 14 h/10 h light/dark cycle.

Table 40. Centrifugation
Conditions

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Centrifugation** | **RCF** | **Time [min]** | **Pellets Dilutions** | **Temperature [°C]** |
| 1. | 100 | 3 | 10­⁶ / 10⁹ | 4 |
| 2. | 500 | 3 | 10­¹ / 10³ | 4 |
| 3. | 1000 | 3 | 10­¹ / 10³ | 4 |
| 4. | 3000 | 3 | 10­¹ / 10³ | 4 |

### Algal DNA Isolation – In-field Method

##### Algal DNA Isolation – In-field Method

**Purpose:** Evaluation of a fast DNA isolation method that could
prospectively be performed in field conditions, simplifying the workflow required for
the SHERLOCK assay. The method was originally described by Zou et al. [4].

The isolation protocol is also available on the Experiments page under the **DNA
isolation - in-field method protocol**.

Two buffers required for this isolation method were prepared in a volume of 50 ml: a
lysis buffer and a wash buffer.

* **Lysis buffer:**
  + 25 mM NaCl
  + 2.5 mM EDTA
  + 0.05% SDS
  + 20 mM Tris (pH=8.0)
  + Water
* **Wash buffer:**
  + 10 mM Tris (pH=8.0)
  + 0.1% Tween 20%
  + Water

Three different isolation sticks were used:

* Citotest swab sticks (C)
* Aptaca swab sticks (A)
* Whatman’s paper cut into 2 mm strips (W), impregnated with wax

To perform this isolation method, two steel ball bearings were used. A 500 µl aliquot of
the liquid culture was added to a 2 ml Eppendorf tube containing 500 µl of lysis buffer.
Two ball bearings were then added, and the tube was shaken for 8 seconds. The isolation
stick was dipped into the mixture three times, followed by dipping the stick three times
into another Eppendorf tube containing 1.75 ml of wash buffer. The material was then
transferred to the previously prepared PCR mix in a PCR tube.

The isolated DNA was directly transferred to the PCR reaction mixture. **Table
41** shows the mixture composition.

Table 41. PCR Samples
Composition. Reaction Volume = 20 ul

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| PCR Mix (A&A Biotechnology) | 10 |
| Primer mix | 1 |
| Water | 9 |

Table 42. PCR Reaction
Conditions

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final Elongation | 72 | 300 |

Number of cycles: 30

DNA electrophoresis of the PCR product will be performed the next day after the PCR.
(Described 17/07 in this notebook).

### DNA Electrophoresis – Multiple PCR Products

##### DNA Electrophoresis – Multiple PCR Products

**Purpose:** The PCR products from the following reactions were subjected to
electrophoresis:

* PCR products from the DNA isolated from July Oder water samples (11/07)
* PCR products from the field DNA isolation (16/07)
* PCR products from the reaction performed on the KAC product (17/07)

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image39.png)
**Figure
38.** Gel electrophoresis of PCR products – results.

**Legend:**

* **1** – O'GeneRuler DNA Ladder Mix
* **2** – Szczecin/F2 Szczecin
* **3** – BOW 2
* **4** – NOW 5.1
* **5** – L1
* **6** – L4
* **7** – L6
* **8** – mistake
* **9** – mistake
* **10** – KAC
* **11** – NC (NOW 5.1 using Citotest stick)
* **12** – NA (NOW 5.1 using Aptaca stick)
* **13** – NW (NOW 5.1 using Whatman stick)
* **14** – MC (BOW 2 using Citotest stick)
* **15** – MA
* **16** – MW
* **17** – LC
* **18** – LA
* **19** – LW
* **20** – O'GeneRuler 1 kb DNA Ladder Mix

##### Conclusions

Clear stripes are visible in rows 2-7, referring to the specific PCR product of the
correct length, indicating that the DNA isolated on 11/07 contains the *Prymnesium
parvum* genome.

The KAC product contains the *Prymnesium parvum* ITS2 sequence (row 10).

No specific product was obtained from any of the samples following field DNA isolation.
However, it can be concluded that the Whatman stick is the most efficient, as primer
dimers are visible, suggesting that the Whatman stick does not absorb the PCR reagents
as much as the other tested sticks [11-19].

### Cultures on Agar Medium – Prymnesium Parvum Pure Culture Attempt

##### Cultures on Agar Medium – *Prymnesium Parvum* Pure Culture Attempt

The agar F/2 medium was prepared in the same manner as described 10/05:

* **Total medium volume:** 400 ml
* **Agar:** 4 g
* **Sea salt:** 1,2 g

5 ml of the NOW 5.1 liquid culture was centrifuged (100 g, 5 min), and the supernatant
was separated. The pellet was diluted in F/2 medium to concentrations of 106
and 109 and spread on agar plates. The supernatant was then centrifuged (500
g, 5 min), the supernatant was separated again, and the pellet was diluted in F/2 medium
to concentrations of 101 and 103 and spread on agar plates.

The process was repeated twice more:

* Centrifugation at 1000 g for 5 min: pellet diluted to 101 and
  103 and spread on plates
* Centrifugation at 3000 g for 5 min: pellet diluted to 101 and
  103 and spread on plates

The plates were secured with parafilm and left in the growth chamber with a 14 h/10 h
light/dark cycle.

### 22.07 - 28.07

### Algal DNA Isolation – In-Field Method, Second Attempt

##### Algal DNA Isolation – In-Field Method, Second Attempt

**Purpose:** Evaluation of a fast DNA isolation method that could
prospectively be performed in field conditions, simplifying the workflow required for
the SHERLOCK assay. The method was originally described by Zou et. al. [4]. In this
second attempt, the isolation method was slightly modified.

**Selected Cultures Based on Macroscopic Observation:**

* NOW 5.1
* Szczecin
* L1
* L4

The isolation protocol is also available on the Experiments page under the **DNA
Isolation - In-Field Method Protocol**.

The isolation was performed using previously prepared lysis and wash buffers in the same
manner as described on 16/07. In addition to Whatman paper dipsticks, two other types of
sticks were tested, sourced from different COVID tests purchased at a pharmacy. These
sticks were labeled as "A" and "C" for this experiment.

After washing the DNA with the wash buffer, the stick was dipped into 15 µl of water for
DNA elution. The eluted DNA was then transferred into the PCR reaction mix.

### PCR After the In-Field Isolation

##### PCR After the In-Field Isolation

The isolated DNA was eluted into water and then transferred into the tube containing the
PCR reaction mix, which included Galluzzi primers.

The PCR protocol is also available on the Experiments page under the **PCR Reaction
on DNA Isolated from Algae Cultures Protocol**.

Table 45: PCR Samples
Composition. Reaction volume = 20 µl

|  |  |
| --- | --- |
| **PCR Reagent** | **Volume [µl]** |
| Water with the isolated DNA | 9 |
| PCR Mix (A&A Biotechnology) | 10 |
| Primer Mix | 1 |

Table 46: PCR Reaction
Conditions Based on Manufacturer's Instructions (A&A Biotechnology)

|  |  |  |
| --- | --- | --- |
| **Reaction Step** | **Temperature [°C]** | **Time [s]** |
| Pre-denaturation | 95 | 60 |
| Denaturation | 95 | 15 |
| Annealing | 67 | 60 |
| Elongation | 72 | 15 |
| Final Elongation | 72 | 300 |

**Number of Cycles:** 30

##### DNA Electrophoresis of the PCR Product

Electrophoresis was performed in a 2% agar gel at 85 V for 45 minutes. 5 µl of each PCR
product was added to the wells of the gel. Two distinct DNA standards were used:
GeneRuler 1 kB DNA Ladder and O'GeneRuler DNA Ladder Mix (SM033).

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image33.png)

**Figure 39.** Gel Electrophoresis of the PCR Product – Results.

##### Conclusions

No specific product was received in any of the rows. The visible stripes are primer
dimers. The reaction should be repeated.

### 29.07 - 04.08

### Disposal of Algae Cultures

##### Disposal of Algae Cultures

**Purpose:** The main goal of the project has been successfully achieved, so
there is no longer a need to supply additional DNA templates for the SHERLOCK lab.
Consequently, most of the algae cultures can be disposed of.

The following liquid cultures were transferred to the Department of Biochemistry and
Plant Physiology JU for further scientific research: 2W111, NOW 511, NOW 5.111, BOW 111,
BOW 211, Szczecin, Szczecin F/2, and Szczecin.

The remaining liquid cultures and all plate cultures were disposed of according to the
protocol available on the Experiments page under the **Algae Liquid Culture
Disposal Protocol**.

### Prymnesium parvum Pure Culture Attempt – Summary

##### Prymnesium parvum Pure Culture Attempt – Summary

**Summary:** Due to limited access to the laboratory, the goal of
establishing a pure culture of *Prymnesium parvum* could not be achieved.
However, the best-maintained cultures, along with the knowledge and experience gained
during the work in the Alga Lab, remain available for future iGEM teams.

### In-field DNA Isolation – Summary

##### In-field DNA Isolation – Summary

**Summary:** Limited laboratory access prevented further work on achieving
successful in-field DNA isolation, and as a result, this goal was not met. Nonetheless,
an important insight has been acquired that remains at the disposal of future iGEM teams
working on the in-field isolation of DNA from *Prymnesium parvum* or similar
harmful species. A potentially valuable method has been identified and needs further
investigation.

### Resources

##### Resources

* Galluzzi, L., Bertozzini, E., Penna, A., Perini, F., Pigalarga, A., Graneli, E. and
  Magnani, M. (2008), Detection and quantification of *Prymnesium parvum*
  (Haptophyceae) by real-time PCR. *Letters in Applied Microbiology*, 46:
  261-266.
* White, T.J., T. Bruns, S. Lee, and J.W. Taylor. (1990). Amplification and direct
  sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322 In: *PCR
  Protocols: A Guide to Methods and Applications*, eds. Innis, M.A., D.H.
  Gelfand, J.J. Sninsky, and T.J. White. Academic Press, Inc., New York.
* Binzer, S.B., Svenssen, D.K., Daugbjerg, N., Alves-de-Souza, C., Pinto, E., Hansen,
  P.J., Larsen, T.O., Varga, E. (2019). A-, B- and C-type prymnesins are clade
  specific compounds and chemotaxonomic markers in *Prymnesium parvum*.
  *Harmful Algae*. 81: 10-17.
* Zou, Y., Mason, M.G., Wang, Y., Wee, E., Turni, C., Blackall, P.J., et al. (2017).
  Nucleic acid purification from plants, animals, and microbes in under 30 seconds.
  *PLOS Biology*, 15(11): e2003916.
* *Raport Kończący Prace Zespołu ds. Sytuacji w Odrze*. Instytut Ochrony
  Środowiska - Państwowy Instytut Badawczy. <https://ios.edu.pl/wp-content/uploads/2022/12/raport-konczacy-prace-zespolu-ds-sytuacji-w-odrze-2.pdf>

# Dry Lab

---

## **PrymChip Lab**

### 12.2023

### Garage of Complexity

The primary goal of the PrymChip Lab was to design and build a portable microfluidic
device for conducting in the field SHERLOCK reaction and fluorescence detection.

We began a collaboration with the Garage of Complexity, part of the Faculty of Physics,
Astronomy, and Applied Computer Science at Jagiellonian University. Through this
partnership, we gained access to two Prusa i3 MK3 3D printers, which use Fused
Deposition Modelling (FDM) technology. Professor Jakub Mielczarek introduced us to 3D
printing software, including TinkerCAD and PrusaSlicer, and trained us in the operation
of the printers. Since then, we have supplemented our learning by exploring various
YouTube videos and websites to deepen our knowledge of design and 3D printing with
filaments.

**Most useful websites:**

* <https://all3dp.com/basics/>
* <https://help.prusa3d.com/>

**Most useful YouTube channels:**

* [https://www.youtube.com/@ItsMeaDMaDe](https://www.youtube.com/%40ItsMeaDMaDe)
* [https://www.youtube.com/@3DPrinterAcademy](https://www.youtube.com/%403DPrinterAcademy)
* [https://www.youtube.com/@SquidmarMiniatures](https://www.youtube.com/%40SquidmarMiniatures)

### 01.2024

### pe3Dish

We began designing a microfluidic system (Figure 2) based on the pe3Dish technology,
which enables the creation of continuous perfusion 3D cell cultures equipped with
channels that mimic vascular systems, as illustrated in Figure 1 (designed in part by
Professor Jakub Mielczarek).

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image15.png)

**Figure 1.** Photo of the pe3Dish (source: https://pe3dish.com/).

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image14.jpg)

**Figure 2.** Primary microfluidic system (designed in Noteshelf).

The first model was designed in TinkerCAD as a square chamber with inlets and outlets for
different fluids (Figure 3A). The second model followed a similar design but was adapted
to fit the bottom of a 90 mm diameter plastic Petri dish, with thicker holders for the
tubes as the thinner ones easily got damaged and fell off (Figure 3B).

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image1.png)

**Figure 3A.** Primary microfluidic chamber based on pe3Dish system.
Model 1.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image16.png)

**Figure 3B.** Primary microfluidic chamber based on pe3Dish system.
Model 2.

Both models were printed using white PLA filament ([Prusament PLA Pristine White](https://www.prusa3d.com/product/prusament-pla-pristine-white-1kg/)). The 3D printing parameters were
based on the previous experience of colleagues with this specific filament. Detailed 3D
printing parameters are available in the files named Model\_1\_print and Model\_2\_print,
while the models themselves can be found as STL files under Model\_1\_stl and Model\_2\_stl.

* Model\_1\_print
* Model\_2\_print
* Model\_1\_stl
* Model\_2\_stl

Photos of the successfully printed models are shown in Figure 4.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image9.png)

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image6.png)

**Figure 4.** Primary microfluidic chambers based on the pe3Dish
system. Printed Model 2 with and without the tubes.

### 02.2024

### PrymChip Design

After considering the small volumes of SHERLOCK assay reagents required for the reaction,
the decision was made to abandon the use of tubes for incorporating fluids into the
reaction chamber.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image6.png)

Figure 5. Model 2 with tubes.

The approach shifted toward designing a 3D-printed device with a smaller reaction
chamber, aimed at detecting fluorescence without the need for using tubes. The detection
system was based on a device described in the paper “Machine Learning-Driven and
Smartphone-Based Fluorescence Detection for CRISPR Diagnostic of SARS-CoV-2”. A simple
schematic of the final device, including its electronic components, is shown in Figure
6.

After conducting research on designing an LED circuit, the necessary parts were ordered.
Additionally, the PLA filaments were replaced with PETG filaments in four colors
reflecting the iGEM JU-Krakow logo, along with a transparent filament for the reaction
chamber. This change was made because PETG is more suitable for environmental
applications, whereas PLA is biodegradable and less durable for such purposes.

**Used filaments are listed below:**

* Jet Black PETG (Prusa PETG, source: X)
* Mango Yellow PETG (Prusa PETG, source: X)
* Purple PETG (Polymaker PolyLite PETG, source: X)
* Transparent PETG (Devil Design PETG, source: X)

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image22.png)

**Figure 6.** A simple schematic of how the PrymChip is designed to
function.

The LEDs emit blue light that excites the fluorescence substrate appended to the reporter
which results in green fluorescence light that goes through a green filter, then is
focused by a lens and captured using a smartphone's camera.

The primary 3D model of the device, now called the PrymChip, is shown in Figure 7. It
consists of two boxes:

* One for the excitation and detection of green fluorescence.
* The other for storing spare parts and reagents required for the SHERLOCK reaction.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image18.png)

**Figure 7.** Primary 3D model of the PrymChip, designed in TinkerCAD.

### 03.2024

### Initial Box Design and Circuit Testing

The initial design of the box consisted of three boxes without lids, printed within the
recommended temperature range for the filaments. The results were unsatisfactory, so it
was decided to print temperature towers for each filament to determine the optimal
printing temperatures.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image4.png)

**Figure 8.** Primary design of the three boxes.

The LEDs and battery were connected to verify the functionality of the electronic
circuit. As shown in the photo below, the circuit operated correctly and did not require
any further adjustments.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image7.png)

**Figure 9.** Electronic circuit.

### 04.2024

### Determining Optimal Printing Parameters and Box Design

To determine the optimal printing parameters for PETG filaments, temperature towers were
printed with each filament. These towers indicate the best temperature settings for each
type of filament and are shown in Figure 10.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image25.png)

**Figure 10.** Temperature towers of PETG filaments.

Improved designs for the black and yellow boxes were prepared, as illustrated in Figure
11.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image5.png)
![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image10.png)

**Figure 11.** Models of the black and the yellow box.

Chambers for the reaction were designed to fit a 14 mm round cover slip. These chambers
were tested for leakage and fluorescence. The chamber with the thinner bottom was found
to leak. It was also found that the transparent PETG filament emitted a green
fluorescent signal. Consequently, the PETG lid was replaced with a lid cut from a
hairspray bottle, which was sized to fit properly into the black box.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image21.png)
![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image24.png)

**Figure 12.** Green signal emitted by a transparent PETG filament.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image28.png)
![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image11.png)

**Figure 13.** Lid from a hairspray bottle.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image26.png)
![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image20.png)

**Figure 14.** Designs of the 3D printed reaction chambers.

### 05.2024

### Box Covers and Colorblind-Friendly Symbols

Covers for the boxes were designed to fit snugly using a sliding mechanism. The black
cover required some modifications to ensure a proper fit, while the yellow lid fit well.
Additionally, colorblind-friendly symbols were added as markings on each box. Concave
and convex symbols were printed at different heights to determine the most effective
design. A height of 1.5 mm and a convex shape were selected for each box.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image12.png)

**Figure 15.** Lids for the black and the yellow box.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image29.png)

**Figure 16.** Colorblind-friendly symbols with different heights
(convex and concave).

### 06.2024

### Improved Cover Design for Detection Box and Reaction Chamber

An improved cover for the black box was designed to fit the green filter and the selected
lens (Figure 17). Additionally, a cover for the reaction chamber was created to securely
hold a glass slide on top and facilitate the removal of liquid from inside the chamber
(Figure 17).

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image13.png)
![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image27.png)

**Figure 17.** Lid for the detection box and a cover for a reaction
chamber.

### 07.2024

### Fluorescein and RNase Testing with the PrymChip

Since the prototype of the device was ready, it was decided to start the tests with
fluorescein.

Fluorescein from the Fluorescence Measurement Kit was used to prepare different
concentrations of the fluorescein. Fluorescein was centrifuged in a table-top minifuge.
It was then
dissolved in 1 ml of PBS. Different concentrations of fluorescein were prepared,
starting from 100 μM down to 0.8 μM. The next concentrations were prepared by serial
2x-dilution. This was done by transferring 250 μl of the higher concentration to a new
Eppendorf and adding 250 μl of PBS. Samples were mixed and transferred with a pipette to
a detection chamber.

The PrymChip was turned on, and a photo of the fluorescence was taken 5 seconds after
turning on the LED lights. The solution was then transferred back to the Eppendorf and
diluted 2 times. The chamber was washed with distilled water and wiped with tissue paper
after each photo. Resulting photos from different concentrations are shown below. They
were used for further analysis in Python, which is described in detail in the “Software”
section. Photos were taken using a chamber with and without the glass holder, with 3
different phones, as indicated in the description of each set of photos.

![](https://static.igem.wiki/teams/5087/pics/software/image1-3.webp)

**Figure 18.** Photos from the PrymChip – fluorescein testing.

By the **naked eye**, the **green signal** was visible at a
concentration
of **6.25 µM**. The **software** was able to detect signals
from the
highest concentration down to **6.25 µM**. Concentrations of **3.13
µM**
and lower appeared as a **black image**. Using a **stronger
light** to excite
the fluorescence signal may improve the detection and yield better results.

As tests using fluorescein fared auspiciously, we decided to conduct tests using the
fluorescent reporter used in the SHERLOCK Lab.

The RNase Control Reagent was tested as follows: 40 µL of RNaseAlert™ substrate (from
RNaseAlert™ Lab Test Kit v2, Invitrogen) was added to 397.5 µL of distilled H₂O. This
solution was then diluted twice, resulting in concentrations ranging from 1 µM to 0.008
µM. To each concentration, 62.5 µL of RNase was added, and the mixture was transferred
to the detection chamber. The PrymChip was activated, and a photo of the fluorescence
was taken 5 seconds after turning on the LED lights. After each photo was taken, the
chamber was washed with distilled water and wiped with tissue paper. The resulting photo for
the
highest concentration is shown below. No signal was visible in the photos, indicating that
the
**light source** was not strong enough to excite the
**fluorescence**,
and perhaps the **wavelength** was not appropriate for the **alert
testing**.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image19.png)

**Figure 19.** Photos from the PrymChip – RNase alert testing.

### 08.2024

### Purple Box Design and Customizable Parts

A purple box was designed and printed based on the known dimensions of the parts it would
contain (Figure 20). Additionally, new covers for the boxes were printed, each featuring
a symbol to aid recognition for colorblind individuals (Figure 20). A customizable
holder for an Eppendorf tube was also printed and can be attached with glue inside the
purple box (Figure 20). Additional ideas for removable parts are shown in Figure 21.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image17.png)

**Figure 20.** Models of lids and a purple box.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image8.png)

**Figure 21.** Removable parts that can be printed for the tool box.
(A) Eppendorf holder 1. (B) Eppendorf holder 2. (C) Syringe holder. Sources:
[Eppendorf holder
1](https://www.thingiverse.com/thing%3A6644660),
[Eppendorf holder
2](https://www.thingiverse.com/thing%3A2999517),
[Syringe holder](https://www.thingiverse.com/thing%3A5797133).

### 09.2024

### Phone Support Design and Adjustment

After adding additional symbols to the boxes' lids, the phone became unstable and
required extra support to remain in place. Movable supports were designed to match the
height of the box. The design of these supports is shown in Figure 22. The printed
support is depicted in Figure X, and the support attached to the box is shown in Figure
X. The addition of the supports did not affect the visibility of the chamber through the
phone camera.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image23.png)

**Figure 22.** Supports printed for the smartphone. (A) Design of the
support. (B) Printed supports. (C) Supports attached to the PrymChip.

## **Genome Lab / Bioinformatics**

### Bioinformatics Support Introduction

The bioinformatics support was conducted in "when-needed" manner and it does not
follow a single workflow or protocol.

For this reason this section might appear less structured that previous
sections, please treat it as supplementary to main project goals.

Main research topics:

* ITS1-5.8S-ITS2 fragment analysis
* Genome from Gdańsk quality assessment
* PCR results analysis (KAC39 emergence from GAL-PCR)
* PCR 2 results analysis (failed KAC39-PCR sequencing)
* RPA primer design/optimization
* Continuous support / literature search (White 1990, ODER1 etc.)

### 10-11.2023

### ITS1-5.8S-ITS2 fragment analysis

### **A-, B-, and C-type prymnesins as chemotaxonomic markers in Prymnesium parvum is a main paper discussed in this section.**

##### **Key Research Questions:**

* Are ITS sequences related to prymnesin types?
* Can ITS sequences be used to differentiate subtypes of Prymnesium?
* Are the 5.8S regions more conserved than ITS regions?
* Can the ITS1-5.8S-ITS2 fragment be used for crRNA design?

##### **Are ITS sequences related to the type of prymnesin?**

ITS (Internal Transcribed Spacer) sequences are often used in phylogenetic
studies to differentiate between species or strains within a species, as they
vary even between closely related organisms. Different strains/clades,
differentiated by their ITS sequences, produce different types of prymnesins,
making ITS sequences indirectly correlated with prymnesin production.

##### **Can ITS sequences be used to differentiate subtypes of Prymnesium?**

To explore this, the ITS1-5.8S-ITS2 fragment of various Prymnesium subtypes was
analyzed in MEGA11 software using the MUSCLE algorithm. There are 26 strains of
prymnesium described in the paper. After aligning them with the “DNA” option,
the MUSCLE algorithm and these, default options:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image1.png)

**Figure 1.** Default MUSCLE setting used in MEGA.

Results were obtained:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image5.png)

**Figure 2.** Alignment of 26 Prymnesium strains with type B
ITS1
highlighted in yellow.

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image3.png)

**Figure 2 enlarged.** Alignment of type B ITS1 end fragment.

On the left text box you can see 26 prymnesium strains, as described in the
paper with the type annotated (A, B or C).
On the right there are colourful
sequences after alignment, the fragment shown is the transition from the ITS1 to
the 5.8S rRNA.
Yellow box is the end of the ITS1 sequence of type B
prymnesium
derived from Prymnesium parvum partial ITS1, 5.8S rRNA gene and partial ITS2,
isolate KAC39 GenBank: AM690999.1 (older annotation, I later switched to the
KJ756812.1). I used the sequences from this position because the 26 prymnesium
strains were not annotated and I wanted to know exactly what is the sequence of
ITS1 etc.

All the 6 yellow highlighted sequences are of type B — this results shows that
it is possible to distinguish type B prymnesium from the rest using even the
short fragment of ITS1 sequence.
Here the custom sequence:

>short ITS1 end fragment from Prymnesium AM690999.1
CGGTCGTTGAGGATCCCCCTCGTCGTGCCCCTGCGCGTTGCGCTCTCGGGCACGCAAGAATTGTTGAA

was used.
Furthermore, based on the map from the paper:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image2.png)

**Figure 3.** Location origins for 26 strains described in A-,
B-, and C-type prymnesins.

All the Prymnesium strains from countries neighbouring Poland are of type B - so
we can suspect that the Prymnesium from the Oder river is also of type B

Further, very short fragment was analysed:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image10.png)

**Figure 4.** Short fragment analysis for type B ITS1.

And similarly, for the ITS2 sequence:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image13.png)

**Figure 5.** Alignment of type B ITS2 sequence (short
fragment).

This shows that it is possible to design crRNA within the ITS2 sequence,
similarly to the ITS1-5.8S from the figures above. It is important to note that
using 5.8S-ITS2 transition fragment might not be desirable because the beginning
of the ITS2 sequence is identical for all the strains and would not be able do
distinguish between the strains:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image4.png)

**Figure 6.** Alignment of 26 prymnesium strains with yellow
highlight showing 5.8S-ITS2 sequence frament.

The above analysis shows that different areas of ITS1-5.8S-ITS2 can be
selected as a potential target for crRNA to specifically detect type B
prymnesium.
Since we are interested in type B prymnesium the analysis didn’t involve finding
sequences specific for type A / type C.

##### **Are 5.8S more and ITS less conservative?**

Here is shown the transition area from ITS1 to 5.8S showing visible change in the
level of similarity, suggesting high level of 5.8S stability and high level of
ITS1 variability:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image14.png)

**Figure 7.** Alignment of 26 prymnesium strains displaying
variability change between ITS1 and 5.8S sequences.

ITS2 follows a similar trend.

##### **Can we use the ITS1-5.8S-ITS2 cluster to design crRNA?**

The variability observed in ITS sequences supports the feasibility of designing
crRNA specific to type B Prymnesium.

##### **Extra Topics:**

We also explored whether the ITS1-5.8S-ITS2 cluster can differentiate Prymnesium
parvum from other algae found in the Oder river.

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image19.png)

**Figure 8.** Alignment of Prymnesium parvum and other algae
species in the Oder river (5.8S area).

Even in more conserved 5.8S region the sequences have few identities (marked with
\*) suggesting high variability between algae-similar species in the Oder river.
There should be no problem using crRNA to specifically detect only Prymnesium.

##### **Are the Galluzzi primers specific to all subtypes of Prymnesium?**

Analysis of Galluzzi primers shows that the forward primer (GalF) is specific to
type B, while the reverse primer (GalR) is specific to all subtypes. Thus, the
primer set is specific only for type B Prymnesium.

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image9.png)

**Figure 9.** Alignment of 26 Prymnesium parvum strains with
GalF primer highlighted.

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image15.png)

**Figure 10.** Alignment of 26 Prymnesium parvum strains with
GalR primer highlighted.

This analysis demonstrates the potential for using ITS sequences in detecting and
differentiating Prymnesium subtypes, particularly type B, in environmental
samples.

### 11-12.2023

### Genome from Gdańsk quality assessment

We received a big genome file with a lot of contings and no additional
explanation.
The primary goal is to find our ITS1-5.8S-ITS2 fragment.
My approach was to use BLAST to search through the sequences looking for our
query (ITS1-5.8S-ITS2 fragment).
I tried to use the typical web BLAST, but the file was way too big and
fragmenting it to contigs resulted in getting too many files.
Ctrl + F is a bad idea since whitespaces/mismatches will render the search
unsuccessful.
You can install BLAST on your PC and omit most of the limitations.
I built a local BLAST database from the Gdańsk genome file.
The query was ITS1-5.8S-ITS2 sequence.

Here are the results:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image17.png)

**Figure 11.** Local BLAST search results from within the
genome from Grańsk using ITS1-5.8S-ITS2 as a query.

Even though it seems something was found, we can immediately see that the length
of a match is only ~119bp.
The expected length of a ITS1-5.8S-ITS2 type B fragment is 672bp.
The percentage of identities is also worrying – we used the query from type B
prymnesium so the strain from the Oder river might have some unexpected
mutations, but 91% identities is alarming.

We really want precision here and can’t rely on low-quality data. We could
theoretically use any type B prymnesium from the database but any unexpected
change in the strain from the Oder river could render our crRNAs useless.
To further investigate the worrying blast results I took the sequences marked as
somewhat similar to the query and used them in the traditional web-blast.

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image18.png)

**Figure 12.** One of the top local BLAST results used in a
web-BLAST search.

And obtained results:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image16.png)

**Figure 13.** Results from using local BLAST top-scoring
sequences in a web-BLAST search.

* This sequence is therefore not specific to Prymnesium.
* It could as well be the fragment from different algae.

Using different BLAST “hits” from the previous local BLAST search gives similar
results.

As a final test I performed BUSCO analysis:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image20.png)

**Figure 14.** BUSCO results showcasing genome assembly
quality.

BUSCO is an algorithm that checks the quality of the genome assembly.

Here we can see that only 59% of highly conserved sequences (sequences we really
expect to see in the genome) are present.
Additionally, 11% of sequences that should not be fragmented are fragmented.

This is not the worst result but combined with the previous BLAST analysis does
not give us any confidence in using this genome.

### 05.2024

### PCR results analysis (KAC39 emergence from GAL-PCR)

Based on sequencing results on Galluzzi primers, online
nucleotide BLAST was performed for forward and reverse sequencing products.

```
PCR product_primerF
TAGCCTAYAGCTCGAGCGTCGCCTGAGGGCAGCGTTGCACGGGAGGATCCTCGGATCTGACGTGTGCCGACGTGCTAGTAGGCCGCCTACCAAGTCGTTGTGCCAAYGGCTTGTCTCGGCCAAA
```

```
PCR product_primerR
TGTCTGCCGTGGACTTAGTGCTGCGCCAGATCAAGGCTCGAGCGTCGCCTGAGGGCAGCGTTGCACGGGAGGATCCTCGGATCTGACGKGCCGACGCWWTCTG
```

**Megablast with default setting for primerF:**

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image7.png)

**Figure 15.** Representation\* of BLAST results from sequencing
product from F\_primer.

\*This screenshot was not done at the time of the original analysis, it presents
the results obtained the same way on 18.08.2024 so some sequences present here
were not publicly available at the time of the original analysis.

Both search results pointed at the presence of a couple high-score results. Top
results included Prymnesium strains:

* KAC39 type B
* UIO223 type B
* ARC140 type B
* SAG18.97 type B
* K0374 type B
* K0081 type B

All of these strains are from: *A-, B- and C-type prymnesins are clade
specific compounds and chemotaxonomic markers in Prymnesium parvum*, and
all produce type-B prymnesins. All are European strains apart from ARC140. KAC39
comes from a Norwegian sample, and SAG18.97 comes from a German sample, hence
they come from countries close to Poland. KAC39 is identical to SAG19.97 (in
terms of available nucleotide sequences).

The consensus sequence FOR FOR\_PRIMER that gives the highest score in BLAST is:

```
seq from primer_F
GCTCGAGCGTCGCCTGAGGGCAGCGTTGCACGGGAGGATCCTCGGATCTGACGTGTGCCGACGTGCTAGTAGGCCGCCTACCAAGTCGTTGTGCCA
```

And that sequence is identical in all 6 strains of prymnesium as shown below:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image11.png)

**Figure 16.** Alignment of 6 type B prymnesium with PCR
product sequence highlighted in yellow.

Additionally, all the 6 type B Prymnesium strains mentioned above and shown in
the BLAST result have IDENTICAL SEQUENCE ALONG THE WHOLE ENTRY. All the 672
nucleotides are the same. Looking only at the available ITS1-5.8S-ITS2 fragment,
the 6 strains are identical. It is therefore impossible to determine which
strain we currently have, and from the point of sequence, it does not matter
since all type B prymnesium are identical when it comes to the ITS1-5.8S-ITS2
fragment available in the database.

We still need to be careful with further analysis, because there is no guarantee
that our Prymnesium from the Oder river doesn't have some surprising changes in
that fragment.

**Megablast with default setting for primerR:**

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image16.png)

**Figure 17.** Representation of BLAST results from sequencing
product from R\_primer.

Same situation here with sequences:

* KAC39 type B
* UIO223 type B
* ARC140 type B
* SAG18.97 type B
* K0374 type B
* K0081 type B

```
seq from primer R
TGTCTGCCGTGGACTTAGTGCTGCGCCAGATCAAGGCTCGAGCGTCGCCTGAGGGCAGCGTTGCACGGGAGGATCCTCGGATCTGACGKG--CCGACG
```

This time there are two gaps present — it might be the result of lower sequencing
quality along the end and beginning of the sequence. Also, there is a "K"
nucleotide, so I propose to keep only the solid sequence:

```
TGTCTGCCGTGGACTTAGTGCTGCGCCAGATCAAGGCTCGAGCGTCGCCTGAGGGCAGCGTTGCACGGGAGGATCCTCGGATCTGACG
```

The entire joined forward and reverse consensus is therefore:

```
sequencing result full
TGTCTGCCGTGGACTTAGTGCTGCGCCAGATCAAGGCTCGAGCGTCGCCTGAGGGCAGCGTTGCACGGGAGGATCCTCGGATCTGACGTGTGCCGACGTGCTAGTAGGCCGCCTACCAAGTCGTTGTGCCA
```

##### **SUMMARY**

BLAST analysis on both products confirmed that we have a type B prymnesium, as
all top results are type B prymnesium. We cannot determine which strain we have
because our product perfectly matches all 6 strains of type B prymnesium. All 6
strains of type B prymnesium (ITS1-5.8S-ITS2 fragment) are identical. We choose
to continue with the KAC39 sequence.

Within the KAC39, our PCR product consensus was marked with green letters (see
below). KAC39 sequence was downloaded and annotated according to the KJ756812.1
*[Prymnesium parvum strain CCAP 946/6]* as this was the most recent
annotated file about prymnesium parvum at the time.

There are differences in what sequence is considered ITS and what is not
(GenBank: AM690999.1 is an old KAC39 sequence and provides a different
annotation). I don't know if this is the most valid way of annotating the
sequence – I assumed the name of a given fragment is not critically important
for us and annotated the KAC39 in a rough way. Below is the resulting KAC39 file
with annotation and consensus PCR product sequence marked in green, displayed in
SnapGene.

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image8.png)

**Figure 18.** KAC39 sequence with consensus (forward and
reverse PCR products) sequence marked in green.

### KAC39 PCR analysis

After agreeing upon KAC39 being the representation of our sequence, we designed
PCR primers at the beginning and the end of the KAC39 sequence.

**KAC39 forward**

```
ATCATTACCGGTCTTTCCACCCAC
```

**KAC39 reverse**

```
GAGTCCAATGGTGCGCGC
```

We had some problems with the PCR that led me to further investigate the
availability of primers for PCR/RPA. Please see the “White 1990 paper section”
for further information.

### White 1990 paper

When I was investigating problems with the KAC39 primers, I found the White 1990
paper describing many sequences for amplifying the ribosomal regions. That's
where we found the ITS1 – ITS4 sequence that was originally used to obtain the
KAC39 sequence in the *A-, B- and C-type prymnesins are clade specific…*
paper.

### RPA primer design/optimisation

##### **Alt F**

This forward primer is designed to specifically detect *Prymnesium parvum*
genomic DNA in an RPA (Recombinase Polymerase Amplification) reaction. It binds
to the ITS2 region. "Alt" is an acronym for alternative - the primer was
designed in the Primer-BLAST online tool using directions from TwistAmp® DNA
Amplification Kits Assay Design Manual [1].

###### **Biology and usage**

The 5.8S rRNA is part of the large ribosomal subunit in eukaryotes, along with
28S and 5S rRNAs, and is encoded within a ribosomal cistron that also includes
18S and 28S rRNAs. The 5.8S rRNA, along with its associated ITS regions, is used
in phylogenetic studies to differentiate species due to the high variability of
the ITS regions between species [2].

###### **Sequence source and design**

This sequence was designed by our team using the KAC39 sequence fragment [3],
with the goal to obtain alternative primers to those from the “Establishment of
methods for rapid detection of *Prymnesium parvum* by recombinase
polymerase amplification combined with a lateral flow dipstick” paper. As
mentioned previously, the TwistAmp® DNA Amplification Kits Assay Design Manual
was used to obtain optimal parameters, and Primer-BLAST was used to obtain
sequences and ensure specificity.

##### **Alt R**

This reverse primer is designed to specifically detect *Prymnesium parvum*
genomic DNA in an RPA reaction. It binds to the end of a 5.8S rRNA region. "Alt"
is an acronym for alternative - the primer was designed in the Primer-BLAST
online tool using directions from TwistAmp® DNA Amplification Kits Assay Design
Manual [1].

###### **Biology and usage**

The 5.8S rRNA is part of the large ribosomal subunit in eukaryotes, along with
28S and 5S rRNAs, and is encoded within a ribosomal cistron that also includes
18S and 28S rRNAs. The 5.8S rRNA, along with its associated ITS regions, is used
in phylogenetic studies to differentiate species due to the high variability of
the ITS regions between species [2].

###### **Sequence source and design**

This sequence was designed by our team using the KAC39 sequence fragment [3],
with the goal to obtain alternative primers to those from the “Establishment of
methods for rapid detection of *Prymnesium parvum* by recombinase
polymerase amplification combined with a lateral flow dipstick” paper. As
mentioned previously, the TwistAmp® DNA Amplification Kits Assay Design Manual
was used to obtain optimal parameters, and Primer-BLAST was used to obtain
sequences and ensure specificity.

##### **Mod F**

This sequence was introduced by the team of Luo et al. [4] and is designed for
ITS sequencing of *P. parvum*.

###### **Biology and usage**

The 5.8S rRNA is part of the large ribosomal subunit in eukaryotes, along with
28S and 5S rRNAs, and is encoded within a ribosomal cistron that also includes
18S and 28S rRNAs. The 5.8S rRNA, along with its associated ITS regions, is used
in phylogenetic studies to differentiate species due to the high variability of
the ITS regions between species [2].

###### **Sequence source and design**

Mod F is an unmodified PR-RPA-4-F primer from the paper mentioned above [4].
PR-RPA-4-F/R were selected as the optimum primers for RPA reactions by the
authors.

##### **Mod R**

This sequence was introduced by the team of Luo et al. [4] and is designed for
ITS sequencing of *P. parvum*. Unlike the forward primer, the Mod R was
modified to better align with our sequence.

###### **Biology and usage**

The 5.8S rRNA is part of the large ribosomal subunit in eukaryotes, along with
28S and 5S rRNAs, and is encoded within a ribosomal cistron that also includes
18S and 28S rRNAs. The 5.8S rRNA, along with its associated ITS regions, is used
in phylogenetic studies to differentiate species due to the high variability of
the ITS regions between species [2].

###### **Sequence source and design**

Mod R is a modified version of the PR-RPA-4-R primer from the paper mentioned
above [4]. PR-RPA-4-F/R were selected as the optimum primers for RPA reactions
by the authors. Because the original PR-RPA-4-R primer had a mismatch of "A"
instead of "G" (with corresponding "C" on the second strand), we decided to
modify this single nucleotide to achieve full identity with our KAC39 sequence
fragment. The modification is as shown below, with the PR-RPA-4-R being the
original sequence from the paper and the PR-RPA-4-R-mod referring to the Mod R
sequence described here:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image12.png)

**Figure 20.** Original and modified PR-RPA-4 reverse primers.

##### **Resources**

* [1] [TwistAmp® DNA Amplification Kits Assay Design Manual](https://www.twistdx.co.uk/wp-content/uploads/2023/12/twistamp-assay-design-manual_rev1_v3.pdf)
* [2] Akhoundi, M., Downing, T., Votýpka, J., et al. (2017). Leishmania
  infections: Molecular targets and diagnosis. *Molecular Aspects of
  Medicine*, 57, 1-29. https://doi.org/10.1016/j.mam.2016.11.012
* [3] Prymnesium parvum strain KAC39 internal transcribed spacer 1, partial
  sequence; 5.8S ribosomal RNA gene, complete sequence; and internal
  transcribed spacer 2, partial sequence GenBank: MK091113.1
* [4] Luo, N., Huang, H., & Jiang, H. (2022). Establishment of methods for
  rapid detection of *Prymnesium parvum* by recombinase polymerase
  amplification combined with a lateral flow dipstick. *Frontiers in Marine
  Science*, 9. https://doi.org/10.3389/fmars.2022.1032847

### Primer table

#### Primer Table

**Table 1:** Primers/crRNAs used in the project.

| Primer set/crRNA | Target | Prymnesium type | For sequence (without T7 promoter etc.) | For sequence (with T7 promoter etc.) | Rev sequence (without T7 promoter etc.) |
| --- | --- | --- | --- | --- | --- |
| ModF & ModR | 5.8S & ITS2 | type B | GAATCATCGAACTTTTGAACGCAACTGG |  | GTAGGCGGCCTACTAGCACGTCGGCACA |
| ModF & AltR | 5.8S & ITS2 | type B | GAATCATCGAACTTTTGAACGCAACTGG |  | GTACCGGTGGGCGGAAACGCGACGACCTA |
| ModF & GalR | 5.8S & ITS2 | all types | GAATCATCGAACTTTTGAACGCAACTGG |  | GGCACAACGACTTGGTAGG |
| AltF & AltR | 5.8S & ITS2 | type B | CTTCCAGGTTCCGCCTGGGAGCATGTTTCTTC |  | GTACCGGTGGGCGGAAACGCGACGACCTA |
| AltF & ModR | 5.8S & ITS2 | type B | CTTCCAGGTTCCGCCTGGGAGCATGTTTCTTC |  | GTAGGCGGCCTACTAGCACGTCGGCACA |
| AltF & GalR | 5.8S & ITS2 | all types | CTTCCAGGTTCCGCCTGGGAGCATGTTTCTTC |  | GGCACAACGACTTGGTAGG |
| GalF & GalR | ITS2 & ITS2 | type B | TGTCTGCCGTGGACTTAGTGCT |  | GGCACAACGACTTGGTAGG |
| GalF & ModR | ITS2 & ITS2 | type B | TGTCTGCCGTGGACTTAGTGCT |  | GTAGGCGGCCTACTAGCACGTCGGCACA |
| GalF & AltR | ITS2 & ITS2 | type B | TGTCTGCCGTGGACTTAGTGCT |  | GTACCGGTGGGCGGAAACGCGACGACCTA |
| prymcrRNA1 | ITS2 | type A and B | GCGCCAGATCAAGGCTCGAGCGTCGCCTG |  | - |
| prymcrRNA2 | ITS2 | type A , B and some C | GAGGGCAGCGTTGCACGGGAGGATCCTCG |  | - |
| KAC39F & KAC39R | ITS1 & ITS2 | type A, B and some C | ATCATTACCGGTCTTTCCACCCAC |  | GAGTCCAATGGTGCGCGC |
| ModF | 5.8S | all types | GAATCATCGAACTTTTGAACGCAACTGG | GAAATTAATACGACTCACTATAGGGGAATCATCGAACTTTTGAACGCAACTGG | - |
| ModR | ITS2 | type B | - |  | GTAGGCGGCCTACTAGCACGTCGGCACA |
| AltF | 5.8S | all types | CTTCCAGGTTCCGCCTGGGAGCATGTTTCTTC | GAAATTAATACGACTCACTATAGGGCTTCCAGGTTCCGCCTGGGAGCATGTTTCTTC | - |
| AltR | ITS2 | type B | - |  | GTACCGGTGGGCGGAAACGCGACGACCTA |
| GalF | ITS2 | type B | TGTCTGCCGTGGACTTAGTGCT | GAAATTAATACGACTCACTATAGGGTGTCTGCCGTGGACTTAGTGCT | - |
| GalR | ITS2 | all types | - |  | GGCACAACGACTTGGTAGG |
| KAC39F | ITS1 | all types | ATCATTACCGGTCTTTCCACCCAC | GAAATTAATACGACTCACTATAGGGATCATTACCGGTCTTTCCACCCAC | - |
| KAC39R | ITS2 | type A, B and some C | - |  | GAGTCCAATGGTGCGCGC |
| ItsF | 18S | - | TCCGTAGGTGAACCTGCGG | GAAATTAATACGACTCACTATAGGGTCCGTAGGTGAACCTGCGG | - |
| ItsR | 28S | - | - |  | TCCTCCGCTTAGTGATATGC |

### 06-07.2024

### SHERLOCK assay visualization

A huge portion of my time from June to July 2024 was used for visualization of
our wet-lab results. I performed some data preparation and visualization using
Plotly (Python library). The Plotly library allows for the implementation of
interactive plots, as well as saving the file as HTML, so that we can use it on
our web page.

### PCR 2 results analysis (KAC39-PCR sequencing)

This was our second sequencing effort. This time we used sequencing products from
KAC39 PCR primers. Using discontinuous BLAST (for less similar sequences) gave
this result:

![](https://static.igem.wiki/teams/5087/pics/bioinfo-not/image21.png)

**Figure 19.** BLAST results from sequencing product from
F\_primer.

Lots of low-quality nucleotides, lots of mismatches. This is probably a distorted
KAC39 sequence. In our case, single nucleotides matter.

### 08.2024

### The discovery of the ODER1 sequence

When I was rerunning the BLAST search on previous PCR products (PCR on Galluzzi primers)
I received an additional
result: *Prymnesium parvum* strain ODER1 chromosome 20 [CP154518.1]. The
ODER1 references the Oder river, which is of interest to us.

The ODER1 sequence was added to the NCBI database on 06.07.2024, so we
unfortunately could not make use of it earlier (although the assembly date
points to December 2023). The article accompanying the data is: “The
haplotype-resolved *Prymnesium parvum* (type B) microalga genome reveals
the genetic basis of its fish-killing toxins.”

KAC39 is identical in 100% with the ODER1 sequence, which reassures us that our
previous analyses were on the basis of merit.

### Key Findings from the ODER1 Paper

* **Sequencing of genomic and chloroplast DNA**
* **Prymnesium from the Oder river is a diploid**
* **Type B genomes are bigger than type A genomes** because of
  repeated regions.
* **There is a deletion in a PKS gene** that explains differences
  between toxins of type A and type B.
* **K0081 from Denmark** is the strain most closely related to
  *Prymnesium* from the Oder river.
* **Haptonema** is used to attach to the prey.

### ITS copies analysis

#### ITS Copies Analysis

**How many copies of ITS does the ODER1 strain hold?**

Since this information isn’t available in the ODER1 paper, I will perform an
analysis to try to determine the number of ITS sequences in the genome.

It is important to note that the article states:

> “As a standard, samples from the Oder catastrophe (n = 80) in summer 2022 were
> counted microscopically (10 x 100) to calculate the correlation factor between
> qPCR-amplicon copy number of a DNA marker (Internal Transcribed Spacer 2: ITS2)
> and Prymnesium cell counts.”

> “Cell equivalents were calculated from the ITS2 copy number (R^2 = 0.87).”

That means they found a correlation between ITS2 copy number and the number of
cells. Unfortunately, the precise calculation is not disclosed.

**First step:** Check if the ITS sequence is present in more than
one chromosome.

I copied the ITS1 sequence from the KAC39 sequence:

```
> KAC39 ITS1
ATCATTACCGGTCTTTCCACCCACACCAGTGCGTACCACTCGTCCCTTTGGGTCCGTCGGTGCTCGCATCGGCGGCTCATCTGTGCTCTTGCTTGAGCTGGCGCTCCGCGCGCCAGATCGGGCGGGACGCAGGCACTGTGTCCCGGACACAGCCACATCTCCTCCTCGGCCCTCGCCGGTCGTTGAGGATCCCCCTCGTCGTGCCCCTGCGCGTTGCGCTCTCGGGCACGCAAGAATTGTTGAA
```

I used it for an online BLAST search limiting organisms to *Prymnesium
parvum*. As expected, ODER1 was among the top-scoring results—only the
entry from chromosome 20 was present, suggesting that the ITS1 sequence is only
present on chromosome 20.

Surprisingly, the number of hits to that sequence was 8 for haplotype CP154518.1
and 4 for haplotype CP154552.1. It is important to note that the matches to the
ODER1 sequence were almost entirely 100% identity matches of the entire ITS1
fragment length, suggesting perfect multiplication of the ITS1 sequence.

**Second step:** I performed the same search for the 5.8S sequence:

```
> KAC39 5.8S rRNA
ACACACAACTCTTGTCGATGGATATCTTGGCTCTCGCAACGATGAAGAACGCAGCGAAATGCGATACGTAATGCGAATTGCAGAATTCAGTGAATCATCGAACTTTTGAACGCAACTGGCGCTTCCAGGTTCCGCCTGGGAGCATGTTTCTTCGAGTGGC
```

Same results were obtained for the 5.8S sequence: 8 hits for CP154518.1 and 4 for
CP154552.1, with 100% identities.

**Final step:** The last fragment is the ITS2 sequence:

```
> KAC39 ITS2
GCCTCCACCCGCCTGGGCGTGCGCCTCGCGCGCACATCCGATCGTGTCTGCCGTGGACTTAGTGCTGCGCCAGATCAAGGCTCGAGCGTCGCCTGAGGGCAGCGTTGCACGGGAGGATCCTCGGATCTGACGTGTGCCGACGTGCTAGTAGGCCGCCTACCAAGTCGTTGTGCCATCGAACGCTGCGATCTCAACCGACGCGGGGACTCGAGGACCTAGGTCGTCGCGTTTCCGCCCACCGGTACGCCTCGCGCGCACCATTGGACTC
```

Same results here: 100% identities, 8 and 4 matches.

{% endblock %}

{% extends "layout\_page.html" %}
{% block title %}Engineering{% endblock %}
{% block banner\_image %}https://static.igem.wiki/teams/5087/pics/banners/engineering.webp{% endblock %}
{% block page\_content %}

## Blueprint for Engineering Excellence: Our DBTL Journey

---

![](https://static.igem.wiki/teams/5087/pics/engineering/dbtl-full.webp)

Our project aimed to develop an **effective and sensitive tool** for detecting
*Prymnesium parvum* blooms. Throughout our journey, we closely embraced the engineering principles
that lie at the heart of iGEM as well as synthetic biology as a scientific discipline.

We applied the engineering cycle to every aspect of our work, even in tasks that might not traditionally be seen as
engineering.
This framework guided us in designing our **hardware**, the **PrymChip**, as well as our
**biological parts**. It also shaped our
**protein purification efforts**, **SHERLOCK reaction design** and the optimization of
**growth conditions** for our *Prymnesium parvum* cultures.

We believe that this comprehensive approach played a crucial role in our project's success.

This page provides an overview of the **Design-Build-Test-Learn (DBTL)** cycles performed throughout
our project.

### CLICK THE CHOSEN BOX TO EXPLORE OUR DBTL CYCLES:

[##### Cycle 1:

Expression and Purification of huLwCas13a and CcaCas13b](#Cycle1)

[##### Cycle 2:

Engineering SHERLOCK for Prymnesium parvum Detection](#Cycle2)

[##### Cycle 3:

Development of SynLOCK – the crRNA Synthesis System for SHERLOCK](#Cycle3)

[##### Cycle 4:

Prymnesium parvum culture: optimisation of lighting conditions](#Cycle4)

[##### Cycle 5:

Optimisation of reaction conditions for PCR performed on Prymnesium parvum genome](#Cycle5)

[##### Cycle 6:

Optimization of an in-field method for DNA isolation](#Cycle6)

[##### Cycle 7:

Development of the PrymChip](#Cycle7)

---

# Cycle 1

![](https://static.igem.wiki/teams/5087/pics/engineering/20.webp)

**Goal:** The goal of this cycle was to establish a Cas13 purification protocol that would be
successful in obtaining
pure Cas13, while also being cost-effective and time-efficient.

## Iteration 1.1

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We based our approach on the protocol suggested by Kellner et al. [1], given the widespread use and study of
huLwCas13a.

For protein expression, we selected the **pC013** (huLwCas13a) [2] and pR0306 (CcaCas13b) [3]
plasmids from
Addgene, which use the **pET system** for high-yield production.
This plasmid includes sequences for purification tags **(6xHis, TwinStrep)** and a
**SUMO** tag to enhance solubility.
The SUMO tag also allows for tag removal, yielding the native form of the protein after cleavage.
We decided to express the protein in the **Rosetta™ 2(DE3)pLysS strain**, which supports the pET
system and
produces T7 polymerase and T7 lysozyme.
The latter suppresses protein expression until induction with IPTG.

**Overview of the initial protocol for Cas13 production and purification:**

1. Protein expression in the Rosetta 2(DE3)pLysS strain.
2. Bacterial cell disruption using sonication.
3. **3-step protein purification** process:
   1. IMAC (Immobilized Metal Affinity Chromatography)
   2. IEC (Ion Exchange Chromatography)
   3. SEC (Size Exclusion Chromatography)

**Accurate purification of Cas13** was crucial because the Bradford assay measures total protein
content, which
can include non-Cas13 proteins and skew concentration readings.
This could result in adding insufficient Cas13, reducing test sensitivity and causing potential false
negatives. To enhance cost-effectiveness, we **substituted** StrepTag affinity chromatography with
Immobilized
Metal Affinity Chromatography **(IMAC)** in Kellner's protocol.

##### Redesign

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

After additional consideration, we noticed that in many protocols, the **6xHis-TwinStrep-SUMO tag at the
N-terminus of the protein is cleaved off after purification** [1], [4], [5].
However, we found no specific data in the literature justifying the removal of this tag, which requires the
use of the relatively costly SUMO protease. We decided to investigate this further and check whether tag
removal is truly necessary.

We compared SUMO-digested and undigested protein fractions after IMAC using:

1. **Circular Dichroism (CD):** A fast, cost-effective method we used to analyze protein secondary
   structure and
   compare spectra of digested and undigested samples.
2. **Nano Differential Scanning Fluorimetry (NanoDSF):** This technique assesses protein stability
   across a wide
   temperature range to monitor denaturation and melting temperature.
3. **SHERLOCK:** We evaluated Cas activity using synthetic test oligonucleotides and the protocol
   from Kellner
   et al. [1], comparing endpoint fluorescence intensities of SUMO-digested and undigested Cas13a to determine
   the most suitable version.

Due to budget constraints, we initially focused on huLwCas13a, postponing ordering synthetic DNA for CcaCas13b
until huLwCas13a's activity was confirmed.
As a result, finally, we decided to apply the redesigned Cas13 purification protocol:

1. Protein expression in the Rosetta 2(DE3)pLysS strain.
2. Bacterial cell disruption using sonication.
3. **3-step protein purification process:**
   1. IMAC (Immobilized Metal Affinity Chromatography)
   2. IEC (Ion Exchange Chromatography)
   3. SEC (Size Exclusion Chromatography)
4. Comparative Analysis of Tagged and Untagged Proteins:
   1. CD (Circular Dichroism)
   2. NanoDSF (Nano Differential Scanning Fluorimetry)
   3. SHERLOCK Activity Tests

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

We carried out protein expression and purification according to the revised protocol (also included under
“Protein Purification” in the “Experiments” page):

**Step 1 – Rosetta 2(DE3)pLysS transformation with pC013 [2] and pR0306 [3] plasmids.**

As a result of transformation we observed colonies on both plated Petri dishes:

![Figure 1](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure1.jpg)

**Figure 1.** Single colony grown after transformation of Rosetta 2(DE3)pLysS cells with pC013.

![Figure 2](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure2.jpg)

**Figure 2.** Colonies grown after transformation of Rosetta 2(DE3)pLysS cells with pR0306.

**Step 2 – Bacterial cultures and Cas proteins expression.**

We used them later to prepare large scale cultures (in
total 9 L) that were induced with IPTG overnight.

**Step 3 – Harvesting bacteria.**

The following day, we harvested the bacteria through centrifugation, obtaining 95.18 g of bacterial pellet
for Cas13a and 96.7 g for Cas13b.

**Step 4 – Sonication.**

A portion of the bacterial pellet for both proteins was disintegrated using sonication. Following
centrifugation, the supernatant was prepared for the first step of purification – Immobilized Metal Affinity
Chromatography (IMAC).

**Step 5 – IMAC.**

We performed SDS-PAGE to investigate the results of IMAC:

![Figure 3](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure3.jpg)

**Figure 3.** SDS-PAGE gel for huLwCas13a after IMAC purification.

![Figure 4](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure4.jpg)

**Figure 4.** SDS-PAGE gel for CcaCas13b after IMAC purification.

Wells key: unind. – uninduced bacterial culture, ind. – culture after 18h IPTG induction, sonic. – supernatant
after sonication and centrifugation, prec. – sediment after sonication and centrifugation, FT – flow-through
from sample application on His-resin column, wash – flow-through from column washing before elution; E1, E2,
E3… – fractions eluted with 150 mM imidazole buffer, \*E1, \*E2, \*E3 – fractions eluted with 300 mM imidazole
buffer

**\*Note:** Some electropherograms show bands shifted downward from their expected positions,
likely due to well
overloading. To confirm the proteins' sequences, mass spectrometry would be ideal, but budget constraints
prevented us from performing this analysis.

**Expression and IMAC Conclusions:**

**The expression step was effective** for both Cas proteins as after induction the bands of a
mass
corresponding
to the produced protein were observed (for Cas13a – 155 kDa, for Cas13a – 160 kDa).

**IMAC was effective in partially purifying both proteins** – percentage content of contaminants
in
the solution decreased (compared to wells ind. and sonic). Although they are still present in significant
amounts. Moreover, fractions were enriched with the produced protein (thicker bands corresponding to the
produced protein – marked by arrows).

**Step 6 – SUMO digestion**

The SUMO digestion step was conducted, as planned, **on half** of the collected IMAC
**fractions**.

SUMO digested and undigested pooled fractions were used in the second purification protocol step – IEC.

**Step 7 – IEC**

SDS-PAGE gels showing results of IEC:

![Figure 5](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure5.webp)

**Figure 5.** SDS-PAGE gel for huLwCas13a – samples after IEC.

Cas13a with the tag is indicated by the violet arrow, Cas13a without the tag – blue arrow. The purple rectangle
marks two bands that correspond to the cleaved 6xHis-TwinStrep-SUMO tag.

Wells key: entry – pooled fractions after IMAC, sample appl. – flow-through from sample application on the
column, wash – flow-through from column washing, E1, E2, E3… – eluted fractions. Samples with \* correspond to
samples after SUMO protease digestion.

![Figure 6](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure6.webp)

**Figure 6.** SDS-PAGE gel for CcaCas13b – samples after IEC.

Cas13b with the tag indicated by yellow arrow, without by orange arrow.

Wells key: eluted fractions of Cas13b (left picture for SUMO protease undigested, right for digested) E6, E8,
E10... – eluted fractions; flow-through – from sample application on the column; wash – flow-through from
column washing. The area where a band for the digested tag was expected is marked with black circles. For the
undigested protein there is one band in this area, while for the digested protein, there are two. Therefore,
the additional band is likely the tag.

**IEC Conclusions**: For Cas13a and Cas13b (both SUMO protease digested and undigested samples)
**reduction in protein contaminants**, approximately 2-fold, can be observed, although they still
remain in significant amounts. Concentration of Cas13a and Cas13b in the collected fractions is visible.
After IEC and concentration of pooled fractions to less than 0.5 ml the final purification step – SEC – could
be conducted.

**Step 8 – SEC**

![Figure 7](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure7.webp)

**Figure 7.** SDS-PAGE gel for huLwCas13a – samples after SEC.

The protein with the tag is marked by the violet arrow, while the protein without the tag is marked by the
blue arrow.

Wells are labeled as follows: E1, E2, E3, etc., representing eluted fractions. Samples marked with \* are those
that underwent SUMO protease digestion.

![Figure 7](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure8.webp)

**Figure 8.** SDS-PAGE gel for Cas13b – samples after SEC\*.

The protein with the tag is marked by the yellow arrow, while the protein without the tag is marked by the
orange arrow.
Wells are labeled as follows: E1, E2, E3, etc., representing eluted fractions.

**SEC Conclusions:** After the 3-step purification process, **we obtained pure huLwCas13a and
CcaCas13b proteins**, as
confirmed by SDS-PAGE (Figures 7 and 8). As expected, a single band was observed for the undigested proteins. In
SUMO-digested fractions, two bands were observed, indicating incomplete tag cleavage.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

As planned, we conducted a comparative analysis of tagged and untagged proteins to test their functionality.
Expected results included:

* **Circular Dichroism (CD)**Anticipated disruption of protein structure by the tag.
* **Nano Differential Scanning Fluorimetry (NanoDSF):**Expected impact of the tag on protein
  stability. On one hand, the presence of the SUMO tag should stabilize it, while additional tag sequences may
  as well stabilize and destabilize the protein due to internal interactions.
* **SHERLOCK Activity Tests:**Expected reduced activity of the protein with the tag. We assessed
  protein activity by measuring endpoint fluorescence intensity.

**Nevertheless, we hoped to not observe significant differences between both digested and undigested
proteins.**

**CD Results:**

![Figure 9](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure9.webp)

**Figure 9.** Dependence of mean residue molar ellipticity for huLwCas13a with and without the
tag in the
far-UV range.

![Figure 10](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure10.webp)

**Figure 10.** Dependence of mean residue molar ellipticity for CcaCas13b with and without a tag
in the far-UV
range.

**Table 1.** Proportions of various secondary structure types determined from Circular Dichroism
spectra for
huLwCas13a.

| huLwCas13a | Mean proportions of secondary structure types determined using algorithms from the CDPro package ± standard deviation | | | |
| --- | --- | --- | --- | --- |
| α-helix | β-sheet | β-turn | unordered |
| undigested | 64.5 ± 2.1 | 3.8 ± 0.9 | 10.2 ± 0.7 | 20.9 ± 2.5 |
| digested | 58.1 ± 10.2 | 4.7 ± 6.3 | 18.2 ± 9.9 | 19.5 ± 7.2 |

**Table 2.** Proportions of various secondary structure types determined from Circular Dichroism
spectra for CcaCas13b.

| CcaCas13b | Mean proportions of secondary structure types determined using algorithms from the CDPro package ± standard deviation | | | |
| --- | --- | --- | --- | --- |
| α-helix | β-sheet | β-turn | unordered |
| undigested | 76.8 ± 11.1 | 1.5 ± 2 | 6.9 ± 4.3 | 15.2 ± 10.5 |
| digested | 80.4 ± 7.4 | 0.5 ± 2 | 4.4 ± 3.3 | 14.7 ± 7.8 |

**CD Conclusion:** The spectra in Figures 9 and 10 overlap significantly, and the contribution of
individual
secondary structures to the protein structure (Tables 1 and 2) does not differ substantially. This indicates
(contrary to our expectations) that **the tag does not have a significant impact on the structure**
of Cas13a and
Cas13b.

**NanoDSF Results:**

![Figure 11](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure11.webp)

**Figure 11.** Dependence of the first derivative of the fluorescence intensity change from
temperature for
huLwCas13a.

![Figure 12](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure12.webp)

**Figure 12.** Dependence of the first derivative of the fluorescence intensity change from
temperature for
CcaCas13b.
 Key: D – sample digested using SUMO protease, UD – sample undigested

**NanoDSF Conclusion:** Contrary to our expectations, NanoDSF results indicated no statistically
significant
difference between tagged and untagged proteins in thermal stability.

**Activity tests in SHERLOCK Results:**

![Figure 13](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure13.jpg)

**Figure 13.** A comparison of the activity of huLwCas13a with and without 6xHis-TwinStrep-SUMO
tag.

Figure key:

1. SUMO protease digested huLwCas13a added to SHERLOCK mix
2. SUMO protease undigested huLwCas13a added to SHERLOCK mix
3. water added instead of Cas13a (negative control)
4. RNase A added instead of Cas13a (positive control)
5. target RNA and crRNA replaced with water in SHERLOCK mix, for SUMO digested huLwCas13a
6. arget RNA and crRNA replaced with water in SHERLOCK mix, for SUMO undigested huLwCas13a

**Activity tests in SHERLOCK Conclusion:** Contrary to our expectations the Cas13a protein, both
digested and
undigested by SUMO protease, showed similar levels of activity.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

Based on our tests, we made several adjustments to the IMAC protocol:

* **Column Washing:** Wash the column with 30 mM imidazole buffer after sample application
  instead of a buffer
  without imidazole. This change will help to remove some weaker-binding impurities.
* **Elution:** Elute the protein using 300 mM imidazole buffer to ensure complete removal of all
  bound proteins.
  We observed that using only 150 mM imidazole buffer results in some Cas13 proteins being retained.

Moreover, we have drawn the following conclusions regarding the entire protocol:

1. **Purification Process:** A **1-step purification using IMAC is insufficient**, as
   only about 15%
   of the protein in
   the collected fractions was Cas13. We recommend a 3-step purification process (IMAC, IEC, and SEC) to achieve
   100% pure Cas13, as can be demonstrated by comparing Figures 4 with 8, and Figures 5 with 9.
2. **Cost-Effectiveness:** IMAC can effectively replace StrepTag affinity chromatography, making
   the purification
   process more cost-effective.
3. **SUMO Protease Tag Cleavage:** The SUMO protease tag cleavage step is unnecessary. This
   conclusion is supported
   by the following tests:
   * **Circular Dichroism (CD):** The mean residue ellipticity spectra (Figures 9 and 10) for
     both tagged and
     untagged proteins overlap, and the proportions of secondary structures (Tables 1 and 2) are similar. This
     indicates that the tag does not significantly impact the proteins' structure.
   * **Nano Differential Scanning Fluorimetry (NanoDSF):** The thermal stability curves for
     digested (D) and
     undigested (UD) proteins overlap (Figures 11 and 12), suggesting that the tag does not significantly
     affect thermal stability. This further supports the decision not to cleave off the 6xHis-TwinStrep-SUMO
     tag.
   * **Activity Tests in SHERLOCK:** huLwCas13a was found to be active. There were no
     statistically significant
     differences in activity between SUMO protease digested (1) and undigested (2) samples (Figure 13).
     huLwCas13a performed as expected and could be used for further SHERLOCK reactions, without the need to
     further test of CcaCas13b.

Overall, this DBTL cycle and the engineering framework applied throughout this procedure enabled us to obtain
**pure and active Cas13** for future experiments.

##### References

[1] Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR
nucleases. Nat Protoc. 2019 Oct;14(10):2986–3012. doi: 10.1038/s41596-019-0210-2.

[2] Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM,
Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F.
Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 13. doi: 10.1126/science.aam9321. PMID:
28408723.

[3] Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid
detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444. doi:
10.1126/science.aaq0179. Epub 2018 Feb 15. PMID: 29449508.

[4] An B, et al. Rapid and sensitive detection of Salmonella spp. using CRISPR-Cas13a combined with recombinase
polymerase amplification. Front Microbiol. 2021 Oct. doi: 10.3389/fmicb.2021.732426.

[5] Khan H, et al. CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2. Chinese
Chem Lett. 2019 Dec;30(12):2201–2204. doi: 10.1016/j.cclet.2019.10.032.

# Cycle 2

![](https://static.igem.wiki/teams/5087/pics/engineering/21.webp)

**Goals:**

1. **Design crRNA Molecules:** Develop crRNA sequences for detecting Prymnesium parvum using the
   SHERLOCK system.
2. **Adapt RPA Primers:** Design or modify RPA primers to amplify a fragment of genome specific to
   *Prymnesium parvum*.
3. **Proof-of-Concept Testing:** Validate the functionality of crRNA designs and RPA primers with
   both fluorescence
   and
   LFA readouts.
4. Identify the most **effective primer-crRNA** combination from all possible combinations.

## Iteration 2.1

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The goal of this iteration was to **design and test initial versions** of RPA primers and
PrymcrRNAs.

In our test, we targeted the **ITS2 (Internal Transcribed Spacer 2)** sequence of *Prymnesium
parvum* for
specific detection. The ITS2 region, located between the 5.8S and nuclear large subunit rRNA genes [1], is
commonly used for species identification due to its **variability between species and conservation
within a
species.** This makes it an ideal target for species-specific primers, including those used to detect
*Prymnesium parvum* [2]. We aimed to leverage these features using our crRNA molecules.

**Gal RPA Primers:**

The first RPA primer pair we designed was based on the primers created by **Galluzzi et al.
[1]**, with a **T7
promoter** appended to the 5' end of the forward primer. This modification allows the sequence
amplified by RPA
(Recombinase Polymerase Amplification) to be transcribed in vitro into RNA, which is necessary since Cas13a
only detects RNA. Since the GalF and GalR primers were proven effective in PCR in previous experiments, we
chose them as a reliable starting point for designing our RPA primers.

![Figure 1](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure1.webp)

**Figure 1.** Binding of Gal primers to the ITS2 region of the *Prymnesium parvum*
genome.

**PrymcrRNA1 and PrymcrRNA2:**

In order to design crRNAs that would bind to a sequence in the genome of *Prymnesium parvum*, we needed
to start from scratch. To establish a starting sequence, we performed PCR on DNA isolated from *Prymnesium
parvum* using the standard Galluzzi [1] primers. We sent the PCR product for **sequencing**
and received
high-quality reads. However, the product was only about 130 bp long, which was too short for designing
multiple crRNA molecules. Since we anticipated that some crRNAs might not be functional, we aimed to design
two initial crRNA sequences to begin with.

At the time of us designing the crRNAs, there were no available *Prymnesium* sequences originating from
the Oder River. Since we needed a reference template for designing crRNAs and primers, we had to find an
alternative *Prymnesium* sequence representation.

Therefore, online nucleotide BLAST was performed for forward and reverse sequencing products revealing. The
search results indicated several high-scoring matches. The top results included *Prymnesium* strains:

* KAC39 type B
* UIO223 type B
* ARC140 type B
* SAG18.97 type B
* K0374 type B
* K0081 type B

Given the high sequence similarity to our samples (PCR products), the production of type-B prymnesins, and
the geographical proximity to the Oder River, we chose **KAC39** as the *Prymnesium*
template. This selection
provides a reference for designing crRNAs and primers for our project.

KAC39 sequence was downloaded from NCBI:

*MK091113.1 *Prymnesium parvum* strain KAC39 internal transcribed spacer 1, partial sequence; 5.8S
ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence*

and annotated based on **KJ756812.1**.

For crRNA design, we opted to use our own sequencing data to ensure complete accuracy with the Oder strain of
*Prymnesium parvum*. Based on this data, we designed two crRNAs, termed **PrymcrRNA1 and
PrymcrRNA2**. The
binding sites for these crRNAs are shown in Figure 2.

![Figure 2](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure2.webp)

**Figure 2.** Binding sites of PrymcrRNA1 and PrymcrRNA2 to the *Prymnesium* genome.

![Figure 3](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure3.webp)

**Figure 3.** Overview – binding of both PrymcrRNAs as well as the Gal primers to the
*Prymnesium* genome.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

**Gal primers:**

We ordered the primers from a manufacturer as the designed DNA sequence. Sequences can be accessed on their
Part Registry page.

1. GalF: BBa\_K5087000
2. GalR: BBa\_K5087001

**PrymcrRNAs:**

We ordered the crRNAs from a manufacturer as the designed DNA sequence. Sequences can be accessed on their
Part Registry page.

1. PrymcrRNA1: BBa\_K5087022
2. PrymcrRNA2: BBa\_K5087023

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

We conducted the SHERLOCK reactions using both fluorescence and LFA readouts, following the protocols
detailed on our “Experiments” page. Each experiment was carried out in multiple replicates, and comprehensive
descriptions of all experiments can be found on the “Notebooks” page. Here, representative results from these
experiments have been included.

![](https://static.igem.wiki/teams/5087/pics/engineering/sherlock-1.webp)

**Figure 4.** SHERLOCK fluorescence readout result 05/06/2024\*.

**Note:** The DNA templates used are: “prymDNA” for ***Prymnesium parvum*
DNA**
and “synDNA” for
synthetic DNA from Kellner et al. [1]. The crRNAs include **“PrymCrRNA1” and “PrymCrRNA2,”**
designed by us for
*Prymnesium parvum* detection, along with “syncrRNA” from Kellner et al. [1]. The sample “synDNA1
syncrRNA” serves as a **positive control**, while “synDNA1 prymcrRNA1” and “synDNA1 prymcrRNA2”
act
as **negative
controls** due to mismatches between the DNA template and crRNAs.

![](https://static.igem.wiki/teams/5087/pics/engineering/sherlock-5-crrna1.webp)

**Figure 5:** SHERLOCK fluorescence readout result 24/06/2024\*.

In this test, the “negative control + prymcrRNA1” produced a fluorescence signal. In this sample, water was
added to an RPA mix containing Gal primers.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure6.jpg)

**Figure 6.** LFA test result 08/07/2024\*.

**LFA strips\*\*:**

1. Negative control + PrymcrRNA1
2. Negative control + PrymcrRNA2
3. *Prymnesium* DNA 200 nM + PrymcrRNA1
4. *Prymnesium* DNA 200 pM + PrymcrRNA1
5. *Prymnesium* DNA 200 fM + PrymcrRNA1
6. *Prymnesium* DNA 200 nM + PrymcrRNA2
7. *Prymnesium* DNA 200 pM + PrymcrRNA2
8. *Prymnesium* DNA 200 fM + PrymcrRNA2
9. Positive control

\*The dates refer to dates of experiments as included in the “Notebook” page. Please refer to the
appropriate Lab Notebooks (SHERLOCK Lab for fluorescence measurements, LFA Lab for LFA readouts) for a more
detailed description of the performed experiments.

\*\*The description of the samples has been simplified for clarity. For a comprehensive overview of their
contents, please refer to the LFA Lab documentation included in the “Notebooks”.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

In our experiments, we confirmed that **PrymcrRNA2 effectively detects *Prymnesium parvum*
DNA** when paired
with Gal primers.

However, **we did not achieve the same success with PrymcrRNA1**, even though it produced positive
results for
samples containing *Prymnesium* DNA. This issue arose because we also obtained **positive results in
the
negative controls** (Figure 5 and 6). In multiple tests using both fluorescence and LFA readouts, we found
that PrymcrRNA1 combined with Gal primers led to false positives (Figure 5), as the negative controls also
showed positive results. This also clarifies why the negative controls in Figure 4 were truly negative—because
syn RPA primers were used in the RPA reaction. This highlighted the **critical importance of including
negative controls
in our
experiments.**

We initially concluded that the Gal primer pair and PrymcrRNA1 combination might be
**incompatible**, so we decided
to temporarily **exclude it from experiments**.
Consequently, we had to
**design new RPA primers** – we aimed to source at least 2 new pairs. Further research was needed
to determine
**whether PrymcrRNA1 is functional**. To investigate this, we planned to test it with the
different, newly
designed pairs of primers. We also decided to later investigate further to determine whether the issue lay
with the forward or reverse primer, and whether one of the Gal primers could still be used in future
experiments.

Our hypothesis was that one or both of the Gal primers might **inadvertently serve as a target for
PrymcrRNA1**.
We suspect this could be the forward primer due to its proximity to the PrymcrRNA1 binding site (Figure 3).

## Iteration 2.2

**Goal:** To design new RPA primer pairs based on the KAC39 sequence for *Prymnesium parvum* in
order
to optimize our SHERLOCK test.

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We designed **2 new RPA primer pairs:**

**1. Mod** (ModF, ModR): we based their sequences on the Luo et al. publication [2]. For ModF, we
used
the PR-RPA-4-F primer from the publication, and we appended the T7 promoter to its sequence. For ModR, we used a
modified version of the PR-RPA-4-R primer, changing an A to a G to better align with the *Prymnesium
parvum*
DNA fragment sequenced by us.

![Figure 7](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure7.webp)

**Figure 7.** Fragment of the ITS1-5.8S-ITS2 region of the *Prymnesium parvum* genome
showing
the simulated binding of the primer suggested by Luo et al. (below) and the primer modified by us – ModR
(above).

![Figure 8](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure8.webp)

**Figure 8.** Binding of Mod primers to the ITS2 region of the *Prymnesium parvum* genome.

**2. Alt** – their sequences were designed using Primer-BLAST, following the guidelines provided
in
the
TwistAmp® DNA Amplification Kits Assay Design Manual.

![Figure 9](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure9.webp)

**Figure 9.** Binding of Alt primers to the ITS2 region of the *Prymnesium parvum* genome.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

We ordered the primers as DNA parts with the designed sequence, which can be accessed on their Part Registry
page.

1. ModF: BBa\_K5087002
2. ModR: BBa\_K5087003
3. AltF: BBa\_K5087004
4. AltR: BBa\_K5087005

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

![Figure 10](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-12-530nm-fix.webp)

**Figure 10.** SHERLOCK fluorescence readout result 16/07/2024. Results for the 530 nM
concentration of *Prymnesium* DNA.

![Figure 11](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-12-200fm-fix.webp)

**Figure 11.** SHERLOCK fluorescence readout result 16/07/2024. Results for the 200 fM
concentration of *Prymnesium* DNA.

![Figure 12](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure12.webp)

**Figure 12.** LFA test result 15/07/2024.

**LFA strips\*\*:**

1. Negative control (Alt) + PrymcrRNA1
2. Negative control (Alt) + PrymcrRNA2
3. *Prymnesium* DNA + PrymcrRNA1
4. *Prymnesium* DNA + PrymcrRNA2
5. Negative control (Mod) + PrymcrRNA1
6. Negative control (Mod) + PrymcrRNA2
7. *Prymnesium* DNA (Mod) + PrymcrRNA1
8. *Prymnesium* DNA (Mod) + PrymcrRNA2
9. Negative control (Gal) + PrymcrRNA2
10. *Prymnesium* DNA (Gal) + PrymcrRNA2
11. Positive control

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The results of both readouts – fluorescence and LFA – were once again consistent with each other.

We confirmed that both pairs of our **newly designed primers successfully allowed amplification using
the RPA
method**. Importantly, no false positives were observed in the negative controls, indicating that
both PrymcrRNA
molecules are potentially compatible with the primers. We also confirmed that **PrymcrRNA1 is
functional**, as it
successfully detects *Prymnesium* DNA. Contrary to our initial assumptions, we were able to design
**two
functional crRNA molecules from the start**, without needing a trial-and-error approach.

We successfully detected *Prymnesium* DNA using the following new primer-crRNA combinations:
**Mod-PrymcrRNA1, Alt-PrymcrRNA1, and Mod-PrymcrRNA2**. However, we were unable to consistently
detect
*Prymnesium* DNA with the Alt-PrymcrRNA2 pair. Additionally, we further confirmed the functionality of
the **Gal-PrymcrRNA2 combination**.

The most effective primer-PrymcrRNA pair was **Mod-PrymcrRNA1**, which achieved high
fluorescence intensity for
both **530 nM and 200 fM** samples. No other combination successfully detected the 200 fM target
concentration,
and this pair also produced the strongest T-line in the LFA readout.

We considered the possibility of **mixing the primers** to see if any combination could be more
effective than
Mod-PrymcrRNA1. However, since this experiment focused on system optimization rather than confirming component
functionality, this approach was **tested in the next cycle**.

We successfully concluded this cycle, having identified **four functional primer-crRNA
combinations**
(Mod-PrymcrRNA1, Alt-PrymcrRNA1, Mod-PrymcrRNA2, and Gal-PrymcrRNA2) for detecting *Prymnesium parvum*
using the SHERLOCK system.

## Iteration 2.3

**Goal:**To identify the most effective primer-crRNA combination from all possible combinations.

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

As described in Iteration 2, we wanted to determine if **mixing forward and reverse primers**
from different sets would enable to achieve a **lower limit of detection** than the previously
established 200 fM.
We also
explored whether
combining the two PrymcrRNAs in equimolar amounts would enhance effectiveness.

To evaluate this, we assessed eight primer combinations using fluorescence readout:

1. ModF + ModR
2. ModF + GalR
3. GalF + ModR
4. ModF + AltR
5. AltF + ModR
6. AltF + AltR
7. AltF + GalR
8. GalF + AltR

Each combination was tested with both **PrymcrRNA1 and PrymcrRNA2**, as well as with negative and
positive
controls, resulting in a total of **41 conditions** being assessed.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

Sequences can be accessed on their Part Registry page, as described in Iteration 1-2. The procedure was
performed according to the established protocol.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

![Figure 13](https://static.igem.wiki/teams/5087/pics/engineering/sherlock-14-480nm.webp)

**Figure 13.** SHERLOCK fluorescence readout result 17/07/2024. Results for the 480 nM
concentration of *Prymnesium* DNA\*.

![Figure 14](https://static.igem.wiki/teams/5087/pics/engineering/sherlock-14-200fm.webp)

**Figure 14.** SHERLOCK fluorescence readout result 17/07/2024. Results for the 200 fM
concentration of *Prymnesium* DNA\*.

*\*The dates refer to dates of experiments as included in the “Notebook” page. Please refer to the
appropriate Lab Notebooks (SHERLOCK Lab for fluorescence measurements, LFA Lab for LFA readouts) for a more
detailed description of the performed experiments.*

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The most effective combination appears to be **ModF + GalR + PrymcrRNA1**, as it produced high
signals for both **480
nM and 200 fM** target DNA concentrations. This result was confirmed in two separate repetitions.

We also discovered that it's not just one Gal primer, but likely the combination of both, that inadvertently
activates PrymcrRNA1. When each Gal primer was paired with a different primer, the results were consistent.
Since our primary
goal was to engineer the most effective combination of primers and crRNAs, and we successfully achieved that, we
decided not to investigate this issue further.

Following the success with mixing the primers, we considered that mixing equimolar amounts of the crRNAs might
also enhance sensitivity, as suggested by a paper [3]. We planned to explore this idea further in the next
iteration using an
LFA readout, while also validating the results obtained so far with LFA.

## Iteration 2.4

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We aimed to **evaluate the effectiveness** of the selected primer combinations in the **LFA
procedure**
based on the
fluorescence readout tests from Iteration 2. The specific combinations tested were: **GalF + ModR, ModF +
GalR,
ModF + ModR**.

Additionally, we investigated whether **combining the two PrymcrRNAs** at a 1:1 ratio
(PrymcrRNA1:PrymcrRNA2 = 1:1)
would improve **detection sensitivity**.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

Sequences can be accessed on their Part Registry page, as described in Iteration 1 and 2. The procedure
was
performed according to the established protocol.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

![Figure 15](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure15.webp)

**Figure 15.** LFA test result 18/07/2024\*\*.

**LFA test strips:**

1. *Prymnesium* DNA (480 nM) Gal-Mod + PrymcrRNA1
2. *Prymnesium* DNA (480 nM) Mod-Gal + PrymcrRNA1
3. *Prymnesium* DNA (480 nM) Mod-Mod + PrymcrRNA1
4. *Prymnesium* DNA (200 fM) Gal-Mod + PrymcrRNA1
5. *Prymnesium* DNA (200 fM) Mod-Gal + PrymcrRNA1
6. *Prymnesium* DNA (200 fM) Mod-Mod + PrymcrRNA1
7. *Prymnesium* DNA (480 nM) Gal-Mod + PrymcrRNA1/2†
8. *Prymnesium* DNA (480 nM) Mod-Gal + PrymcrRNA1/2†
9. *Prymnesium* DNA (480 nM) Mod-Mod + PrymcrRNA1/2†
10. *Prymnesium* DNA (200 fM) Gal-Mod + PrymcrRNA1/2†
11. *Prymnesium* DNA (200 fM) Mod-Gal + PrymcrRNA1/2†
12. *Prymnesium* DNA (200 fM) Mod-Mod + PrymcrRNA1/2†
13. Negative control Gal-Mod + PrymcrRNA1/2
14. Negative control Mod-Gal + PrymcrRNA1/2
15. Negative control Mod-Mod + PrymcrRNA1/2

**†***PrymcrRNA1/2 indicates the equimolar mixture of PrymcrRNA1 and PrymcrRNA2.*

*\*\*The description of the samples has been simplified for clarity. For a comprehensive overview of their
contents, please refer to the LFA Lab documentation included in the “Notebooks”.*

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

Combining these results with ones from the previous iteration, it can be stated that the most optimal
primer-crRNA combination for our test is **ModF + GalR + PrymcrRNA1**. We decided to use this
combination for all
subsequent tests, and it later underwent precise **Limit of Detection (LOD)** determination (as
described in the
“Measurement” page).

Combining the **two PrymcrRNAs did not enhance specificity**, as samples with only PrymcrRNA1
showed more intense
T-lines.

The negative control for the GalF + ModR primer combination yielded a positive result. As a result, all other
findings using this primer pair were not considered. However, the negative controls for the ModF + GalR and ModF
+ ModR combinations were negative, indicating that the results from these combinations are reliable and can be
interpreted with confidence.

##### References

[1] Galluzzi L, Bertozzini E, Penna A, Perini F, Pigalarga A, Graneli E, Magnani M. Detection and
quantification
of *Prymnesium parvum* (Haptophyceae) by real-time PCR. *Lett Appl Microbiol.* 2008 Feb;46(2):261-6.
doi:10.1111/j.1472-765X.2007.02294.x. Epub 2007 Dec 13. PMID: 18086191.

[2] Luo N, Huang H, Jiang H. Establishment of methods for rapid detection of *Prymnesium parvum* by
recombinase polymerase amplification combined with a lateral flow dipstick. *Front Mar Sci.* 2022 Oct.
doi:10.3389/fmars.2022.1032847.

[3] Fozouni P, Son S, Derby MDDL, Knott GJ, Gray CN, D’Ambrosio MV, Zhao C, et al. Amplification-free detection
of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. *Cell.* 2021 Jan;184(2):323-333.e9.
doi:10.1016/j.cell.2020.12.001.

# Cycle 3

![](https://static.igem.wiki/teams/5087/pics/engineering/22.webp)

**Goals:**
Engineer a Standardized System for Customizable crRNA Synthesis for the SHERLOCK Platform.

## Iteration 3.1

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

After recognizing the need for a user-friendly crRNA synthesis system, we designed our initial **SynLOCK
Cassette**.
We imagined our system as a set of elements that could accept the custom crRNA spacer of a user’s choice.

The initial SynLOCK Cassette design (shown in Figure 1.) includes the **T7 promoter** for *in
vitro*
transcription, the
**DR loop** for huLwCas13a binding to the crRNA molecule, **SapI** sites for cloning
the
custom spacer
sequence, and a
**BbsI** site for vector linearization prior to in vitro transcription.

![](https://static.igem.wiki/teams/5087/pics/engineering/kloc-1.webp)

**Figure 1.** Initial SynLOCK Cassette Design

Initially, our team used the **PSB1C5C** plasmid backbone for assembly due to its size, high copy
number, and
availability in the 2024 iGEM Distribution kit, which was accessible to us and other teams.

Throughout our design stage, we used **SnapGene** software to simulate the integration of crRNA
spacers into the
cassette and the process of crRNA synthesis. This confirmed that in silico, molecules of the correct length and
sequence could be obtained using our system, giving us confidence to proceed with the lab work.

We chose to use **standard E. coli strains** as the foundation for our design, specifically the NEB
5-alpha competent cells provided by our sponsors, New England Biolabs, and the commonly available Top10
competent cells from ThermoFisher.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

We ordered the Cassette DNA, assembled it by hybridizing two DNA strands to create sticky ends, and
successfully cloned it into the pSB1C5C plasmid backbone using cloning with BsaI enzyme.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle3/figure2.webp)

**Figure 2.**Results of cloning the initial cassette design into the
pSB1C5C plasmid backbone:
Plates showing colonies post-transformation, with the negative control marked in the image.

![](https://static.igem.wiki/teams/5087/pics/engineering/plazmid-1.webp)

**Figure 3.** The schematic representation of the initially assembled device.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

We verified the sequence of our design using **Sanger sequencing** to ensure that the clones we
selected contained
the Cassette. The results confirmed that the **Cassette was present and capable of accepting the crRNA
spacers**.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle3/figure4.webp)

**Figure 4.** Sequencing data confirming the successful integration of the Cassette spine into the
pSB1C5C plasmid backbone, with no detected mutations.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

In this iteration, and after consulting with specialists, we learned the following:

* Currently, **time-inefficient sequencing is required** to verify if the Cassette is integrated
  into the plasmid
  backbone, which is
  **not time-efficient**. This is because our system doesn’t have a method to distinguish between
  bacteria with the
  empty plasmid backbone and those that have incorporated the Cassette. A **reporter** within the
  Cassette could solve
  that problem.
* The **plasmid backbone** we used is **not ideal**. It is designed for storing CDS
  parts and is not a
  LVL1 plasmid
  suitable for Type IIS assembly. Additionally, **it lacks the biobrick prefix and suffix**, making
  it impossible to
  transfer the Cassette between vectors after cloning.
* Both *E. coli* strains performed well and could be used to host our system.

## Iteration 3.2

**Goal:** To resolve the issues encountered in the first iteration by incorporating a
**different
plasmid backbone and adding a reporter**.

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We designed an improved version of our Cassette with a **reporter unit**. We tested several
reporter
combinations
assembled from basic parts available in the Distribution Kit. The reporter was strategically placed
**between the
SapI sites** to ensure it would be excised when a custom crRNA spacer was incorporated, allowing for
**easy
visual
screening** of colonies.

![](https://static.igem.wiki/teams/5087/pics/engineering/kloc-2.webp)

**Figure 5.** Improved SynLOCK Cassette Design.

Additionally, we chose the **pSB1C3** plasmid as the new backbone. This plasmid is a standard for
storing BioBrick
parts, meeting our criteria for being **high-copy and accessible**, and it supports cassette
transfer
using BioBrick
prefix and suffix cutting sites.

We used **SnapGene’s** Golden Gate cloning simulator to predict our assembly process for the
reporter. This involved
simulating the construction of the reporter from basic parts. We then simulated the PCR reaction to amplify the
reporter module with primers that introduced sticky ends, facilitating cloning into the backbone. Finally, we
assessed whether the assembly would be successful by simulating the cloning of the product into a new backbone.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

We introduced the reporter unit into the Cassette by amplifying it via PCR to obtain compatible sticky ends.
Then we amplified
the complete Cassette with the reporter and transferred it into the pSB1C3
plasmid backbone.

![](https://static.igem.wiki/teams/5087/pics/engineering/plazmid-2.webp)

**Figure 6.** The schematic representation of the final assembled
device.

Then we assembled
the system by cloning in the custom crRNA spacers used in our PrymDetect
test. We employed the
**Type IIS assembly standard** to insert the crRNA spacers, ensuring that there were **no
scar
sites** in
our final IVT
template construct that could impact the length or sequence of the crRNA.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle3/figure7.webp)

**Figure 7.** Schematic depiction of the improved system in action. On the right, a fragment of a
Petri dish showing visible white and purple bacteria, indicating which colonies are ready for picking.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

We conducted IVT on the plasmids according to a protocol included in the “Experiments” page.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle3/figure8.webp)

**Figure 8.** RNA electrophoresis – IVT results. "SVR" indicates
that
the crRNA from that well
was
stored in the pSB1C5C backbone, while "USV" signifies that it was stored in the pSB1C3 backbone.

**Conclusions:** We **successfully obtained crRNAs** of the correct length, as
indicated
by the single
band on the gel for
most wells at around 64 bp. Some contamination is visible in certain wells, likely due to the presence of the
plasmid IVT template.

We also tested the **Cassette’s transferability** between different backbones by excising it from
the
pSB1C3 backbone
using the enzymes present in the BioBrick prefix and suffix. This step added to the system’s standardization.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle3/figure9.webp)

**Figure 9.** Electrophoresis results showing the digestion of USV plasmid with the enzymes
marked
in
the image.

**Table 1.** Expected band size the digestion of the USV plasmid.

| Enzyme/s used | Expected Band Size (bp) |
| --- | --- |
| EcoRI & SpeI | 2047 & 1031 |

**Conclusion:** The correct bands were obtained, demonstrating that the **Cassette can now be
transferred** between other
systems compatible with the BioBrick assembly standard.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

In this iteration, we learned that:

* Incorporating the reporter **streamlined** the screening process and eliminated the need for
  sequencing our constructs.
* Changing the plasmid backbone **did not affect the system’s performance**, as shown in Figure
  7.
  It made our
  system **compatible with the BioBrick assembly standard**, enhancing its
  **usability**
  and **modularity**.
* Our system **worked effectively** and allowed us to synthesize crRNAs of the correct length.
* We learned that adopting a better and **more efficient purification method** for the crRNAs
  would
  be beneficial to
  eliminate the plasmid DNA, which served as the IVT template.

# Cycle 4

![](https://static.igem.wiki/teams/5087/pics/engineering/23.webp)

**Goals:** Determining optimal lighting conditions for algae cultures containing Prymnesium parvum.

## Iteration 4.1

##### Design & Build

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)
![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

After setting up the initial algal cultures, we stored them in a growth chamber under **constant light
conditions**,
following the suggestion from our advisor – Dr Paweł Jedynak.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

This environment proved **suboptimal** for *Prymnesium parvum*, as we observed a
**decline in
cell numbers and reduced
cell movement** after about a month.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The **lighting conditions** that we utilize for our algae cultures should be
**optimized**. It would be
beneficial to
contact a **specialist** who has expertise in *Prymnesium parvum* culture.

## Iteration 4.2

##### Design & Build

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)
![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

Seeking a solution to the problem of reduced algae growth, we consulted **Dr Ewa Górecka** from
the Institute of
Marine and Environmental Sciences, University of Szczecin, to learn about her algae culture conditions.

Dr. Górecka advised us to place some of our algal cultures in a growth chamber with a **14-hour
light/10-hour dark
cycle**. We followed her recommendation and changed the lighting conditions of our cultures.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

As a result, we observed **improved algae growth** after about a month, with an **increase
in cell
numbers and
enhanced movement**.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

A **14-hour light/10-hour dark cycle** is more beneficial for algae growth compared to
**constant light conditions**.

##### References

[1] Galluzzi L, Bertozzini E, Penna A, Perini F, Pigalarga A, Graneli E, Magnani M. Detection and
quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol. 2008 Feb;46(2):261-6.
doi: 10.1111/j.1472-765X.2007.02294.x. Epub 2007 Dec 13. PMID: 18086191.

[2] Raport Kończący Prace Zespołu ds. Sytuacji w Odrze. Instytut Ochrony Środowiska – Państwowy Instytut
Badawczy. Available at:
https://ios.edu.pl/wp-content/uploads/2022/12/raport-konczacy-prace-zespolu-ds-sytuacji-w-odrze-2.pdf

# Cycle 5

![](https://static.igem.wiki/teams/5087/pics/engineering/24.webp)

## Iteration 5.1

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

PCR was performed to **confirm the presence of the ITS2** sequence in **DNA isolated from
algal
cultures**. The ITS2
sequence is specific to *Prymnesium parvum*, allowing for its **identification among other algal
species**.
Primers
targeting the ITS2 sequence were designed by Galluzzi et al. [1]. The goal of this iteration was to
**optimize
conditions** for this PCR reaction.

To determine the initial PCR conditions, we referred to the Final Report of the Team on the Situation in the
Oder River [2], where Galluzzi primers were used in a standard PCR.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

We conducted a PCR reaction on the Prymnesium parvum genome using Galluzzi primers and the reaction conditions
sourced from the Final Report of the Team on the Situation in the Oder River [2]. Table 1 shows the implemented
reaction conditions.

**Table 1.** Reaction conditions sourced from the Final Report of the Team on the Situation in the
Oder River [2].

| **Reaction step** | **Temperature [°C]** | **Time [s]** |
| --- | --- | --- |
| Pre-denaturation | 98 | 30 |
| Denaturationn | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final elongation | 72 | 300 |

Number of cycles: 40

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

Figure 1 presents the results of DNA electrophoresis of the PCR product.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle5/figure1.jpg)

**Figure 1.** Gel electrophoresis of the PCR product – results.

Legend:

1. O'GeneRuler DNA Ladder Mix
2. High-volume culture
3. 2W1D
4. 1BW11
5. 2W11
6. colony PCR
7. O'GeneRuler 1 kB DNA Ladder Mix

“High-volume”, “2W1D”, “1BW11”, and “2W11” are names of DNA samples derived from **different
*Prymnesium
parvum*
cultures**. For a more detailed description, please refer to the AlgaeLab documentation included in the
“Notebooks.”

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

Not all PCR reactions yielded a specific product, even though identical DNA samples and preparation conditions
were used for each reaction. This inconsistency suggests that the experiment **cannot be reliably
reproduced** under
the current conditions, indicating a need for **optimization**.

Nevertheless, in the case of sample 3 the PCR reaction did produce a specific product of the correct length, so
we adopted these evaluated reaction conditions for subsequent PCR assays following each algal DNA isolation.

## Iteration 5.2

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The PCR conditions were
**optimized** for the Taq polymerase used, following the manufacturer's instructions (A&A
Biotechnology). This polymerase was used due to its availability in the lab.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

Table 2 presents the PCR conditions after optimization.

**Table 2.** PCR reaction conditions based on the manufacturer’s instruction (A&A Biotechnology).

| **Reaction step** | **Temperature [°C]** | **Time [s]** |
| --- | --- | --- |
| Pre-denaturation | 95 | 60 |
| Denaturationn | 95 | 15 |
| Annealing | 67 | 60 |
| Elongation | 72 | 15 |
| Final elongation | 72 | 300 |

Number of cycles: 30

We conducted a PCR with Galluzzi primers using the **optimized reaction conditions**.
Simultaneously, we performed
another PCR with the same reagents but under the **previous conditions to compare** the results.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

Figure 2. presents the results of a DNA electrophoresis of the PCR product.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle5/figure2.webp)

**Figure 2.** Gel electrophoresis of PCR product – results. Conditions A – PCR conditions before
optimization. Conditions B – optimized PCR conditions.

Legend:

1. O'GeneRuler DNA Ladder mix
2. negative control (water)
3. P
4. BOW 2
5. NOW 5
6. Gdańsk 2
7. negative control (water)
8. P
9. BOW 2
10. NOW 5
11. Gdańsk 2
12. GeneRuler 1kB DNA Ladder Mix

P, BOW 2, NOW 5, and Gdańsk 2 are names of DNA samples derived from different algal cultures. For a more
detailed description, please refer to the AlgaeLab documentation included in the “Notebooks.”

PCR performed under Conditions A produced one product of the correct length, but this result was confounded by a
product of the same size appearing in the negative control sample. In contrast, PCR under the optimized
conditions yielded two specific products and did not show any visible product in the negative control sample.

The PCR under the optimized conditions was performed repeatedly, consistently **yielding specific
products**.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The optimized PCR conditions are **suitable for the reaction**, as they consistently yielded a
**specific product of
the correct length** and the experiment demonstrated **reproducibility**.

##### References

[1] Galluzzi L, Bertozzini E, Penna A, Perini F, Pigalarga A, Graneli E, Magnani M. Detection and
quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol. 2008 Feb;46(2):261-6.
doi: 10.1111/j.1472-765X.2007.02294.x. Epub 2007 Dec 13. PMID: 18086191.

[2] Raport Kończący Prace Zespołu ds. Sytuacji w Odrze. Instytut Ochrony Środowiska – Państwowy Instytut
Badawczy. Available at:
https://ios.edu.pl/wp-content/uploads/2022/12/raport-konczacy-prace-zespolu-ds-sytuacji-w-odrze-2.pdf

# Cycle 6

![](https://static.igem.wiki/teams/5087/pics/engineering/25.webp)

## Iteration 6.1

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We evaluated a rapid **DNA isolation method** that could potentially be performed in **field
conditions**, simplifying
the workflow required for the SHERLOCK assay. This method was originally described by Zou et al. [1]. The goal
was to develop the most efficient DNA isolation procedure using an in-field method.

We implemented the in-field DNA isolation method for our algae cultures, following the original protocol
described by **Zou et al. [1]**. This protocol has also been included in the “Experiments”
page.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

We performed DNA isolation followed by PCR according to the following steps:

1. A 500 µl aliquot of liquid culture was added to a 2 ml Eppendorf tube containing 500 µl of lysis buffer.
2. Two ball bearings were added to the tube, and it was shaken for 8 seconds.
3. An isolation stick was dipped into the mixture three times and then dipped three times into another
   Eppendorf tube containing 1.75 ml of wash buffer.
4. The material collected on the isolation stick was transferred directly to the prepared PCR mix in a PCR tube
   for amplification.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle6/figure1.webp)

**Figure 1.** DNA electrophoresis after the PCR – results.
Electrophoresis performed after three
separate PCR reactions. Results for the in-field isolation are labeled 11-19.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The PCR **did not yield a specific product**. The bands visible on the gel were determined to be
primer dimers.

The evaluated in-field DNA isolation method proved ineffective, and improvements to the process are necessary.
We hypothesize that the unsuccessful amplification may be due to a portion of the reaction mixture being
absorbed by the dipstick and subsequently removed from the PCR tube.

## Iteration 6.2

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The originally described in-field DNA isolation protocol was **modified** as follows:

1. The tube was shaken for 30 seconds instead of 8 seconds.
2. The isolation stick was dipped into the mixtures five times instead of three.
3. After washing the DNA with the wash buffer, the stick was dipped into 15 µl of water for DNA elution. The
   eluted DNA was then transferred into the PCR reaction mix, eliminating the risk of the PCR mixture being
   absorbed into the dipstick.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

We made a second attempt at DNA isolation using the in-field method, applying the modified protocol to improve
the efficiency of DNA recovery for subsequent PCR amplification.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle6/figure2.webp)

**Figure 2.** DNA electrophoresis after the PCR – results.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The subsequent PCR reaction **still did not yield a specific product**. The visible bands on the
gel were identified
as primer dimers, indicating that the DNA amplification was unsuccessful despite the modifications to the
in-field isolation protocol.

Increasing the shaking time and number of dips with the isolation sticks **did not improve the
efficiency** of DNA
isolation. Thus, the method requires **further optimization**. Due to limited laboratory access, we
were unable to
continue working on the in-field DNA isolation process, and consequently, this goal was not achieved. However,
the insights gained during this process provide a **valuable foundation for future iGEM teams**
aiming to optimize
in-field DNA isolation from *Prymnesium parvum* or other harmful species. We believe that using **more than
two ball
bearings**, as well as **reducing the initial volume** of the
buffers used for the isolation are
approaches worth
testing in the further optimization process.

##### References

[1] Zou Y, Mason MG, Wang Y, Wee E, Turni C, Blackall PJ, et al. Nucleic acid purification from plants, animals,
and microbes in under 30 seconds. PLoS Biol. 2017 Nov;15(11). doi: 10.1371/journal.pbio.2003916.

# Cycle 7

![](https://static.igem.wiki/teams/5087/pics/engineering/26.webp)

**Goals:**
Design and 3D-print a functional **device** to excite fluorescence, then **quantify**
the detected signal by analyzing the
resulting image with a **Python script**.

## Iteration 7.1

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We began designing a **3D-printed device** aimed at detecting the green fluorescence signal
generated by our
SHERLOCK detection system. Our initial concept was inspired by the pe3Dish model (Figure 1A), which consists of
a 3D-printed chamber with a network of channels formed by flexible tubes, allowing continuous perfusion of 3D
cell cultures. These channels are designed to mimic the human vascular system. We **adapted** this
approach for our
project.

Figure 1B shows the **preliminary** model we developed, featuring a central chamber with several
channels. Two
channels function as **inlets**, separately delivering the SHERLOCK reagents and the DNA target,
while a third
channel acts as an **outlet** to remove waste liquid after the reaction.

Figure 1C illustrates the primary 3D-printed system, which was designed in **TinkerCAD**. Unlike
the five inlets and
outlets in the original pe3Dish model, our design incorporates **one outlet** for waste removal and
**three inlets** for
reagent delivery. This configuration allows us to separate the SHERLOCK reagents into two distinct channels. The
exact dimensions of the design are detailed in Figure 1C. The STL file was processed using **Prusa Slicer
2.7.1.**
This software was used in all following iterations.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure1.webp)

**Figure 1.** Initial designs of the 3D-printed system. (A) Photo of the pe3Dish. (B) Scheme of
the initial design of the 3D-printed device. (C ) Initial 3D-printed device designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The initial model of the device was successfully 3D-printed using a **Prusa i3 MK3** printer with
**Prusament PLA
Pristine White filament**.
The same printer was used in all following iterations. The general print
settings were
based on the '0.10mm DETAIL' preset from the Prusa Slicer system. The specific modifications made to the default
system presets are as follows: Infill: 20%; Brim: enabled; Supports: none; Fill pattern: Rectilinear; Top fill
pattern: Rectilinear; Bottom fill pattern: Rectilinear; Perimeters: 20 mm/s; Small perimeters: 15 mm/s; External
perimeters: 15 mm/s; Top solid infill: 20 mm/s; Nozzle (first and other layers): 215 ⁰C; Bed (first and other
layers): 60 ⁰C; Filament type: PLA; Retraction length: 2 mm; Retraction speed: 40 mm/s.
The printing process was completed in 2 hours and 6 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The chamber was printed successfully, maintaining **good structural integrity**. However, the tube
holders proved to
be too thin, making them **fragile** and prone to breaking or detaching from the model. They were
easily **damaged** by
light pressure from a finger or by being dropped. The damaged tube holders are highlighted in Figure 2.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure2.webp)

**Figure 2.** Photo of the 3D-printed initial 3D-printed device with damaged tube holders.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The most critical improvement needed for the model was to thicken the walls of the tube holders to
**enhance their
durability**. Additionally, the chamber required an external **protective enclosure** to
secure and protect
its
components.

## Iteration 7.2

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

A new model with thicker walls for the tube holders was designed to **improve durability**.
Additionally, drawing
inspiration from the pe3Dish system, the model was adapted to fit into the bottom of a Petri dish, allowing the
dish to function as an external **protective enclosure**. Figure 3 shows the updated model with,
designed in **TinkerCAD**.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure3.webp)

**Figure 3.** Improved 3D-printed device designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The new model was successfully printed using the **Prusament PLA Pristine White filament**. The
general print
settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. The specific modifications made
to the default system presets were the same as for Iteration 1.

The printing process was completed in 2 hours and 26 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The chamber was **printed successfully** and fit well within the 90 mm Petri dish, allowing for
secure **closure and
protection** of the entire chamber. The tubes fit properly into the inlets and outlets of both the
chamber and the
tube holders, as shown in Figure 4.
The thicker tube holders proved to be **sturdy** and did
not
break. The
integrity of the channel system was tested using water and a 5 ml syringe, confirming that the design was
**functional and robust**.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure4.webp)

**Figure 4.** Photos of the improved 3D-printed device. (A) Model in Petri dish without the
tubes.
(B) Model in Petri dish with the tubes

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

Although the design improvements were successful, we concluded that in future use, **transferring
reagents** into
the chamber with a syringe and fitted tubes would be **challenging** due to the small microliter
volumes required
for the SHERLOCK assay. The 3 mm diameter tubes necessitated a **larger volume** to fill the
chamber, indicating the
need for a different approach to the chamber design.

Additionally, it was noted that the PLA filament used to print the chamber is **biodegradable**,
which could
potentially affect biochemical reactions from environmental samples. This suggests that a **different
material** or
coating may be necessary to prevent interference with the assay.

## Iteration 7.3

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We decided to **abandon the use of tubes** and redesign the chip. Our focus shifted to developing a
**smaller reaction
chamber without tubes**, drawing inspiration from the device described in the paper "Machine
Learning-Driven and
Smartphone-Based Fluorescence Detection for CRISPR Diagnostic of SARS-CoV-2" [1]. The new design features
**three
separate compartments** that can be easily attached and detached for convenient transportation.
Additionally, we
replaced the initial PLA filaments with **PETG filaments**, which are non-biodegradable but more
suitable for
preventing interference with biochemical reactions.

Each box was designated a specific function and in different colors of PETG filament, chosen to align with the
colors in our team logo. The **reaction chamber** was placed in the black **detection
box**, as this
was where
fluorescence was expected to be induced. A special compartment for the chamber was designed within this box
(Figure 5 A).

The box containing the electronics necessary for generating blue light to induce green fluorescence was
labelled the yellow **electronics box**. This box
also included a separate
compartment for the **battery** to power
the LEDs and a hole for the **LED switch** (Figure 5B). Both the black and yellow boxes featured
three holes for
LEDs, designed to allow the LEDs to be inserted between the boxes within LED holders (Figures 5A and 5B).

The final box, intended for storing **tools and reagents** required for the experiment, was named
the purple **tool
box**. The initial box included designated spaces for a reaction chamber, glass cover slips
and
a 500 µL syringe
(Figure 5 C).

The overall dimensions of the boxes were determined by the size of the phone intended to be placed on the
device. The height of the boxes was minimized to accommodate the battery. Dedicated compartments within the
boxes were designed to fit the dimensions of components prepared for the chip-based assay, including
**LED
holders, battery, light switch, 500 µL syringe, and cover slips**. Initially, the boxes were designed
without lids
to test the dimensions for the listed elements and to assess the printing quality of the filaments.

Figure 5 presents all the boxes, with exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure5.webp)

**Figure 5.** Models of the boxes designed in TinkerCAD

**(A)** The black detection box with a compartment for the chamber and holes for the LED holders.

**(B)** The yellow
electronics box with a separate space for a battery and holes for the light switch and LED holders.

**(C)** The
purple tool box with designated spaces for a reaction chamber, glass cover slips and a 500 µL syringe.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The models were printed with the following filaments:

* Jet Black Prusa PETG – detection box
* Mango Yellow Prusa PETG – electronics box
* Purple Polymaker PolyLite PETG – tool box

The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications
to the default system were the same as for Iteration 1, except for: Supports: everywhere (grid); Top fill
pattern: Archimedean Chords; Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s;
Infill: 40 mm/s; Gap fill: 20 mm/s; Nozzle (first and other layers): 250 ⁰C (black), 250 ⁰C (yellow), 240 ⁰C
(purple); Bed (first and other layers): 70 ⁰C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed:
50 mm/s.

The printing processes were completed in 4 hours and 35 minutes (black box), 7 hours and 6 minutes (yellow box)
and in 8 hours and 8 minutes (purple box).

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The boxes were printed, but the **results were not optimal**. The walls were **too
thin**, resulting in
areas that
exhibited elasticity and transparency rather than stability and sturdiness (Figure 6A). Additionally, the
supports inside the holes for the LED holders in the black and yellow boxes left **filament
residue**, distorting
the circular shapes of the holes (Figure 6B). The bottoms of each box did not print well; the layer height was
**uneven**, leading to filament blobs, and stringing was particularly noticeable in the elements
within the purple
box. In some areas of the purple box, there were even **holes** in the bottom. Additionally, in the
yellow box, the
wall designed to create a separate space for the battery was too **fragile** due to its thickness
and came off
during the removal of the supports (Figure 6C).

The sizes of the boxes were adequate and did not require adjustments; however, increasing the height of the
boxes would likely be beneficial.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure6.webp)

**Figure 6.** Photos of the 3D-printed initial boxes.

**(A)** Yellow box displaying elastic and transparent walls.
**(B)** Holes in the
black and yellow
boxes showing residue
left after support removal.
**(C)** Poorly printed bottoms of the boxes.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The **thickness** of the walls should be **increased** to enhance the durability of
the boxes.
Additionally, after
investigating the issues, we found that the problems with the printed boxes may have resulted from an
**incorrectly chosen nozzle temperature**. To determine the optimal printing temperature for these
filaments, it is
essential to print **calibration models**, such as **temperature towers**. Increasing
the wall thickness will further
contribute to the overall durability of the boxes.

## Iteration 7.4

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

Since it was unclear which **glass cover slip** among the three ordered options (circular 9 mm,
circular 15 mm,
square 16 mm) would be the best fit for the lid of the reaction chamber, a **box with dedicated holders
for each
sli**p was designed. This box was constructed to match the height of the boxes designed in Iteration 5,
allowing
us to **evaluate which distance between the phone and the detection chamber** would be optimal for
capturing images.
Figure 7 presents the model of the box, with exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure7.webp)

**Figure 7.** Model of the box with holders for different glass
coverslips designed in
TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The new model was **successfully printed with Prusament PLA Galaxy Silver filament**. The general
print settings
were based on the '0.20mm QUALITY' preset from the Prusa Slicer system. Modifications to the default system
presets were the same as for Iteration 1, except: Supports: on build plate only (grid); Top fill pattern:
Octagram Spiral; Bottom fill pattern: Octagram Spiral; Nozzle (first and other layers): 220°C.

The printing process was completed in 3 hours and 13 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The box was **printed successfully**. Upon testing, the square coverslip appeared too large for the
final chamber,
while the 9 mm circular coverslip was **too small**. The medium-sized **15 mm circular
coverslip**
emerged as the **best
option for the detection chamber** (Figure 8). However, the box's height was insufficient, making it
difficult to
achieve optimal camera focus.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure8.webp)

**Figure 8.** The gray box with holders for the coverslips in various sizes and shapes.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The circular **15 mm glass coverslip was selected for further improvements** to the detection
chamber.

It was decided to **remodel** the black box and **increase its height** from 29 mm to
41 mm to
facilitate better camera
focus and overall functionality.

## Iteration 7.5

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

Since the size of the glass coverslip was selected, the **reaction chamber was designed** in
TinkerCAD specifically
to accommodate it. The exact dimensions of the design are presented in the scheme shown in Figure 9.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure9.webp)

**Figure 9.** Model of the reaction chamber designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed using the **Prusa i3 MK3 printer with Transparent Devil Design PETG
filament**. The general
print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the
default system presets were the same as for Iteration 1, except: Supports: on build plate only (organic);
Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s; Infill: 40 mm/s; Gap fill: 20
mm/s; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG;
Retraction length: 5.5 mm; Retraction speed: 50 mm/s.

The printing process was completed in 32 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The chamber was printed, but the **results were unsuccessful**. Significant
**stringing** occurred, and
the overall
structure of the model was **uneven**. Additionally, the **supports** did not print
correctly and
remained inside the
chamber, as shown in Figure 10.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure10.webp)

**Figure 10.** The 3D-printed design of the initial reaction
chamber.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

Since the printing of the chamber was unsuccessful, it was decided that the filament needed to be printed at a
**more optimal temperature**. Therefore, there was a need to print a **temperature
tower** for this
filament as well.

## Iteration 7.6

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

**Temperature towers** for optimizing the printing of PETG filaments were found online. The design
selected from the
website Printables.com
(https://www.printables.com/model/20652-temp-tower-pla-petg-absasa-for-prusa-mini-mk3s-and/files) was chosen for
optimizing the printing of all four previously mentioned PETG filaments. The file named ‘Temp\_Tower\_PETG.stl’
was downloaded and sliced using Prusa Slicer (Figure 11).

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure11.webp)

**Figure 11.** The temperature tower opened in Prusa slicer.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The models were printed with all four **PETG filaments**. The general print settings were based on
the '0.10mm
DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets were the same as for
Iteration 1, except: Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s; Infill: 40
mm/s; Gap fill: 20 mm/s; Nozzle: 220°C – 260°C; Bed (first and other layers): 70°C; Filament type: PETG;
Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of each temperature tower was completed in 9 hours and 35 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

Each of the printed temperature towers was carefully examined for **stringing, even edges, detail
quality**, and the
**clarity** of the circular holes and numbers to determine the **optimal printing
temperatures** for
each filament. The
whole towers and their close-ups are shown in Figure 12. Generally, **higher temperatures resulted in
increased
stringing**, while lower temperatures produced clearer numbers and smoother circular holes, though the
edges
appeared more bent.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure12.webp)

**Figure 12.** The 3D-printed temperature towers of the PETG filaments.

(A) Black temperature tower. (B) Close-up of the black temperature tower. (C) Yellow temperature tower. (D)
Close-up of the yellow temperature tower. (E) Purple temperature tower. (F) Close-up of the purple temperature
tower. (G) Transparent temperature tower. (H) Close-up of the transparent temperature tower.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

Based on a careful evaluation, the following temperatures were chosen for further printing with each filament:

* Jet Black PETG: 240 °C
* Mango Yellow PETG: 225 °C
* Purple PETG: 230 °C
* Transparent PETG: 220 °C

These temperatures were selected based on achieving minimal stringing, good circularity of the holes, and
minimal bending of the edges while maintaining overall print quality.

## Iteration 7.7

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The **black box was redesigned** with **higher walls** and a dedicated **space
for a cover lid**, which
will close the box
using a **sliding mechanism**. The walls surrounding the chamber were also **raised**.
Additionally,
the holes for the
LED holders were designed with **organic supports** this time. Figure 13 presents the model of the
box, with exact
dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure13.webp)

**Figure 13.** Model of the detection box designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **black PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except:

Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern:
Monotonic Lines; Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s; Infill: 40 mm/s;
Gap fill: 20 mm/s; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type:
PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.

The printing process of the model was completed in 11 hours and 48 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The box was **printed successfully**. The change in nozzle temperature significantly improved the
quality of the
design, and the results are satisfactory, as shown in Figure 14. Switching from grid supports to organic
supports helped maintain the **smooth**, circular shape of the holes for the LED holders. The
thickness of the walls
was ideal, making the box **sturdy**, and the walls were no longer elastic. The spot for the lid
also appeared
well-constructed. However, one issue remained—the **height of the box was still too low** for
taking clear photos.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure14.webp)

**Figure 14.** Photo of the improved 3D-printed detection box.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The **improvements made to the box were successful** and will be incorporated into future
iterations of the black
box model. However, the **height of the box needs to be increased** by approximately 10 mm to
address the issue with
photo clarity.

## Iteration 7.8

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The **yellow box was redesigned** with higher walls and a **dedicated space** for a
cover lid, which
will close using a
**sliding mechanism**. The wall adjacent to the battery was also **raised and
thickened**.
Additionally, the hole for
the light switch was redesigned to be circular, offering better protection, and the holes for the LED holders
were designed with organic supports this time. Figure 15 presents the model of the box with exact dimensions,
designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure15.webp)

**Figure 15.** Model of the electronics box designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **yellow PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill
pattern: Monotonic Lines; Nozzle (first and other layers): 225°C; Bed (first and other layers): 70°C; Filament
type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 16 hours and 11 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The box was **printed successfully**. The change in **nozzle temperature**
significantly improved the
design quality,
yielding satisfactory results, as shown in Figure 16. Switching from grid supports to organic supports helped
maintain the smooth, circular shape of the holes for the LED holders. The **wall thickness was
ideal**, making the
box sturdy without elasticity, and the wall adjacent to the battery was well-constructed. Additionally, the
circular spot for the light switch **fits perfectly**. However, one issue persisted—the height of
the box was still
**too low**, as the simultaneously printed black box also proved to be too low.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure16.webp)

**Figure 16.** Photo of the improved 3D-printed electronics box.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The improvements made to the box were **successful** and will be incorporated into future
iterations of the yellow
box model. However, the **height of the box needs to be increased** by approximately 10 mm to match
the height of
the improved version of the black box.

## Iteration 7.9

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The **height of the black box was increased** by 10 mm. Figure 17 presents the updated model of the
box with exact
dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure17.webp)

**Figure 17.** Model of the improved detection box designed in
TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed using black PETG filament. The general print settings were based on the '0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill
pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament
type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 14 hours and 49 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The box was **printed successfully**, as shown in Figure 18. The increase in height yielded
satisfactory results,
providing the **proper height** for the detection box, making it suitable for further testing.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure18.webp)

**Figure 18.** Photo of the improved 3D-printed detection box.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The improvement was **successful**, providing a properly designed detection box for further tests.
The new height
of the box can now be applied to the yellow and purple boxes, which will be printed next.

## Iteration 7.10

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The **height of the yellow box was increased** by 10 mm. Figure 19 presents the updated model of
the box with exact
dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure19.webp)

**Figure 19.** Model of the improved electronics box designed in
TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **yellow PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets were the same as for Iteration
1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill
pattern: Monotonic Lines; Nozzle (first and other layers): 225°C; Bed (first and other layers): 70°C; Filament
type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 19 hours and 18 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The electronics box was **printed successfully**, as shown in Figure 20. The increased height
yielded **satisfactory
results**, aligning perfectly with the height of the black box and providing the **proper
dimensions** for
further
testing.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure20.webp)

**Figure 20.** Photo of the improved 3D-printed electronics box.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The **improvement was successful**, providing a properly designed electronics box for further
tests.

## Iteration 7.11

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The properly printed boxes required **well-designed lids** that would function as a
**sliding
mechanism**. Figure 21
presents the yellow cover lid with exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure21.webp)

**Figure 21.** Model of the yellow cover lid designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **yellow PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: none; Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines;
Nozzle (first and other layers): 225°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction
length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 3 hours and 36 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The lid was **printed successfully** as shown in Figure 22. It fit the yellow box well, and the
sliding mechanism
operated perfectly.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure22.webp)

**Figure 22.** Photo of the yellow cover lid in the yellow box.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The lid was **well designed** and could be implemented in both the black and purple boxes.

## Iteration 7.12

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

Based on the design of the yellow lid, the **black lid** was created for the black detection box.
It required an
additional hole for the green filter and a magnifying lens. Figure 23 presents the lid with exact dimensions,
designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure23.webp)

**Figure 23.** Model of the black cover lid designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **black PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill
pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament
type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 3 hours and 55 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The lid was **printed successfully**. However, some of its dimensions were **not precise
enough**,
resulting in the
filter **protruding** from the lid. This issue prevented the lid from being properly inserted into
the designated
space in the black box, as shown in Figure 24.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure24.webp)

**Figure 24.** The photo of the black lid and the black box.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The lid had an **appropriate size** and did not require major improvements. However, the hole in
the center needed
to be **widened slightly**, and the protruding element in the center of the lid needed to be
**removed**.

## Iteration 7.13

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The black lid was **redesigned** to smoothly fit the green filter. Figure 25 presents the lid with
exact dimensions,
designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure25.webp)

**Figure 25.** Model of the improved black lid designed in
TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **black PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill
pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament
type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 3 hours and 55 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The lid was **printed successfully**. This time, the **dimensions were perfect**,
allowing it to fit
the filter well.
The lid was smoothly inserted into the black box using the sliding mechanism, as shown in Figure 26.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure26.webp)

**Figure 26.** The improved black lid and the black box.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The black lid was printed successfully and did not require any further improvements.

## Iteration 7.14

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The reaction chamber from Iteration 5 was **printed again** without any changes in the design.
Figure 27 presents
the model of the chamber, with exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure9.webp)

**Figure 27.** Model of the reaction chamber designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **transparent PETG filament**. The general print settings were based on
the '0.10mm
DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for
Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines;
Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 220°C; Bed (first and other layers):
70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 34 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The chamber was **printed successfully**, as presented in Figure 28A. It was tested in the black
detection box to
verify its **visibility** when the LEDs were turned on. The chamber was well illuminated, but a
**green signal was
also observed within it**, as shown in Figure 28B. To determine whether the filament emitted a green
fluorescence
signal, it was examined under a fluorescent microscope and excited with blue light. A fragment of the photo
taken by the microscope is shown in Figure 28C, confirming that **the filament did emit some green
fluorescence**.
It also became apparent that the chamber **did not hold water effectively** and was leaking due to
its thin bottom.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure28.webp)

**Figure 28.** Reaction chamber printed with transparent PETG filament.

(A) Well-printed chamber. (B) Green signal produced by the chamber in the detection box. (C) Green fluorescence
produced by the chamber, recorded by the fluorescence microscope.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

Since the **filament produced green fluorescence**, it could not be used for creating the reaction
chamber in the
PrymChip. Therefore, the **approach had to be revised**.

## Iteration 7.15

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

It was decided to **discontinue printing the reaction chamber**. After exploring different options,
it was
determined to use a **cap from a hair spray bottle as the reaction chamber**, as the material did
not produce a
green fluorescence signal. The cap needed to be cut to a proper size, similar to that of the printed chamber as
Figure 29 indicates.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure29.webp)

**Figure 29.** The chosen hairspray bottle cap and the printed
reaction chamber.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The cap was carefully cut to the proper height using scissors.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The cap had the proper height, and after placing it in the detection box, it was easy to **capture a clear
photo**.
It also **did not leak and did not produce green fluorescence**. The cut cap is shown in Figure 30
A. The reaction
chamber made from the hair spray bottle was tested with fluorescein in the detection box. However, it was found
that the green fluorescence of the fluorescein solution was uneven at the edges of the chamber, creating an
**intense green ring** (Figure 30 B). Additionally, since the cap was inserted into the black box
made in iteration
7, it **did not fit well** in the designated space for the reaction chamber.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure30.webp)

**Figure 30.** The reaction chamber made out of the cut cap. (A) The cut cap and 3D-prited
reaction chamber. (B) Green fluorescence of the fluorescein in the chamber with a visible intense green ring.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The cut cap was a **good choice** for the reaction chamber. However, a **cover for the
edges** needed
to be designed to
even out the registered fluorescence signal. Additionally, the black box required adjustments to fit the chamber
perfectly. In iteration 9, this issue was addressed, resulting in a larger space for the chamber in the box.

## Iteration 7.16

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The **cover for the reaction chamber** was designed. Figure 31 presents the model of the cover,
with exact
dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure31.webp)

**Figure 31.** Model of the chamber cover designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **black PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: none; Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines;
Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction
length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process was completed in 10 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The cover was **printed successfully**. It fit the chamber well and the glass cover slip that was
chosen to fit the
reaction chamber fit well as Figure 32 A indicates. It was tested with the fluorescein solution and as Figure 32
B indicates it removed the need for further improvements of the chamber.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure32.webp)

**Figure 32.** The 3D-printed cover for the chamber.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The printed lid cover was **satisfactory** and did not require further improvements. The issue with
the intense
green fluorescent ring was **resolved**.

## Iteration 7.17

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The purple box was redesigned to feature an **increased height** that aligns with the improved
black and yellow
boxes. Additionally, a space was designated for the **cover lid**, incorporating a sliding
mechanism. Figure 33
presents the model of the box with exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure33.webp)

**Figure 33.** Model of the tool box designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed using **purple PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill
pattern: Monotonic Lines; Nozzle (first and other layers): 230°C; Bed (first and other layers): 70°C; Filament
type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 24 hours and 12 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The purple box was **printed successfully**, matching the height and width of the black and yellow
boxes, as
indicated in Figure 34.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure34.jpg)

**Figure 34.** The three printed boxes aligned with each other.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The box was **well designed and did not require any further improvements**.

## Iteration 7.18

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

We concluded that the boxes may require **additional symbols for easier recognition**. Symbols
**indicating the use of
each box** were designed with varying heights to determine which would look best on the boxes. Figure
35 presents
the plates with the chosen symbols, including their exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure35.webp)

**Figure 35.** Models of the plates with symbols designed in
TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The models were printed with **black and yellow PETG filaments**. The general print settings were
based on the
'0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as
for Iteration 1, except: Infill: 30%; Supports: none; Top fill pattern: Monotonic Lines; Bottom fill pattern:
Monotonic Lines; Nozzle (first and other layers): 240°C (black), 225°C (yellow); Bed (first and other layers):
70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the concave model was completed in 1 hour and 32 minutes and the convex model in 49
minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The printing process was **successful**, as shown in Figure 36. The symbols were evaluated to
determine which
version looked better: **concave or convex**. Subsequently, the **convex** symbols
were assessed to
identify which
height appeared most suitable.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure36.webp)

**Figure 36.** Convex and concave 3D-printed symbols.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The convex versions of the symbols were chosen for their **better appearance**, and a height of 1.5
mm was selected
as the most suitable. These symbols can be incorporated into the designs of the cover lids in future steps.

## Iteration 7.19

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The lids were **redesigned** to implement the convex 1.5 mm height symbols. The size of the lids
was not changed.
Figure 37 presents the models of the improved cover lids, with exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure37.webp)

**Figure 37.** Models of the cover lids with incorporated symbols
designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The models were printed with **black, yellow and purple PETG filaments**. The general print
settings were based on
the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the
same as for Iteration 1, except: Infill: 30%; Supports: none (purple, yellow), on build plate only (black); Top
fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C
(black), 225°C (yellow), 230°C (purple); Bed (first and other layers): 70°C; Filament type: PETG; Retraction
length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the black model was completed in 3 hours and 58 minutes, yellow in 3 hours and 38
minutes, purple in 6 hours and 9 minutes.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The cover lids were **printed successfully and fit the designated boxes**, as shown in Figure 38.
However, the
smartphone placed on the connected boxes experienced **stability issues** due to the **uneven
height**
created by the
protruding symbols.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure38.jpg)

**Figure 38.** The three boxes are connected to each other with
cover lids that feature symbols.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

**Additional support** was required on the opposite side of the boxes to stabilize the phone
without removing the
symbols, which are beneficial for **colorblind individuals**.

## Iteration 7.20

##### Design

![](https://static.igem.wiki/teams/5087/pics/engineering/design.webp)

The **supports for the smartphone** were designed to stabilize the device when placed on the
PrymChip. They are
**easily detachable** and fit conveniently in the purple toolbox. Figure 39 presents the model of
the support, with
exact dimensions, designed in TinkerCAD.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure39.webp)

**Figure 39.** Model of the support designed in TinkerCAD.

##### Build

![](https://static.igem.wiki/teams/5087/pics/engineering/build.webp)

The model was printed with **black PETG filament**. The general print settings were based on the
'0.10mm DETAIL'
preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration
1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill
pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament
type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process was completed in 1 hour.

##### Test

![](https://static.igem.wiki/teams/5087/pics/engineering/test.webp)

The supports were **printed successfully** and fit the boxes, as shown in Figure 40. After placing
the phone on the
PrymChip with the supports, the **level was even**.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle7/figure40.webp)

**Figure 40.** The supports attached to the PrymChip.

##### Learn

![](https://static.igem.wiki/teams/5087/pics/engineering/learn.webp)

The design of the supports was **effective and did not require further improvements**.

##### References

[1] Samacoits A, Nimsamer P, Mayuramart O, Chantaravisoot N, Sitthi-amorn P, Nakhakes C, Luangkamchorn L,
Tongcham P, Zahm U, Suphanpayak S, Padungwattanachoke N, Leelarthaphin N, Huayhongthong H, Pisitkun T,
Payungporn S, Hannanta-anan P. Machine learning-driven and smartphone-based fluorescence detection for CRISPR
diagnostic of SARS-CoV-2. ACS Omega. 2021;6(4):2727-2733. doi: 10.1021/acsomega.0c04929.

[2] Temp tower: PLA/PETG/ABS/ASA for Prusa Mini/MK3s and more. Available at:
https://www.printables.com/model/20652-temp-tower-pla-petg-absasa-for-prusa-mini-mk3s-and/files.

{% endblock %}

{% extends "layout\_page.html" %}
{% block title %}Experiments{% endblock %}
{% block banner\_image %}https://static.igem.wiki/teams/5087/pics/banners/experiments.webp{% endblock %}
{% block page\_content %}

## Algae Lab

---

[DNA isolation in-field method](https://static.igem.wiki/teams/5087/pics/protocols/isolation-field.pdf)

[Algal DNA isolation with QIAwave Kit](https://static.igem.wiki/teams/5087/pics/protocols/isolation2.pdf)

[Increasing the DNA concentration](https://static.igem.wiki/teams/5087/pics/protocols/increase.pdf)

[Algal DNA isolation with the NEB kit](https://static.igem.wiki/teams/5087/pics/protocols/neb-isolation.pdf)

[F/2 medium preparation](https://static.igem.wiki/teams/5087/pics/protocols/f-2-medium.pdf)

[Setting up liquid algal cultures](https://static.igem.wiki/teams/5087/pics/protocols/setting-up.pdf)

[Passaging liquid algal cultures](https://static.igem.wiki/teams/5087/pics/protocols/passage.pdf)

[Establishing algae cultures on solid medium](https://static.igem.wiki/teams/5087/pics/protocols/solid.pdf)

[Preparing culture samples for imaging](https://static.igem.wiki/teams/5087/pics/protocols/imaging.pdf)

[PCR on DNA from algae cultures](https://static.igem.wiki/teams/5087/pics/protocols/pcr-alga.pdf)

[Algae culture disposal](https://static.igem.wiki/teams/5087/pics/protocols/disposal.pdf)

## Protein Lab

---

[Cas13 purification](https://static.igem.wiki/teams/5087/pics/protocols/cas13-puri-prot.pdf)

[Plasmid isolation](https://static.igem.wiki/teams/5087/pics/protocols/plasmid.pdf)

## SHERLOCK Lab

---

[RNA purification](https://static.igem.wiki/teams/5087/pics/protocols/rna-purification.pdf)

[RPA (Recombinase Polymerase Amplification)](https://static.igem.wiki/teams/5087/pics/protocols/rpav2.pdf)

[SHERLOCK assay [1]](https://static.igem.wiki/teams/5087/pics/protocols/sherlock01.pdf)

[SHERLOCK assay [2]](https://static.igem.wiki/teams/5087/pics/protocols/sherlock02.pdf)

[IVT (In Vitro Transcription)](https://static.igem.wiki/teams/5087/pics/protocols/ivt-prot.pdf)

## LFA Lab

---

[PCR on Prymnesium parvum genome](https://static.igem.wiki/teams/5087/pics/protocols/pcr.pdf)

[SHERLOCK LFA](https://static.igem.wiki/teams/5087/pics/protocols/lfa.pdf)

## PrymChip Lab

---

[PrymChip Protocol](https://static.igem.wiki/teams/5087/pics/protocols/prymchip.pdf)

## SynLOCK Lab

---

[Oligo Annealing and Ligation](https://static.igem.wiki/teams/5087/pics/protocols/oligo-anneal.pdf)

[Golden Gate Assembly](https://static.igem.wiki/teams/5087/pics/protocols/golden-gate.pdf)

[Transformation [1]](https://static.igem.wiki/teams/5087/pics/protocols/trans-1.pdf)

[Transformation [2]](https://static.igem.wiki/teams/5087/pics/protocols/trans-2.pdf)

[Plasmid Isolation [1]](https://static.igem.wiki/teams/5087/pics/protocols/plasmidiso1.pdf)

[Plasmid Isolation [2]](https://static.igem.wiki/teams/5087/pics/protocols/plasmidiso2.pdf)

[DNA gel Extraction](https://static.igem.wiki/teams/5087/pics/protocols/dna-extract.pdf)

[DNA Ligation For Cohesive Ends](https://static.igem.wiki/teams/5087/pics/protocols/ligation.pdf)

[Post Enzymatic Reaction Clean-Up](https://static.igem.wiki/teams/5087/pics/protocols/clean-up.pdf)

{% endblock %}

{% extends "layout\_page.html" %}
{% block title %}Results{% endblock %}
{% block banner\_image %}https://static.igem.wiki/teams/5087/pics/banners/results.webp{% endblock %}
{% block page\_content %}

Results

## Unveiling the Findings: Successes and Lessons Learned

---

Our project set out to develop a **sensitive and reliable tool for detecting** ***Prymnesium
parvum*** **blooms**. We can confidently say that we have achieved many successes in
this endeavor. However, like any scientific journey, we have also encountered challenges and areas that require
further refinement.

On this page, we share the results we have achieved during our iGEM year, as interpreted and understood by our
team. We have included findings from **each phase of the project**, along with a brief **summary
of successes and areas needing improvement**, followed by our plans for **future work** to
build on these outcomes.

## SHERLOCK detection of Prymnesium parvum

---

We can confidently say that we have **succeeded** in **engineering functional SHERLOCK reaction
components** that allow for the **detection of** ***Prymnesium parvum***
DNA.

To program the system to target a specific region of the *Prymnesium parvum* genome, we needed to design an
effective **combination of RPA primers** and a **crRNA molecule**. We carefully considered
which region of the genome to focus on, ultimately selecting the **ITS2 (Internal Transcribed Spacer
2)** sequence for specific detection. The ITS2 region, located between the 5.8S and nuclear large subunit
rRNA genes [1], is commonly used for **species identification** due to its **variability between
species** and **conservation within a species**. This makes it an ideal target for
species-specific primers, including those used to detect *Prymnesium parvum* [2].

### Design and validation of SHERLOCK components

#### Design of crRNAs

We designed crRNA molecules to target ITS2, which we named **PrymCrRNA1** (BBa\_K5087022) and
**PrymCrRNA2** (BBa\_K5087023). Anticipating that some crRNAs might not be functional, we designed
**two** initial crRNA sequences to start our experiments.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle2/figure2.webp)

**Figure 1**. Binding sites of PrymCrRNA1 and PrymCrRNA2 to the
*Prymnesium parvum* genome.

#### Design of RPA primers

We finalized **three** primer pairs, some of which were adapted from literature, while others were
designed from scratch:

1. **Gal** primers: **GalF** (BBa\_K5087000), **GalR** (BBa\_K5087001) –
   the first RPA primer pair we designed was based on the primers created by Galluzzi et al. [1], with a T7 promoter
   appended to the 5' end of the forward primer. Since the GalF and GalR primers were proven effective in PCR in
   previous experiments, we chose them as a reliable starting point for designing our RPA primers.
2. **Mod** primers: **ModF** (BBa\_K5087002), **ModR** (BBa\_K5087003) – we based their sequences on the
   Luo et al. publication [2]. For ModF, we used the PR-RPA-4-F primer from the publication, and we appended the T7
   promoter to its sequence. For ModR, we used a modified version of the PR-RPA-4-R primer, changing an A to a G to
   better align with the *Prymnesium parvum* DNA fragment sequenced by us.
3. **Alt** primers: **AltF** (BBa\_K5087004), **AltR** (BBa\_K5087005) – their sequences were designed
   using Primer-BLAST, following the guidelines provided in the TwistAmp® DNA Amplification Kits Assay Design
   Manual.

With our three primer pairs and two crRNA designs, we were left with **18 possible combinations** of
SHERLOCK components.

![](https://static.igem.wiki/teams/5087/pics/results/startery-fig-3-results.png)

**Figure 2**. Positioning of all designed primers and crRNAs on the
ribosomal cistron of *Prymnesium parvum* genomic DNA.

We validated the functionality of **all but one** of our primer-crRNA combinations using both
fluorescence and Lateral Flow Assay (LFA) readouts.

The one combination we found to be **non-functional** was **GalF-GalR + PrymCrRNA1**, as
it consistently produced **false positives** (positive results in negative controls), such as what can
be observed in Figure 3. In this test, the “negative control + prymcrRNA1” sample produced a
fluorescence signal. This sample included water added to an RPA mix containing Gal primers. This outcome underscored
the critical importance of **including negative controls** in our experiments, which we had been
diligently maintaining.

We hypothesize that **both Gal** primers **inadvertently serve as a target for
PrymCrRNA1**, likely due to the forward primer’s proximity to the PrymCrRNA1 binding site (Figure 2).
However, we did not consistently observe the same effect when the GalF primer was paired with a different reverse
primer, leading us to conclude that GalF can be used in combination with other reverse primers.

![](https://static.igem.wiki/teams/5087/pics/engineering/sherlock-5-crrna1.webp)

**Figure 3**. SHERLOCK fluorescence readout result – false positives given
by the GalF-GalR + PrymCrRNA1 combination (red line).

The rest of primer-crRNA combinations were validated through a mixture of preliminary testing using both LFA and
fluorescence readouts. We selected only representative results from the multiple tests conducted (Figures 4-6).

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-11.webp)

**Figure 4**. SHERLOCK fluorescence readout result – GalF-GalR +
PrymCrRNA2, performed on decreasing concentrations of *Prymnesium parvum* DNA.

The chart in Figure 4 shows that **PrymCrRNA2** in combination with the **Gal primers**
was effective in detecting *Prymnesium parvum* DNA, even at a **200 fM** concentration. The
detection of 200 fM by the assay was a promising sign for the LOD value.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-12-530nm-fix.webp)

**Figure 5**. SHERLOCK fluorescence readout result – Mod, Alt and
Gal primers, performed on a 530 nM sample of *Prymnesium parvum* DNA.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-12-200fm-fix.webp)

**Figure 6**. SHERLOCK fluorescence readout result – Mod, Alt and
Gal primers, performed on a 200 fM sample of *Prymnesium parvum* DNA.

All of the combinations shown in Figure 5 were **effective** in detecting *Prymnesium parvum*
DNA. The best primer-PrymcrRNA pair appeared to be **ModF-ModR + PrymCrRNA1**, as it achieved high
fluorescence intensity for **both 530 nM and 200 fM samples**. No other combination was effective in
detecting the 200 fM target concentration.

### Optimization of SHERLOCK components

After **validating** our designs, we could have concluded the testing process. However, we aimed to
**optimize** the system further by identifying the **most efficient primer-crRNA combination
among all the possible configurations**. We performed fluorescence-based assays where we assessed all of
our candidate combinations in one assay so that we could compare their performance to each other (Figures 7-9).

Specifically, in this test we aimed to determine if **mixing forward and reverse primers** as well as
**PrymCrRNAs** between the sets could achieve a lower limit of detection than the preliminarily
observed LOD of 200 fM (ModF-ModR-PrymCrRNA1).

For simplicity, negative controls are not included in the graphs on Figures 7-9, as they showed no significant
signal and their fluorescence intensity values have been subtracted from the obtained results.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-14-480nm.webp)

**Figure 7**. SHERLOCK fluorescence readout result – primer and
crRNA mixing screening, performed on a 480 nM sample of *Prymnesium parvum* DNA.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-14-200fm.webp)

**Figure 8**. SHERLOCK fluorescence readout result – primer and
crRNA mixing screening, performed on a 200 fM sample of *Prymnesium parvum* DNA.

As shown in Figures 7-8, the best combination was **ModF + GalR + PrymCrRNA1** because it yielded high
signals for both **480 nM** and **200 fM target DNA concentrations**.

To **ultimately select the most promising primer pair for optimization,** an assay was conducted using
the primers that produced the most satisfactory results in the previous test: ModF-GalR; ModF-ModR; AltF-GalR.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-15-fix.webp)

**Figure 9**. SHERLOCK fluorescence readout result – primer and crRNA
mixing screening, additional experiment.

Based on the results shown in Figure 9 and the previous assay (Figure 8), the best primer combination was indeed
**ModF-GalR + PrymCrRNA1**. This combination provides the **highest fluorescence intensity**and **effectively detects a 200 fM target DNA concentration**, as demonstrated in the previous
test.

Having identified our chosen pair, our next step was to **quantify the SHERLOCK system** to determine
if the fluorescence signal corresponds to the amount of target *Prymnesium parvum* DNA and, consequently, the
number of algal cells.

### Limit of Detection (LOD) determination and SHERLOCK quantification

The **Limit of Detection (LOD)** was defined as the lowest concentration of PCR-amplified
*Prymnesium parvum* DNA that yielded a positive test result. This was indicated by a significant increase in
fluorescence intensity over time, with the measured values exceeding those of the corresponding negative control.

**Initial experiments** on assessing the LOD were carried out using the **GalF-GalR +
PrymCrRNA2** combo, as they were performed before the adaptation of Mod and Alt primers.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-10-fix.webp)

**Figure 10**. SHERLOCK fluorescence readout result – preliminary LOD
determination, repeat 1.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-11.webp)

**Figure 11**. SHERLOCK fluorescence readout result – preliminary LOD
determination, repeat 2.

Based on the charts in Figures 10-11, we concluded that the initial template DNA concentration does not show a
linear relation with fluorescence intensity. We hoped that **optimizing RPA primer concentration** may
solve the issue. We thought the detection of **200 fM** by the assay to be a promising sign for the LOD
value. From the first SHERLOCK test (Figure 10), it was concluded that the LOD is between **200 pM and 200
fM**. The second SHERLOCK test (Figure 11) was performed to assess the LOD more precisely, and it was
unexpectedly able to detect a **200 fM** concentration, which was not the case for the previous test.

We conducted experiments to **optimize the RPA primer concentration** and therefore **make
SHERLOCK quantifiable**; the details are described on the [**"Measurement"**](https://2024.igem.wiki/ju-krakow/measurement) page. Unfortunately, these
efforts were **not successful**, as we were unable to define a range of target DNA concentrations that
consistently correlated with the final fluorescence intensity. As a result, we were also unable to establish a
universal **standard curve** for the assay.

After **ModF-GalR + PrymCrRNA1** was established as the most effective combination, an additional
assay was conducted to determine the **final LOD** for our test.

This experiment was repeated twice. In Figures 12-15, the data were split into two charts (for higher and lower
concentrations) to facilitate easier visual analysis. However, it's important to note that all samples were measured
in the same experiment, allowing for direct comparison between them (the two charts simply reflect an approach to
data presentation).

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-1-higher-nopoz.webp)

**Figure 12**. SHERLOCK fluorescence readout results – final LOD
determination, higher DNA concentrations, repeat 1.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-1-lower-nopoz.webp)

**Figure 13**. SHERLOCK fluorescence readout results – final LOD
determination, lower DNA concentrations, repeat 1.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-2-higher-nopoz.webp)

**Figure 14**. SHERLOCK fluorescence readout results – final LOD
determination, higher DNA concentrations, repeat 2.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/sherlock-17-2-lower-nopoz.webp)

**Figure 15**. SHERLOCK fluorescence readout results – final LOD
determination, lower DNA concentrations, repeat 2.

Despite optimizing the RPA primer concentration, it was **not possible to define a range of target DNA
concentrations** that are **proportional** to the **final fluorescence
intensity**.

The SHERLOCK reaction proved to be **unrepeatable**. Different fluorescence intensities were observed
for samples from the same RPA, and **varying LODs** were determined for test duplicates: **1
pM** for the first test and **200 fM** for the second. This **inconsistency**
undermines the ability to compare fluorescence intensities between tests.

Given these observations, the test **does not appear suitable for quantitative measurements**. If a
range of *Prymnesium parvum* DNA concentrations proportional to fluorescence intensity could be established, then creating a standard curve for each test would provide reference points for quantifying DNA
concentration.

After conducting these experiments, we discovered that commercial RPA kits are optimized for **rapid
amplification**, but diluting the kit (e.g., 2x) may improve the proportional relationship between the
amplified DNA and the initial target concentration. Therefore, **future efforts** to quantify the
reaction could involve testing with a **diluted RPA kit**. Additionally, it would be beneficial to
measure the DNA yield after RPA using more direct methods, such as the use of EvaGreen DNA dye [3], rather than
relying on SHERLOCK, which depends on the activity of two proteins: T7 polymerase and Cas13.

Nevertheless, based on the collected data, we can conclude that the limit of detection (LOD) of our test is
**1 pM**. This concentration corresponds to **50 billion** ***Prymnesium
parvum*** **cells per liter of water** (see calculations below). Additionally, we
successfully detected *Prymnesium parvum* genomic DNA in the sample taken from our algal cultures. These
findings suggest that SHERLOCK can be used for screening the presence of *Prymnesium parvum* in water with a
detection limit of **1 pM**.

Based on bioinformatic analysis we learned that *Prymnesium parvum* from the Oder River has ITS1-5.8S-ITS2
sequences exclusively on chromosome 20, present in **two haplotypes**: one with **8**
repetitions of the ITS1-5.8S-ITS2 sequence and another with **4** repetitions. Statistically, this
means that 25% of the Prymnesium parvum population should have 8 copies of the ITS1-5.8S-ITS2 sequence (haplotypes 4
+ 4), 50% should have 12 copies (8 + 4), and 25% should have 16 copies (8 + 8).

$$ 48 \text{ ITS1 - 5.8S - ITS2 copies} - x \text{ mole} $$

$$ 6.02 \times 10^{23} = 1 \text{ mole} $$

$$ x = 7.97 \times 10^{-23} \text{ moles of ITS1 - 5.8S - ITS2 correspond to } 4 \text{ algal cells in } 1 \,
\text{L of water} $$

$$ 7.97 \times 10^{-23} \text{ moles of ITS1 - 5.8S - ITS2} - 4 \, \textit{Prymnesium parvum}\text{ in 1 L of water}
$$

$$ 10^{-12} \, \text{M (1 pM)} = y \, \text{cells} $$

$$ y = 5 \times 10^{10} = 50 \, \text{billion } \textit{Prymnesium parvum} \text{ cells in 1 L of water} $$

### Lateral Flow Assay

The LFA procedure was used to support and validate the conclusions gained from tests that utilized the
fluorescence-based readout. However, there were also experiments conducted which were notable or specific to the LFA
procedure.

#### Direct Prymnesium parvum detection

We were successful in directly detecting *Prymnesium parvum* DNA isolated from **algal culture
samples** as well as **water samples** taken from **bodies of water affected by*****Prymnesium parvum*** **blooms** using the LFA procedure. The following
test was repeated twice.

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image8.jpg)

**Figure 16**. LFA test result on *Prymnesium parvum* DNA.

The strip numbers in Figure 16 correspond to the following samples (“Water” – sample of
*Prymnesium* genomic DNA isolated directly from a water body affected by *Prymnesium parvum* blooms;
“Culture” – sample of *Prymnesium* gDNA isolated from our laboratory cultures):

1. “Water” ModF-GalR + PrymCrRNA1
2. “Culture” ModF-GalR + PrymCrRNA1
3. Negative control ModF-GalR + PrymCrRNA1
4. “Water” AltF-GalR + PrymCrRNA1
5. “Culture” AltF-GalF + PrymCrRNA1
6. *Prymnesium* DNA (480 nM) AltF-GalR + PrymCrRNA1
7. Negative control AltF-GalR + PrymCrRNA1
8. Positive control

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image4.jpg)

**Figure 17**. LFA test result on *Prymnesium parvum* DNA – repeat.

The strip numbers in Figure 17 correspond to the following samples (“Water” – sample of
*Prymnesium* genomic DNA isolated directly from a water body affected by *Prymnesium parvum* blooms;
“Culture” – sample of *Prymnesium* gDNA isolated from our laboratory cultures):

1. “Water” Mod-Gal + PrymCrRNA1
2. “Culture” Mod-Gal + PrymCrRNA1
3. Negative control Mod-Gal + PrymCrRNA1
4. *Prymnesium* DNA (480 nM) Mod-Gal + PrymCrRNA1
5. Positive control

The tests **successfully detected** ***Prymnesium*** **genomic DNA**
in samples isolated from our **algal culture** as well as in samples **directly obtained from a
water body affected by** ***Prymnesium parvum*** **blooms**. This
confirmed the **efficacy** and **utility** of our test for **screening water bodies
for the presence of** ***Prymnesium parvum***.

#### Body heat incubation of RPA

Once we optimized our reaction components, we aimed to simplify the SHERLOCK procedure as much as possible. In this
experiment, we compared the effectiveness of incubating the RPA and SHERLOCK reactions using body heat versus
thermocycler incubation. This was done to assess whether the reaction can be effectively carried out by the user
holding it in their hand, therefore making the LFA assay portable and possible to use for in the field screening.

![](https://static.igem.wiki/teams/5087/pics/lfa-lab-not/image18.jpg)

**Figure 18**. LFA test results – body heat vs thermocycler incubation
experiment. Samples incubated using the thermocycler are labeled in red, while those incubated by hand (palm of the
hand) are denoted in black.

The strip numbers in Figure 18 correspond to the following samples:

1. *Prymnesium* DNA (480 nM) Mod-Gal + PrymCrRNA1
2. *Prymnesium* DNA (200 fM) Mod-Gal + PrymCrRNA1
3. “Culture” Mod-Gal + PrymCrRNA1
4. “Water” Mod-Gal + PrymCrRNA1
5. Negative control Mod-Gal + PrymCrRNA1

The test results for both thermocycler and hand incubation methods **do not show significant differences in
band intenstity**, suggesting that **hand incubation is as effective as thermocycler incubation for
this protocol**. However, only test one from the tested conditions yielded a positive result, indicating
that there may be issues with either the RPA reaction reagents or the DNA templates used in the other tests, since
“water” and “culture” samples have been positive in previous tests (Figures 16 and 17). This inconsistency in
results points to the need for **further optimization** or **troubleshooting** of the
reaction components to ensure reliable outcomes across all tests.

#### Limit of Detection (LOD) determination

The **Limit of Detection (LOD)** was determined as the lowest concentration of PCR-amplified
*Prymnesium parvum* DNA for which the test result remained positive, indicated by the presence of a
**visible T-line**. For this experiment, samples were incubated in a PCR thermocycler.

![](https://static.igem.wiki/teams/5087/pics/results/lfa-fig-19-results.jpg)

**Figure 19**. LFA test results – LOD determination experiment.

The strip numbers in Figure 19 correspond to the following samples:

1. *Prymnesium* DNA (200 nM) + ModF-GalR + PrymCrRNA1
2. *Prymnesium* DNA (20 nM) + ModF-GalR + PrymCrRNA1
3. *Prymnesium* DNA (2 nM) + ModF-GalR + PrymCrRNA1
4. *Prymnesium* DNA (200 pM) + ModF-GalR + PrymCrRNA1
5. *Prymnesium* DNA (40 pM) + ModF-GalR + PrymCrRNA1
6. *Prymnesium* DNA (20 pM) + ModF-GalR + PrymCrRNA1
7. *Prymnesium* DNA (10 pM) + ModF-GalR + PrymCrRNA1
8. *Prymnesium* DNA (5 pM) + ModF-GalR + PrymCrRNA1
9. *Prymnesium* DNA (2 pM) + ModF-GalR + PrymCrRNA1
10. *Prymnesium* DNA (1 pM) + ModF-GalR + PrymCrRNA1
11. *Prymnesium* DNA (200 fM) + ModF-GalR + PrymCrRNA1
12. *Prymnesium* DNA (20 fM) + ModF-GalR + PrymCrRNA1
13. *Prymnesium* DNA (2 fM) + ModF-GalR + PrymCrRNA1
14. *“Water”* + ModF-GalR + PrymCrRNA1
15. *“Culture”* + ModF-GalR + PrymCrRNA1
16. **Negative control* ModF-GalR + PrymCrRNA1*
17. *Positive control*

By comparing strips 1-13 with test 16, a negative control, it can be estimated that the limit of detection (LOD)
for our Sherlock assay is approximately **10 pM**, as indicated by test 7. However, this conclusion is
somewhat **subjective**, given that band intensity was not quantified.

To obtain a more precise and reliable LOD determination, this test should be **repeated**. The
positive control (stripe 17) yielded a positive result. However, the test performed on older RPA samples previously
provided a more definitive positive result (lower C-line was not visible). This suggests there may be
**inefficiencies** in the RPA procedure or potential contamination of its reagents, which could be
affecting the consistency of the results.

The tests for both the “Water” and “Culture” samples returned **negative
results**. This is likely due to unidentified issues within the procedure, possibly linked to the RPA step
or sample handling.

## Protein purification

---

We successfully developed a **Cas13 purification protocol** that efficiently yields **pure
Cas13** while also being **cost-effective** and **time-efficient**. We purified
both huLwCas13a and CcaCas13b proteins on the first attempt. This page presents the results of the
**purification process** and the **functionality** of **Cas13a** as this is
the protein we used in all our SHERLOCK assays (detailed results for CcaCas13b can be found in our [Notebook: ProteinLab section](https://2024.igem.wiki/ju-krakow/notebook#protein-lab)).

### Overview of key steps in the protein purification process

1. **Protein Expression**: Expression in *E. coli* strain Rosetta 2(DE3)pLysS using the pET
   system.
2. **Bacterial Cell Disruption**: Sonication was used to lyse the bacterial cells.
3. **Protein Purification**:

1. **IMAC**(Immobilized Metal Affinity Chromatography): Evaluated HisTag versus StrepTag
   purification efficiency. This step was followed by **SUMO digestion** on half of the collected
   fractions.
2. **IEC**(Ion Exchange Chromatography): Removed remaining contaminants and concentrated the sample
   before SEC.
3. **SEC**(Size Exclusion Chromatography): Achieved highly pure Cas13a (Figure 22).

4. **Comparative Analysis**: The tag’s effect on structure, thermal stability, and activity was
   evaluated via Circular Dichroism (Figure 23), NanoDSF (Figure 24), and SHERLOCK activity tests (Figure 25).

After each step of the protein purification process, results were visualized using SDS-PAGE (Figures 20-22).

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure3.jpg)

**Figure 20**. SDS-PAGE gel for huLwCas13a – samples after IMAC\*.

Wells key: unind. – uninduced bacterial culture, ind. – culture after 18h IPTG
induction, sonic. – supernatant after sonication and centrifugation, prec. – sediment after sonication and
centrifugation, FT – flow-through from sample application on His-resin column, wash – flow-through from column
washing before elution; E1, E2, E3… – fractions eluted with 150 mM imidazole buffer, \*E1, \*E2, \*E3 – fractions
eluted with 300 mM imidazole buffer.

\***Note**: Some electropherograms show bands shifted downward from their expected positions, likely
due to well overloading. To confirm the proteins' sequences, mass spectrometry would be ideal, but budget
constraints prevented us from performing this analysis.

The expression step was **effective** as after induction the band of a mass corresponding to the
produced protein was observed (for Cas13a – 155 kDa).

IMAC was effective in **partially purifying** Cas13a – percentage content of contaminants in the
solution decreased (compared to wells ind. and sonic), although they are still present in significant amounts.
Moreover, fractions were enriched with the produced protein (thicker bands corresponding to the produced protein
– marked by violet arrows).

The SUMO digestion step was conducted, as planned, on half of the collected IMAC fractions. SUMO digested and
undigested pooled fractions were used in the second purification protocol step – IEC.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure5.webp)

**Figure 21**. SDS-PAGE gel for huLwCas13a – samples after IEC.
Cas13a with the tag is indicated by the violet arrow, Cas13a without the tag – blue arrow. The purple rectangle
marks two bands that correspond to the cleaved 6xHis-TwinStrep-SUMO tag.

Wells key: entry – pooled fractions after IMAC, sample appl. – flow-through from
sample application on the column, wash – flow-through from column washing, E1, E2, E3… – eluted fractions. Samples
with \* correspond to samples after SUMO protease digestion.

For Cas13a, for both SUMO protease digested and undigested samples, a reduction in protein contaminants,
approximately 2-fold, can be observed, although they still remain in significant amounts. Concentration of Cas13a in
the collected fractions is visible.

After IEC and concentration of pooled fractions to less than 0.5 ml the final purification step – SEC –
could be conducted.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure7.webp)

**Figure 22**. SDS-PAGE gel for huLwCas13a – samples after SEC.
The protein with the tag is marked by the violet arrow, while the protein without the tag is marked by the blue
arrow.

Wells are labeled as follows: E1, E2, E3, etc., representing eluted fractions.
Samples marked with \* are those that underwent SUMO protease digestion.

After the 3-step purification process, we obtained **pure huLwCas13a**, as confirmed by SDS-PAGE
(Figure 22). As expected, a single band was observed for the undigested protein. In SUMO-digested fractions, two
bands were observed, indicating incomplete tag cleavage.

As planned, we conducted a comparative analysis of tagged and untagged proteins to test their functionality.

![](https://static.igem.wiki/teams/5087/pics/sherlock-plots/cd-cas13a-fix.png)

**Figure 23**. Dependence of mean residue molar ellipticity for huLwCas13a
with and without the tag in the far-UV range.

**Table 1**. Proportions of various secondary structure types determined
from Circular Dichroism spectra for huLwCas13a.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **huLwCas13a** | **Mean proportions of secondary structure types determined using algorithms from the CDPro package ± standard deviation** | | | |
| α-helix | β-sheet | β-turn | unordered |
| undigested | 64.5 ± 2.1 | 3.8 ± 0.9 | 10.2 ± 0.7 | 20.9 ± 2.5 |
| digested | 58.1 ± 10.2 | 4.7 ± 6.3 | 18.2 ± 9.9 | 19.5 ± 7.2 |

The spectra in Figure 23 overlap significantly, and the contribution of individual secondary structures to the
protein structure (Table 1) does not differ substantially. This indicates (contrary to our expectations) that the
**tag does not have a significant impact on the structure** of Cas13a.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle1/figure11.webp)

**Figure 24**. Dependence of the first derivative of the fluorescence
intensity change from temperature for huLwCas13a.

Key: D – sample digested using SUMO protease, UD – sample undigested

Contrary to our expectations, NanoDSF results indicated **no** statistically significant
**difference** between tagged and untagged proteins in thermal stability.

![](https://static.igem.wiki/teams/5087/pics/sher-lab-not/5-sher.png)

**Figure 25**. A comparison of the activity of huLwCas13a with and without
6xHis-TwinStrep-SUMO tag.

Figure key: 1 – SUMO protease digested huLwCas13a added to SHERLOCK mix; 2 – SUMO
protease undigested huLwCas13a added to SHERLOCK mix; 3 – water added instead of Cas13a (negative control); 4 –
RNase A added instead of Cas13a (positive control); 5 –target RNA and crRNA replaced with water in SHERLOCK mix,
for SUMO digested huLwCas13a; 6 – target RNA and crRNA replaced with water in SHERLOCK mix, for SUMO undigested
huLwCas13a.

Contrary to our expectations the Cas13a protein, both digested and undigested by SUMO protease, showed
**similar levels of activity.**

### Final conclusions

Our optimized protocol successfully produced **pure, active Cas13 for future experiments**. We
obtained 3.6 mg of purified protein, sufficient for nearly **29,000 SHERLOCK reactions**.

During the purification process, we observed that a single-step purification using IMAC yielded only around 15%
Cas13 in the collected fractions, making it **insufficient** for our needs. We recommend a
**three-step purification process**—IMAC, IEC, and SEC—to achieve pure Cas13 detailed
protocol can be found [here](https://static.igem.wiki/teams/5087/pics/protocols/cas13-purification.pdf).
This improvement builds upon existing protocols and offers a **valuable contribution for future iGEM
teams**.

Notably, we concluded that the **SUMO protease cleavage** step is **unnecessary**. This
conclusion was supported by several tests:

* **Circular Dichroism (CD)**: The mean residue ellipticity spectra (Figure 23) for tagged and
  untagged protein overlap, and the proportions of secondary structures (Table 1) are similar. This indicates that
  the **tag does not significantly impact the protein's structure**.
* **Nano Differential Scanning Fluorimetry (NanoDSF)**: Contrary to our expectations, NanoDSF results
  indicated **no statistically significant difference** between tagged and untagged protein **in
  thermal stability**. The thermal stability curves for digested (D) and undigested (UD) proteins overlap
  (Figure 24), suggesting that the tag does not significantly affect thermal stability. This further supports the
  decision not to cleave off the 6xHis-TwinStrep-SUMO tag.
* **Activity Tests in SHERLOCK**: huLwCas13a was found to be active. Contrary to our expectations the
  Cas13a protein, both digested and undigested by SUMO protease, showed similar levels of activity. There were
  **no statistically significant differences in activity** between SUMO protease digested (1) and
  undigested (2) samples (Figure 25).

Omitting the SUMO digestion step makes the purification protocol more cost-effective and time-efficient.

We produced and purified **Cas13b** as a **backup** in case Cas13a faced challenges such
as inefficient purification or loss of activity. (The data presented here pertains to Cas13a, with a full
description of the purification process for both proteins available in the Protein Lab documentation in
"Notebooks"). This strategy ensured the project could continue without delays. However, due to budget constraints,
we did not order synthetic DNA for Cas13b testing, as Cas13a's activity had already been confirmed.

Looking ahead, **future work** could involve testing our purified **CcaCas13b** and using
it in SHERLOCK assays. This could allow for **multiplex detection** of the ITS2 region of
*Prymnesium parvum* alongside other genomic fragments.

In conclusion, our **Cas13a** protein **met our purity standards** and was used for
**all SHERLOCK tests** throughout the project.

## Prymnesium parvum culture

---

*Prymnesium parvum* is a non-model organism, and there are no established protocols for culturing this
species. Through a comprehensive system of trial and error, combined with guidance from our supervisors and external
collaborators, we **developed a method for maintaining** ***Prymnesium
parvum*** **under laboratory conditions**. Additionally, we tested various techniques
for isolating DNA from *Prymnesium parvum* samples.

### Culture conditions

We successfully set up **cultures** from various **water samples** collected from bodies
of water affected by *Prymnesium parvum* blooms.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image32.jpg)

**Figure 26**. Microscoping image of water sample containing
*Prymnesium parvum* cells.

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image18.jpg)

**Figure 27**. Fluorescent microscopic image of water sample containing
*Prymnesium parvum* cells.

Characteristic morphological features of *Prymnesium parvum,* such as the **haptonema** and the
**two chloroplasts**, are visible in Figure 26.

Despite observing these features, differentiating *Prymnesium parvum* cells from other phytoplankton in the
water samples proved **challenging**. Therefore, we confirmed its presence via PCR using
**primers designed by Galluzzi et al.** [1], which target the ITS2 sequence specific to *Prymnesium
parvum*. This PCR product is **132 bp** in length.

We isolated DNA and performed PCR, both following the [protocol](https://static.igem.wiki/teams/5087/pics/protocols/pcr-alga.pdf).

![](https://static.igem.wiki/teams/5087/pics/alga-lab-not/image36.png)

**Figure 28**. Gel electrophoresis of the PCR product – result.

Legend to Figure 28:

* O'GeneRuler DNA Ladder Mix
* Gz primer concentration 200 nM
* Gz primer concentration 400 nM
* Kz primer concentration 200 nM
* Kz primer concentration 400 nM
* GeneRuler 1kb DNA Ladder Mix

“Gz” and “Kz” are names of DNA samples derived from different *Prymnesium parvum*
cultures. Samples Gz and Kz contain the ITS2 sequence of 132 bp, as indicated by the clear **bands visible
between the 200 bp and 100 bp markers**. No specific product was observed in row 2. Based on these results,
we can conclude that the **algal DNA isolated contains** ***Prymnesium
parvum*** **genomic DNA**, confirming the presence of *Prymnesium parvum* cells
in the respective water samples.

Based on our optimization of culture conditions, we **developed a method for maintaining*****Prymnesium parvum*** **under laboratory conditions**. A detailed
description of the recommended culture conditions can be found on the “Contribution” page, as we hope it
will be useful for future iGEM teams working with *Prymnesium parvum*.

### DNA isolation

Throughout our project, we used three **different DNA isolation method**s:

* isolation with DNeasy Blood and Tissue Kit (QIAGEN)
* isolation with Monarch Genomic DNA Purification Kit (NEB)
* in-field isolation using sticks made from cellulose paper

Isolation using the NEB kit yielded a slightly higher DNA concentration compared to the QIAGEN kit. Additionally,
the NEB isolation kit proved to be more convenient, as it did not require a lengthy initial incubation step of 5-7
hours like the QIAGEN kit. Tables 2 and 3 display the DNA yields obtained for selected samples with the NEB and
QIAGEN kits, respectively.

**Table 2**. Concentration of the DNA isolated with the NEB kit.

|  |  |  |  |
| --- | --- | --- | --- |
| **Sample name** | **DNA concentration [ng/µl]** | **A260/A280** | **A260/A230** |
| NOW 5 | 16 | 1,68 | 1,34 |
| BOW 2 | 24,1 | 1,79 | 1,56 |

**Table 3**. Concentration of the DNA isolated with the QIAGEN kit.

|  |  |  |
| --- | --- | --- |
| **Sample name** | **Concentration [ng/µl]** | **A260/A280** |
| NOW 5 | 5,8 | 1,66 |
| NOW 5.1 | 4,9 | 1,65 |
| P | 12,7 | 1,67 |
| BOW 1 | 5,6 | 1,53 |
| BOW 2 | 6,4 | 1,55 |
| 2W | 11,2 | 1,28 |
| Gdańsk 2 | 5,2 | 1,8 |

We also tested a nucleic acid purification method designed for use outside the laboratory setting [4]. Our
objective was to adapt this method for field-based nucleic acid isolation within the PrymChip. The technique
involves preparing cellulose dipsticks coated with wax for nucleic acid purification. The dipsticks we created are
presented in Figure 29.

![](https://static.igem.wiki/teams/5087/pics/contribution/image2-5.webp)

**Figure 29**. The dipsticks made out of Wattman paper.

![](https://static.igem.wiki/teams/5087/pics/contribution/image3-4.webp)

**Figure 30**. Equipment for the in-field isolation with instructions. In
Eppendorf tubes lysis buffer (number 1) and wash buffer (number 2) are presented.

Despite our efforts, we were **unable to successfully isolate DNA using this method** within the
PrymChip system. However, we highlight that this **cost-effective field-based method for nucleic acid
isolation** exists and could be further optimized by future iGEM teams for environmental testing purposes.

## SynLOCK

---

During the design of our crRNA spacers for detecting *Prymnesium parvum*, we recognized that the process was
quite **inefficient**. While we were only changing a small portion of the crRNA sequence with each
design, we still had to order the full sequence each time. To simplify this process, we aimed to create a system
where users would only need to focus on designing their specific spacer, while the T7 and direct repeat (DR)
fragments—common to all crRNAs used with Cas13a—would remain unchanged. This led to the creation of
SynLOCK, a user-friendly crRNA synthesis system for SHERLOCK. **SynLOCK – the crRNA Synthesis System for
SHERLOCK** – is envisioned as a modular set of components that allows users to easily insert a custom
crRNA spacer of their choice.

To do this, we initially designed the SynLOCK Cassette (BBa\_K5087017) as shown in the schematic in Figure 31.

![](https://static.igem.wiki/teams/5087/pics/engineering/kloc-1.webp)

**Figure 31**. Initial SynLOCK Cassette Design.

Initially, our team used the **PSB1C5C** plasmid backbone for assembly due to its size, high copy
number, and availability in the 2024 iGEM Distribution kit, which was accessible to us and other teams. We ordered
the Cassette DNA, assembled it by hybridizing two DNA strands to create sticky ends, and successfully cloned it into
the pSB1C5C plasmid backbone using cloning with BsaI.

![](https://static.igem.wiki/teams/5087/pics/engineering/plazmid-1.webp)

**Figure 32**. The schematic representation of the initially assembled
device.

We verified the sequence of our design using **Sanger sequencing** to ensure that the clones we
selected contained the Cassette. The results confirmed that the Cassette was present and capable of accepting the
crRNA spacers.

However, we realized that the sequencing is required to verify if the Cassette is integrated into the plasmid
backbone, which is not **time-efficient**. Including a **reporter** within the Cassette
could solve that problem. Moreover, the plasmid backbone we used is not ideal. It is designed for storing CDS parts
and is not a LVL1 plasmid suitable for Type IIS assembly. Additionally, it lacks the biobrick prefix and suffix,
making it impossible to transfer the Cassette between vectors after cloning.

Therefore, we **refined our design** and included a reporter unit. The reporter was strategically
placed between the SapI sites to ensure it would be excised when a custom crRNA spacer was incorporated, allowing
for easy visual screening of colonies.

![](https://static.igem.wiki/teams/5087/pics/engineering/kloc-2.webp)

**Figure 33**. Improved SynLOCK Cassette Design.

Additionally, we chose the **pSB1C3** plasmid as the new backbone. This plasmid is a standard for
storing BioBrick parts, meeting our criteria for being high-copy and accessible, and it supports cassette transfer
using BioBrick prefix and suffix cutting sites.

We introduced the reporter unit into the Cassette by amplifying it via PCR to obtain compatible sticky ends. We
then amplified the complete Cassette with the reporter and transferred it into the pSB1C3 plasmid backbone.

![](https://static.igem.wiki/teams/5087/pics/engineering/plazmid-2.webp)

**Figure 34**. The schematic representation of the final assembled device.

We then assembled the system by cloning in the custom crRNA spacers used in our PrymDetect test. We employed the
Type IIS assembly standard to insert the crRNA spacers, ensuring that there were no scar sites in our final IVT
template construct that could impact the length or sequence of the crRNA.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle3/figure7.webp)

**Figure 35**. Schematic depiction of the improved system in action. On
the right, a fragment of a Petri dish showing visible white and purple bacteria, indicating which colonies are ready
for picking.

We conducted IVT on the linearized plasmids and performed RNA electrophoresis to visualize the results.

![](https://static.igem.wiki/teams/5087/part-registry-images/ivtgelresult.png)

**Figure 36**. RNA electrophoresis – in vitro transcription (IVT) results
using SVR and USV plasmids containing the Cassette with crRNA spacers. "SVR" indicates that the crRNA from that well
was stored in the pSB1C5C backbone, while "USV" signifies that it was stored in the pSB1C3 backbone.

We also tested the **Cassette’s transferability** between different backbones by excising it
from the pSB1C3 backbone using the enzymes present in the BioBrick prefix and suffix. This step added to the
system’s **standardization**.

![](https://static.igem.wiki/teams/5087/pics/engineering/cycle3/figure9.webp)

**Figure 37**. Electrophoresis results showing the digestion of USV
plasmid with the enzymes marked in the image.

**Table 5**. Expected band size the digestion of the USV plasmid.

|  |  |
| --- | --- |
| **Enzyme/s used** | **Expected Band Size (bp)** |
| EcoRI & SpeI | 2047 & 1031 |

The correct bands were obtained, demonstrating that the Cassette can now be transferred between other systems
compatible with the BioBrick assembly standard. This allows our design to be used with a variety of plasmid
backbones, giving users the flexibility to choose the one that best suits their specific needs.

In our results, we **successfully obtained crRNA transcripts of the correct length**, as indicated by
the single band on the gel for most wells at around 64 bp. Some **contamination** is visible in certain
wells, likely due to the plasmid IVT template remaining in the sample. Additionally, some bands appeared
**faint**, suggesting that the vectors may not have been fully digested with BbsI or that the DNase I
treatment was only partially effective. For future experiments, it would be ideal to include an RNA ladder with
bands below 200 bp in the electrophoresis samples; unfortunately, the RiboRuler High Range RNA Ladder (Thermo Fisher
Scientific) was the only option available to us.

The correct single band at an expected transcript length confirms that the system is functioning as expected,
establishing a **proof-of-concept for SynLOCK**. Overall, while further technical optimization is
necessary to obtain purer molecules, our results support the system's **functionality** and
**potential for future development**.

## PrymChip

---

We aimed to address the need for **accessible**, **field-based environmental sample
testing** by creating a device that allows individuals, regardless of research experience, to test their
water samples for *Prymnesium parvum* near water sources. Leveraging the versatility and cost-effectiveness
of **3D printing**, we focused on developing a **low-cost prototype** as part of our iGEM
project. Our efforts culminated in the development of a device called the **PrymChip**.

The **PrymChip** is a **3D-printed device** specifically designed to excite, detect, and
quantify **green fluorescence signals**, with data processed using a **Python script**.
The primary objective was to create an affordable, field-deployable tool capable of detecting *Prymnesium parvum*in environmental water samples using the SHERLOCK detection system.

We successfully designed all the 3D-printed parts in TinkerCAD and printed using filament fused deposition modeling
(FDM) technology with a Prusa i3 MK3 Printer. We went through multiple iterations of the **DBTL
(Design-Build-Test-Learn) cycle** before landing on a final design (Figure 38). The design was carefully
planned to incorporate all the components necessary for detecting *Prymnesium parvum*. Consequently, the
PrymChip consists of **three** **main parts** (Figure 38):

* **Detection Box:** This section houses the reaction chamber where the test solution is placed. It
  is also the area where a smartphone is used to capture a photo for analysis.
* **Electronics Box:** This contains the core electronic components, including the circuit
  responsible for exciting the sample's fluorescence using blue LED lights.
* **Tool Box:** A storage compartment designed to hold necessary experimental equipment and reagents
  for the procedure.

![](https://static.igem.wiki/teams/5087/pics/hardware/image6-2.webp)

**Figure 38**. Schematic of the PrymChip.

A comprehensive description of the PrymChip has been included in the ‘Hardware’ page.

Due to time constraints and limited reagent availability, we were **unable to demonstrate algae detection
using SHERLOCK** within the PrymChip. Instead, we successfully performed **proof-of-concept
experiments**, demonstrating the device’s functionality with **fluorescein solutions**.
Testing the software on fluorescein solutions was the most **convenient** and **reliable**
option as the excitement (**~490 nm**) and emission maximums (**~520 nm**) suggest that
the fluorescein or its derivative is used in formation of the **RNase Alert** utilized normally in
SHERLOCK tests conducted by our team.

Fluorescein from the Fluorescence Measurement Kit was used to prepare different concentrations of the fluorescein.
A series of **photos** were then taken, corresponding to **fluorescein solutions**
prepared in **PBS**, with concentrations ranging from **0.78 µM** to **100
µM**, including a **blank sample** containing only **PBS solution (Figures
39-40)**.

![](https://static.igem.wiki/teams/5087/pics/software/image4-3.webp)

**Figure 39**. Series of fluorescein solutions and the PBS solution.
Photos taken by a Google Pixel smartphone.

![](https://static.igem.wiki/teams/5087/pics/software/image1-3.webp)

**Figure 40**. Series of fluorescein solutions and the PBS solution.
Photos taken by a Samsung smartphone.

As illustrated in Figures 39-40, the fluorescent signal from fluorescein can be detected using both smartphone
devices, although minor differences in fluorescence intensity are observed between the models.

The calibration curve images served as training data for a linear regression model in the Python software. After
training, the model was tested to evaluate its ability to predict the concentration of green fluorescence based on
fluorescence intensity values obtained from images with unknown fluorescein concentration.

**Table 6**. Concentrations of the tested fluorescein solutions and the
results obtained from the calibration curves using different phones.

|  |  |  |
| --- | --- | --- |
| **Real fluorescein concentration (µM)** | **Concentration detected by Samsung phone (µM)** | **Concentration detected by Google Pixel phone (µM)** |
| 8 | 7.22 | 0 |
| 2 | 2.84 | 0 |
| 50 | 53.60 | 42.88 |

The results indicated that the software accurately analyzed the photos, yielding fluorescein concentration values
that closely matched the actual concentrations. This demonstrates that our PrymChip software is **effective
and reliable**. However, the sensitivity of measurement highly depends on the smartphone’s camera. It would be advisable to test the software amongst a wider range of devices.

Encouraged by the promising results from the fluorescein tests, we decided to test the RNaseAlert™ substrate
(from the RNaseAlert™ Lab Test Kit v2, Invitrogen), which is the RNA probe used in our fluorescence-based
SHERLOCK assays. We introduced RNase A to cleave the probe and emit a fluorescent signal.

Unfortunately, this experiment did not yield successful results, as we did not detect a fluorescent signal (Figure
41). Several factors may have contributed to this outcome, including the possibility that the LEDs used in the
PrymChip design may not effectively excite the fluorophore in RNaseAlert. This experiment needs to be repeated to
further investigate the cause of this issue; however, we were unable to do so due to limited availability of
RNaseAlert™.

![](https://static.igem.wiki/teams/5087/pics/3dlab-not/image19.png)

**Figure 41**. Photos from the PrymChip – RNase alert testing.

The current PrymChip serves as a **proof-of-concept** but requires **several
improvements** to detect *Prymnesium parvum* in environmental samples. One key enhancement is
incorporating **nucleic acid isolation via cellulose dipsticks**, which could be seamlessly integrated
into the design. Looking ahead, key future experiments will include **further testing with
RNaseAlert**, as well as final experiments utilizing all optimized **SHERLOCK reaction
components** to detect *Prymnesium parvum* DNA.

In terms of device design, several modifications can be made. Adding a **drawer** to the black
detection box would simplify solution insertion and reduce spillage risk. Redesigning the lid to house an
**external camera**, connecting to a phone via USB, would standardize image capture and eliminate the
need for calibration curves from individual phone cameras. However, the current lid option should remain for those
preferring phone-based photography. A **mobile app** for image analysis is essential to avoid the
inefficiency of manual photo transfer. Finally, the connection between the boxes could be improved by using a
**magnetic mechanism**, which would be easier to incorporate into the thick PrymChip walls and would
enhance overall usability.

In summary, at the current development stage, the PrymChip **excites green fluorescence** signals,
**captures images** via a smartphone, and **analyzes the data** using a Python script to
calculate fluorescein concentration from the captured images. We have estimated the final cost of the functional
device at **€20.38**. We envision this device as a promising, **reusable**, and
**cost-effective tool** that enables individuals, regardless of their research experience, to
**test water samples for** ***Prymnesium parvum*** near water sources.
Additionally, it represents a valuable **contribution to future iGEM teams**, providing an excellent
foundation for innovation. We anticipate seeing numerous exciting applications and modifications in the future.

## Modeling of Cas13a Reaction Kinetics

---

Our modeling approach is focused on **qualitative intuition** enabling for grasping the key concepts
of the enzyme dynamics in the PrymDetect system. The model and calculations were divided into 4 sections:

1. Model based on the **law of mass action**.
2. **Michaelis-Menten** kinetics model.
3. Determination of a **time scale** for the SHERLOCK reaction.
4. Qualitative visualization of the **RPA and IVT progression**.

We **successfully** developed a model based on the law of mass action that allowed us to relate the
**chemical reaction rate** to **concentration** for all reactants in the SHERLOCK
reaction, aiding the understanding of the **overall dynamics** of the system. This system of 6 ordinary
differential equations was subsequently simplified to a single **Michaelis-Menten equation** using a
series of assumptions based on literature findings such as the **quasi-steady-state** assumption. The
time scale which can be interpreted as an **estimate of time to detection** was calculated for
literature values for the same CRISPR enzyme and fluorescent reporter as used in the PrymDetect system giving an
estimate of **18 minutes**. However, this estimate is likely to be **longer** due to an
anticipated **higher value of the Michaelis constant** in our assay compared to the assay in the
publication. Finally, the qualitative progression of the RPA and IVT reactions was modeled to illustrate the
dynamics of these reactions specifically in the **PrymDetect** system.

## Summary

---

### Successes

#### SHERLOCK reaction components functionality

**17 out of the 18 primer-crRNA** combinations we engineered are **viable and
functional**, which has exceeded our expectations. Contrary to what we anticipated, **both crRNA
designs proved to be effective**, as confirmed by both fluorescence and LFA readouts. Additionally, after
encountering issues with false positive results from one of the combinations, we feared this might be a widespread
issue across many others; fortunately, that was not the case. Ultimately, we selected the **most effective
combination** – **ModF-GalR + PrymCrRNA1** – to focus our efforts on. However, we
believe that the wide array of combinations offers numerous opportunities for future optimization and fine-tuning of
the system.

#### Cas13 purification

Our protein purification was **successful on the first attempt**, allowing us to move forward with the
project. Additionally, we made **adaptations to the protocol** that we hope will help future iGEM teams
purify this protein more effectively and at a lower cost.

#### Algae cultures

Working with an **understudied**, **non-model organism**, relying on environmental
samples, proved to be extremely challenging—something we did not fully anticipate when brainstorming our
project idea. Nevertheless, we successfully **maintained our cultures**, **isolated** and
**sequenced** the ***Prymnesium parvum*** **DNA** as well as
**optimized culture conditions** and **DNA isolation methods**.

#### SynLOCK

We successfully **engineered a system** that will simplify the work of future iGEM teams and other
researchers using SHERLOCK. We established a **proof-of-concept** for this
**user-friendly** and **reliable system** for **crRNA synthesis**.

#### PrymChip

We developed an **inexpensive**, **3D-printed device** that has potential to be used with
the SHERLOCK method to test for the presence of *Prymnesium parvum* in water samples. We established a
**proof-of-concept** for the device and successfully engineered **Python software** that
allows a smartphone camera to detect fluorescence signals using the device.

#### Mathematical modeling

We developed a **mathematical model** that accurately represents SHERLOCK reaction kinetics and
conducted **back-of-the-envelope checks** on published literature data to enhance our understanding of
the SHERLOCK assay and inform its practical application and refinement.

### Failures and areas that need improvement

As an iGEM team and as young researchers, we place great importance on **honesty** and
**scientific integrity**. In evaluating our results and the project as a whole, we have identified
several **areas for improvement**, along with elements that could be considered
**failures**.

#### Fluorescence intensity calibration

Firstly, all of our fluorescence-based SHERLOCK data present **fluorescence intensity** on the y-axis.
This poses a problem because absolute fluorescence values can vary between plates and instruments, making it
impossible to directly compare results across different setups. Unfortunately, **we did not perform a
calibration** using a fluorescence standard curve, which we acknowledge as a mistake on our part. By the
time we recognized this issue, near the end of our project, we had already used up all the fluorescein from the
Fluorescence Measurement Calibrants Kit to calibrate our PrymChip. Although we ensured that **comparisons
between experimental samples** were made **within the same read** from the start, we recognize
that the lack of calibration may complicate the reproducibility of our results for future teams.

#### Field applicability

From the outset, we aimed to make our method **field-applicable**. This goal was a constant
consideration as we worked to optimize the test for maximum efficiency. We explored practical solutions, including
incubation using body heat, and simplified the overall procedure as much as possible. However, one key challenge
remained: the **DNA isolation step**. While we identified a protocol that could be used for in-field
DNA isolation and began testing it, we were unable to fully optimize it due to time constraints, and the method did
not function as intended. Nevertheless, we believe there is potential for **further optimization**, and
our proposed approach can still be adapted for use with samples containing *Prymnesium parvum*.

#### High LOD

Another challenge affecting the full in-field application of our test is the relatively **high Limit of
Detection (LOD)** that we were able to establish. In our fluorescence-based test, the LOD for detecting
*Prymnesium parvum* DNA was determined to be **1 pM**, which corresponds to
approximately **50 billion** ***Prymnesium parvum*** **cells** per
liter of water. This is significant, as fish kills typically occur at cell counts exceeding **50-100 million
cells per liter** [5]. To address this issue, we propose **centrifuging** the water sample
before applying the SHERLOCK detection procedure. However, this complicates field application since a centrifuge
would require access to electricity. Another possible solution is using a syringe with a filter. On a positive note,
Cas13-SHERLOCK fluorescence detection has been shown to demonstrate even **attomolar sensitivity**[6].
Therefore, we believe that with further optimization of the procedure and more thorough LOD determination, it would
be possible to eliminate the need for condensing the samples and enable direct application of the method.

#### One-pot SHERLOCK

In the original paper by Kellner et al., the authors describe a **one-pot SHERLOCK** procedure that
**combines the RPA and Cas13 detection** reactions into a single mixture [6]. We aimed to test this
method, as its success would further simplify the LFA detection procedure. However, the authors also note that
one-pot SHERLOCK is **less sensitive** and **more challenging to optimize** [6]. Our
findings confirmed this – after testing the procedure twice, both with the original reagent proportions and a
modified version, we received **completely negative results** for all samples, including the positive
controls.

#### PrymChip testing

Another area of improvement concerns testing our **PrymChip hardware** with **actual SHERLOCK
reagents** and***Prymnesium parvum*** **DNA**. Unfortunately, we were
unable to carry out these tests due to time constraints and limited availability of reagents, which meant we had to
conclude our work at the proof-of-concept stage. The test we did perform, using the **RNaseAlert™ probe**typically used in SHERLOCK (which was digested by an RNase), did not yield the expected results, as no
fluorescence was detected. Despite this, we believe there is still **room for optimization**, and we
encourage future iGEM teams to pursue this line of experimentation.

#### Lack of quantifiability

Lastly, we were disappointed that our efforts to optimize RPA primer concentrations for **quantifiable
SHERLOCK fluorescence detection** did not yield the desired results. From the outset, we recognized the
potential quantifiability of the SHERLOCK method as a **significant advantage** in applying it to
detect *Prymnesium parvum*. Despite our attempts to **optimize the RPA primer concentration**,
we found it **challenging** to establish a **range of target DNA concentrations** that
would **correlate proportionally** with the final fluorescence intensity or any other measurement
point. On the [“Measurement”](https://2024.igem.wiki/ju-krakow/measurement) page we provided our insights
what changes in conducting experiments can be done to make SHERLOCK quantifiable.

### Considerations for repeating experiments

#### Poor repeatability of SHERLOCK

Firstly, throughout our experiments, we observed that our SHERLOCK method exhibited **poor
repeatability**, particularly in the **fluorescence-based readout**. Although we cannot
directly compare intensity values between different reads for the reasons mentioned earlier, we noted that the
trends exhibited by the samples sometimes varied across reads. Additionally, the limit of detection (LOD) differed
between test duplicates, with one test showing an **LOD of 1 pM** and another yielding **200
fM**. Consequently, establishing a universal standard curve for the assay was not feasible.

#### Controls

Our results have underscored the importance of including relevant controls, particularly **negative
controls**, when validating SHERLOCK reaction components. We recommend using a sample in which
**water is added to the RPA reaction instead of DNA**, with all other components—especially
primers—retained, followed by SHERLOCK detection. We believe this control is preferable to simply adding water
to an 'empty' RPA reaction mix for the SHERLOCK reaction. While the latter control can also be useful, we emphasize
that the former is more critical due to the risk of **primers inadvertently serving as templates for the
Cas13:crRNA complex**, which can lead to false positive results, as we observed in our project. As a
positive control, we recommend including the synthetic oligonucleotides described in the Kellner et al. paper [6].
In our experience, these oligonucleotides have proven to be a reliable positive control for both fluorescence-based
and LFA detection methods.

#### RNase contamination issues

Secondly, we advise any future iGEM teams intending to use SHERLOCK in their projects to be mindful of**RNase contamination** and to seek supervisors and lab space with experience handling RNA. As a team of
students, most of whom lacked prior experience with RNA samples, we were often concerned that some of our seemingly
‘strange’ results might be attributed to RNase contamination in our samples or reagents. Therefore, we
recommend regularly **checking for RNA contamination** using kits like the RNaseAlert™ Lab Test
Kit (Invitrogen), which we also utilized during our project. Additionally, we strongly suggest implementing
**preventative measures**, such as using RNase-free reagents and materials, and thoroughly wiping down
surfaces, pipettes, and other equipment with RNase decontamination solutions like RNaseZap™ (Thermo Fisher
Scientific).

## Future of PrymDetect

---

We believe our project establishes a **solid foundation** for future efforts to rapidly and reliably
detect *Prymnesium parvum* at scale. However, we recognize that several aspects need further investigation
before the test can be implemented on a larger scale.

To ensure **project advancement beyond iGEM**, we have spoken with **Prof. Dariusz
Dziga** from the Laboratory of Metabolomics at Jagiellonian University. He has shown enthusiasm for
potentially **continuing the project**.

Key areas for future research and improvement could include:

* **Multiplexing the assay**: Detecting two DNA targets (with Cas13b) or both toxins' genes and a
  genetic marker of *Prymnesium parvum*.
* **Refining the limit of detection (LOD)**: Conducting more detailed assessments and working to
  lower the LOD.
* **Correlating fluorescence readout**: Further developing conditions to quantify fluorescence
  signals in relation to the amount of *Prymnesium parvum* DNA.
* **PrymChip testing**: Expanding its testing to SHERLOCK reagents and *Prymnesium parvum* DNA
  samples.
* **Field-ready DNA isolation**: Optimizing DNA extraction methods to enable the entire test to be
  conducted directly in the field.
* **One-pot SHERLOCK procedure**: Simplifying the process for a more efficient, single-step readout.
* **Optimizing culture conditions**: Establishing methods to maintain long-term liquid and solid
  cultures of *Prymnesium parvum*.

## PrymDetect in Numbers

---

Overall, we consider the PrymDetect project a **significant success**. What began as an initial idea
to apply SHERLOCK for detecting *Prymnesium parvum* expanded to include many unexpected aspects, such as the
development of the PrymChip or the mathematical modeling of SHERLOCK reaction kinetics. It’s important to
highlight that all idea development and planning were carried out by our student team members.

**29 000** SHERLOCK reactions possible using Cas13 we purified

**650** individual SHERLOCK reactions performed

**160** hours of 3D-printing

**112** LFA tests performed

**30** parts added to the Registry

**25** passages of algae cultures

All of this was carried out during… **304** days

By **10** undergraduate and graduate students fully planning
and executing the project!

## References

---

[1]L. Galluzzi *et al.*, “Detection and quantification of Prymnesium parvum (Haptophyceae) by real‐time
PCR,” *Letters in Applied Microbiology*, vol. 46, no. 2, pp. 261–266, Feb. 2008, doi:
10.1111/j.1472-765X.2007.02294.x.

[2]N. Luo, H. Huang, and H. Jiang, “Establishment of methods for rapid detection of Prymnesium parvum by
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