iGEM 2023 — Team stockholm

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{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/4658/wiki/headers/lab2.png)

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{% block header %}Throughout our experiments we have kept a detailed log of what was performed for each day. Bellow you can find the
lab notebooks for cloning and transformation, protein purification and assay development.
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##### Cloning and transformation

Read about what we did for each day of cloning
 [here.](https://static.igem.wiki/teams/4658/wiki/lab/cloning-lab-notebook.pdf)

##### Protein purification

Read about how we produced our fusion proteins
 [here.](https://static.igem.wiki/teams/4658/wiki/lab/protein-purification-lab-notebook.pdf)

##### Assay development

Read about how we developed our assay
 [here.](https://static.igem.wiki/teams/4658/wiki/lab/assay-development.pdf)

![](https://static.igem.wiki/teams/4658/wiki/project/ocean.png)

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{% block title %}Engineering Success{% endblock %}
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![](https://static.igem.wiki/teams/4658/wiki/header-bolder.png)

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{% block header %} Our project aimed to develop a method for detecting microplastics in water samples through the strategic design and utilisation of fusion proteins.
Two fusion proteins were meticulously crafted based on an anchor peptide from previous research, with one incorporating NLuc as a reporter molecule
and the other employing enhanced green fluorescent protein (eGFP) for simplicity in detection. Although initial attempts to validate the correct
insertion of the protein sequences using colony PCR proved challenging, the constructs were cloned successfully. Subsequent phases focused on
protein expression and purification and we were able to demonstrate specific binding to polypropylene surfaces, a crucial step
towards microplastic detection. Our results showcase an innovative approach to microplastic detection, highlighting the complexities and
achievements in fusion protein design, expression, and purification. {% endblock %}
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- Cycle 1
- Cycle 2
- Cycle 3

![](https://static.igem.wiki/teams/4658/wiki/project/design-circle.png)

Throughout our project we were constantly looking to improve our design. To achieve this, we followed the four stages of the engineering design cycle:
design, build, test and learn. By implementing and reflecting on this engineering philosophy, not only were we able to improve our project as we moved forward, but we
were also able to learn from the process. Here, we outline each cycle of our project and how we moved from the design stage to the learning stage.

Cycle 1: Protein design

### Design

To accomplish our goal, the detection of microplastics in water samples, we designed two different fusion proteins based on the anchor
peptide published by Rübsam et al. (2017). We chose pET45b as a vector plasmid because it could be provided in-house and because it contains a 6x His
tag for purification purposes. To be able to detect the bound anchor peptides, two different molecules were used. Our main construct is based on NanoLuc (NLuc), a
luciferase from the marine shrimp *Oplophorus gracilirostris*. The second construct is based on eGPF (enhanced green fluorescent protein). It is used
to simplify the proof of concept and as a control, given that an eGFP fusion protein has been successfully developed for microplastic detection by Ji et al. (2021).
The two constructs only differ in the reporter molecule.

To separate the anchor peptide and the eGFP or NLuc, respectively, a spacer sequence was designed. In their paper, Rübsam et al. used a sequence of ten alanine
residues as a spacer. We followed their lead and did the same but used different codons optimised for *E. coli*. Furthermore, we included a TEV cleavage site
to be able to cleave the fusion protein in anchor peptide and eGFP/NLuc later on. This has two advantages, firstly we can use the anchor peptide alone to build a
sandwich assay as described later and secondly, we would be able to use the separated eGFP or NLuc as a negative control since it should not bind to plastics.
For the sandwich binding assay, an Avi tag was included for biotinylation of the anchor peptide as described in later sections. An additional spacer of five
alanines was added between the Avi tag and the anchor peptide.

As part of our protein design we performed *in silico* analysis, visualising our folded fusion proteins in space. With structural prediction software, we were able to
observe our reporter molocules in relation to the anchor peptide and linker.

Discover more about our fusion proteins on our [experiments](https://2023.igem.wiki/stockholm/experiments)  or [model](https://2023.igem.wiki/stockholm/model) pages.

### Build

Both constructs were developed in house. The protein sequence was inserted into the vector plasmid using restriction enzymes (SacI and HindIII) and in-fusion cloning.

### Test

The ligation product was transformed into *E. coli* Top10 cells to select and amplify the plasmid. Several colonies were obtained. Colony PCR was performed and, after several attempts,
we were able to verify the sequence and cloning success through sequencing.

![](https://static.igem.wiki/teams/4658/wiki/project/luci-surf.png)

The predicted folding of our NLuc construct. In red you can see NLuc luciferase and in yellow the anchor peptide. These are joined by a linker in blue.

### Learn

The cloning process presented unanticipated challenges. Following multiple attempts, with changes made to the protocol and extra controls, we were able to identify the error in our
ordered sequence insert. We took corrective action by procuring the correct sequence, which had been meticulously tailored with appropriately designed overhang sequences for
in-fusion cloning methodology. The in-fusion protocol allowed for rapid cloning with high accuracy.

![](https://static.igem.wiki/teams/4658/wiki/project/design-circle.png)

Cycle 2: Protein expression and purification

### Design

To express our protein we used *E.coli* BL21 and a lac operon induced by IPTG. The *lacI* gene was part of the vector backbone.
The start and stop codons were adapted according to the protein sequence and the desired tags on the plasmid. The 6xHis tag was included to
facilitate purification after protein expression and TEV cleavage respectively.

Discover more about our fusion proteins on our [experiments](https://2023.igem.wiki/stockholm/experiments)  or [results](https://2023.igem.wiki/stockholm/results) pages.

### Build

Different incubation temperatures were chosen to find the optimum for protein expression of the fusion proteins. Purification methods were chosen because of their availability (batch vs. HPLC).
In order to determine the protein concentration with the photometer, the extinction coefficient was calculated using the ExPASy ProtParam web tool based on the protein sequence.

### Test

The purified plasmid (after cloning) was transformed into *E. coli* BL21 and protein expression was induced with IPTG. To confirm protein expression, SDS-PAGE and Western blot were performed.
Sonication was used to lyse the cell, followed by batch purification or HPLC (ÄKTA) using a HisTrap column. Purification success was confirmed by SDS-PAGE and protein concentration was determined
with the photometer. TEV cleavage was performed using in-house produced enzymes and buffers followed by purification of the cleaved anchor peptide. Its success was investigated using SDS-PAGE.

### Learn

Both fusion proteins were successfully expressed and purified. However, the TEV cleavage did not work as expected and due to logistical constraints we were not able to trouble shoot this in time.

![](https://static.igem.wiki/teams/4658/wiki/project/design-circle.png)

Cycle 3: Assay development

3.1 Proof of concept

### Design

Specific binding with a polypropylene surface was tested by using our fusion proteins and the separated reporter molecule, eGFP and NLuc respectively, as a negative control.
The fusion proteins were designed to be cleaved at their TEV cleavage site to separate the anchor peptide from the linker connected to the reporter molecule. Without the anchor peptide,
it would not be able to bind polypropylene.

Discover more about our assay development on our [results](https://2023.igem.wiki/stockholm/results)  or [proof of concept](https://2023.igem.wiki/stockholm/proof-of-concept) pages.

### Build

To validate the binding of the anchor peptide to polypropylene we designed a binding assay to be analysed with a plate reader. Both constructs can be analysed with the plate reader due to its ability to measure luminescence,
as well as fluorescence intensity and absorbance. The TEV cleavage product, including the separated linker and eGFP/NLuc, could serve as a negative control to normalise for non-specific binding. The assay was designed to test
different concentrations of our fusion proteins to validate if it could be used for quantification purposes. The same assay was performed with polypropylene and polystyrene plates to confirm binding specificity to polypropylene.

![](https://static.igem.wiki/teams/4658/wiki/project/post3.png)

### Test

Both constructs were tested using 96 well plates made of polypropylene and polystyrene respectively. The signal was measured using the plate reader with programs measuring fluorescence intensity and luminescence to optimise detection.
The assay was repeated several times to eliminate errors. R Studio was used to analyse the data and to display the results in graphical form. In the figure above you can see the post-wash luminescence for different concentrations of our
NLuc fusion protein. The increase in luminescence following washing steps would suggest that our protein binds successfully.

### Learn

Given that the TEV cleavage was not successful, our assay lacked a clear negative control. To confirm whether it is the anchor peptide that is responsible for the binding to polypropolene, further optimisation would be needed. Nevertheless,
by performing an innitial binding test with our NLuc fusion protein on polystyrene plates, we were able to confirm the specific binding of our fusion protein to polypropylene.

3.2 Microplastic binding

### Design

The construct was designed to bind to polypropylene surfaces and with this we were able to design an assay for the detection of microplastics in water samples. To remove any unbound fusion
proteins we designed a sandwich assay. The constructs contain a TEV cleavage site to separate the anchor peptide from the reporter molecule connected to it and an Avi tag to enable biotinylation of the
anchor peptide to a magnetic bead.

Discover more about our assay design on our [project description](https://2023.igem.wiki/stockholm/description) page.

### Build

To build the sandwich assay, the anchor peptide had to be cleaved off and biotinylated using the enzyme BirA. Due to problems
from the manufacturers side we had to produce the enzyme in-house. Streptavidin coated magnetic beads were used to separate bound
protein from unbound protein. The signal could be detected using the plate reader and the fluorescence microscope.

### Learn

We were not able to test the assay due to time constraints and logistic problems. Nevertheless we learnt a lot about assay design by speaking to
experts in the field.

Learn more about the experts we reached out to on our [human practices](https://2023.igem.wiki/stockholm/human-practices) page.

![](https://static.igem.wiki/teams/4658/wiki/project/sandwich.png)

Schematic overview of the sandwich assay principle. The separated and biotinylated anchor peptide (orange, biotin in green) is
immobilised on streptavidin coated magnetic beads (grey) and binds to the microplastic surface (light blue). The fusion protein binds
to the microplastic particle as well, creating the sandwich. Washing removes all unbound proteins and NLuc (dark blue) gives a detectable
luminescent signal after adding its substrate.

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![](https://static.igem.wiki/teams/4658/wiki/headers/lab2.png)

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{% block header %}We were fortunate enough to have two full months to optimise our protocols and design our experiments. This provided us with a solid foundation on which to base our proof-of-concept. Below, you can find the experiments we performed, from cloning through to bioluminescence detection, and their corresponding protocols.
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- Experiments
- Protocols

[Construct design and cloning](#construct-design-and-cloning)
[Protein expression and purification](#protein expression-and-purification)
[Assay development](#assay-development)
[References](#references)

### Construct design and cloning

Two fusion proteins of an anchor peptide with the ability to bind polypropylene (PP)
together with enhanced green fluorescent protein (eGFP) and NanoLuc luciferase (NLuc)
respectively were created to ease detection and quantification of anchor peptides bound to PP.
A spacer sequence of 10 alanines, a TEV protease cleavage site, an Avi tag and another
spacer sequence of 5 alanines separates eGFP or NLuc from the anchor peptide sterically.
The TEV-protease cleavage site allows for the isolation and purification of the anchor
peptide (without eGFP or NLuc) for the sandwich bead assay as described below. The plasmid
pET45b served as vector backbone for the two different fusion constructs. The sequences for
the anchor peptide and the spacer sequence were obtained from Rübsam et al. (2017) and were
later codon optimised for *E. coli*. The insert was cloned into pET45b using restriction
enzymes, HindIII and SacI, and restriction cloning. The full construct was transformed into
*E. coli* Top10 cells and the success of the cloning was verified by sequencing.

![](https://static.igem.wiki/teams/4658/wiki/lab/pet45b-nluc-cloned.png)
See our eGFP construct

### Protein expression and purification

The fusion proteins were expressed in *E. coli* BL21 cells and sonication was used for cell lysis.
Purification was performed using chromatography with batch purification and Äkta protocols. The fusion proteins were stored in a 1x PBS buffer
at 4°C until further usage. Protein concentrations were determined with photometer (280 nm) and
protein homogeneity was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDSPAGE). Western blot was performed to confirm the presence of the target protein. One third of
the purified proteins were digested with TEV protease to separate the anchor peptide from the eGFP
and NLuc respectively. Chromatography was used to purify the anchor peptide. The cleaved eGFP and NLuc
were kept to serve as a negative control for binding. The AviTag in our construct designs was included to enable targeted biotinylation
to the magnetic beads. We produced the BirA enzyme from glycerol stocks to facilitate biotinylation. However, due to time
constraints we were not able to perform the biotinylation itself.

### Assay development

The eGFP and NLuc fusion proteins were tested on polypropolene surfaces to confirm slective binding to polypropolene.
For eGFP, this involved the incubation of our eGFP fusion protein in a black polypropolene 96 well plate. The fluorescence intensity
was measured with a plate reader prior to and post washing with 1x PBS buffer. For NLuc, we used the Promega Nano-Glo® Luciferase Assay.
Our NLuc fusion protein was incubated in a black polypropolene 96 well plate. Follwing the addition of the NLuc substrate and washing 1x PBS, luminescence was
measured. The same assay was performed on a polystyrene plate and the differences in luminescence intensity were observed.

![](https://static.igem.wiki/teams/4658/wiki/lab/lumi.png)

Figure showing our NLuc binding assay in a polypropolene plate. Imaged with a smart phone camera.

### References

Rübsam, K., Stomps, B., Böker, A., Jakob, F., & Schwaneberg, U. (2017). Anchor peptides: A green and versatile method for polypropylene functionalization. Polymer, 116, 124-132.

![](https://static.igem.wiki/teams/4658/wiki/lab/long-jellyfish.png)

Restriction digestion

Restriction digestion process is used to precisely cleave a DNA fragment at a defined location using restriction enzymes.
Upon cleavage at the recognition sites, there is the formation of 'sticky ends' which aids in downstream ligation.
The use of two different restriction enzymes at the respective ends of insert and plasmid
backbone ensure that the insert is introduced into the vector in the desired orientation.

---

###### Materials

* Nuclease-free water (MiliQ)
* 10x FastDigest Buffer
* FastDigest Enzymes Hind III HF and Sac I HF
* Plasmid DNA pET45b

###### Procedure

1. Thaw enzymes and FastDigest Buffer on ice.
2. Add the following reagents in the following order:

| Component | Amount |
| --- | --- |
| Water, Nuclease-Free | Adjust to a final volume of 20 μl |
| 10x FastDigest Buffer | 2 μl |
| Plasmid DNA 1 μg | (0.5 μl to 10 μl depending onthe DNA concentration) |
| Hind III HF | 1 μl |
| Sac I HF | 1 μl |
| Total volume | 20 μl |

3. Mix gently and spin down.
4. Incubate at 37°C in a heat block for 1-2 h.
5. *Optional,* inactivate the enzymes by incubating for 20 min at 80°C.

Gel electrophoresis

To purify, separate and identify DNA fragments, agarose gel electrophoresis is performed.

---

###### Materials

* Tris base
* Acetic acid
* EDTA
* dH2O
* Agarose
* GelRed
* 1x loading dye
* DNA ladder

###### Procedure

1. For 50x TAE Buffer, mix the following:

| Component | Amount |
| --- | --- |
| Tris base | 242 g (in water) |
| Acetic acid | 57.1 ml |
| EDTA | 100 ml |
| dH2O | Fill up to 1 l |

2. For 1x TAE buffer, add 40 ml of 50x TAE buffer to a measuring cylinder and dilute with dH2O to make it upto 2 L in a glass
   container.
3. Gel preparation:

| Component | Amount |
| --- | --- |
| 1% Agarose | 1 g / 2 g |
| 1x TAE buffer | 100 ml / 200 ml |

4. Mix the agarose with buffer in a conical flask and heat till the agarose melts and the solution turns transparent.
   Be careful not to burn yourself as the flask may get too hot. OBS! boiling delay
5. Add 10 μl / 20 μl of GelRed into this agarose solution and swirl lightly.
6. Cast the agarose gel on a cast and place the comb on the top part of the gel. Let the gel solidify at room
   temperature for 20-30 min. Then place the gel onto the electrophoresis apparatus ensuring that it is totally
   submerged in 1x TAE buffer.
7. Meanwhile, prepare your DNA samples by adding 1X loading dye solution to the samples (1:1). For example: 1 μl
   of loading dye into 1 μl of DNA sample.
8. Carefully load each sample into its designated wells. To determine the DNA fragment sizes on the gel, load a
   DNA Ladder into one of the wells (5 μl).
9. Run an electric current of 180 V for 30 minutes.
10. Visualize gel using a gel reader or under UV light.

Gel extraction and PCR clean-up

To extract DNA fragments from the agarose gel, visible bands are cut out on an UV transilluminator.
The QIAGEN Gel extraction and PCR Clean-up Kit is used to extract the DNA according to manufacturer's specifications.

---

###### Materials

* QIAGEN Gel extraction and PCR clean-up kit
* Isopropanol

###### Procedure

1. Excise the DNA fragment from the agarose gel with a scalpel (Make sure to wear protective gear while working in UV)
2. Weigh the empty tube.
3. Weigh the gel slice in a tube.
4. Add 3 volumes of QG Buffer to the gel slice tube - 100 mg ~ 100 μl
5. Incubate at 50°C for 10 mins until the gel has completely dissolved. Vortex every 2-3 mins to dissolve the gel.
   The colour of the solution should be yellow.
6. Add 1 gel volume Isopropanol to this mixture and mix.
7. Place a QIAguick spin column in a 2 ml collection tube. Add the sample mixture to this column and centrifuge
   for 1 minute (13000rpm).
8. Discard the flowthrough and place the QIAguick column back in the same tube. (Sample volume should be less than 800 μl)
9. Keep repeating the previous step until all of the sample has been used.
10. Add 500 μl of QG buffer to the column and centrifuge for 1 minute. Discard the flow-through and place the
    column back in the same tube.
11. Washing - add 750 μl of PE buffer to the column. Let the column stand for 2-5 minutes.
12. Centrifuge for 1 minute and discard the flowthrough and place the column back into the same tube.
13. Centrifuge the column containing the tube once again for 1 minute to remove the residual wash buffer.
14. Place the column in a 1.5 μl microcentrifuge tube and add 30 μl EB Buffer to the centre of the column.
15. Let the column stand for 4 minutes and centrifuge for 1 minute.
16. Measure the DNA concentration using NanoDrop or Qubit.

Sticky end ligation

To combine the vector DNA with the insert DNA, T4 DNA ligase is used to link the fragments. The T4 ligase connects the
corresponding sticky ends to create the new plasmid. For the ligation, 100 ng of vector DNA were used, the mass of insert was
calculated using the following formula (e.g. using NEBcalculator): minsert = bpinsert/bpvector ∙ mvector∙ 5/1

---

###### Materials

* Linear vector DNA (digested)
* Insert DNA (digested)
* 10x T4 DNA Ligase buffer
* T4 DNA Ligase
* Nuclease free water

###### Procedure

1. Prepare the following reaction mixture:

| Component | Amount |
| --- | --- |
| Linear vector DNA | 20-100 ng |
| Insert DNA | 1:1 to 5:1 molar ratio (insert:vector) |
| 10x T4 DNA ligase buffer | 2 μl |
| T4 DNA ligase | 1 μl |
| Nuclease free water | to 20 μl |
| Total volume | 20 μl |

2. Incubate for at least 10-20 minutes at 22 °C (room temperature).
3. Use 8 μl of the mixture for transformation of 10 μl of competent cells.

Transformation Top10

We used two *E. coli* strains for our experiments: Top10 and BL21.
This protocol outlines how Top10 was transformed with the plasmid of interest for amplification.
The protocol for BL21 can be seen below.

---

###### Materials

* 5x KCM stock (0.5 M KCl, 0.15 M CaCl2, 0.25 M MgCl2)
* Ligation mix
* 1 aliquot competent cells
* TSB media

###### Procedure

1. Mix 8 μl Ligation mix and 2 μl 5x KCM in a sterile Eppendorf tube.
2. Chill on ice for 2-5 minutes.
3. Add 10 μl competent cells.
4. Incubate on ice for 20 minutes.
5. Incubate at room temperature for 10 minutes.
6. Heat shock: 60 sec at 42°C, then incubate for 2 min on ice.
7. Add 200 μl preheated TSB media (37°C), phenotype on rotamixer 150 rpm at 37 ̊C for 1 hour.
8. Centrifuge for 3 min 3000 rpm, discard the supernatant and resuspend the pellet in 200 μL TSB.
9. Plate the 200 μl on agar plate with appropriate antibiotic.
10. Incubate at 37 ̊C overnight or at room temperature over the weekend.

Miniprep

To purify the amplified plasmid of interest, miniprep is performed. The protocol is adapted from the manufacturers.

---

###### Materials

* QIAGEN Miniprep kit

###### Procedure

1. Pellet 1-5 ml bacterial overnight culture by centrifugation at 4600 rpm for 5 min.
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a 1.5 mL Eppendorf tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do
   not allow the lysis reaction to proceed formore than 5 min.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 60 s and
   discard the flow-through.
7. Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60 s and discard the flow-
   through.
8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 60 s and discard the flow-
   through.
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 μl Buffer EB (10
    mM TrisCl, pH 8.5) to the center of the QIAprep 2.0 spin column, let it stand for 4 min, and centrifuge for
    1 min.
11. Load the flowthrough on the column and incubate for 4 min before centrifugate for 1 min.
12. Determine concentration with NanoDrop.
13. Send it for sequencing.

Transformation BL21

The *E. coli* strain BL21 was transformed for the expression of the protein of interest.

---

###### Materials

* 5x KCM stock (0.5 M KCl, 0.15 M CaCl2, 0.25 M MgCl2)
* Ligation mix
* 1 aliquot competent cells
* TSB media

###### Procedure

1. Heat TSB to 37°C and let cell aliquots thaw on ice.
2. Gently mix the cell aliquots and pipettes 50 μL of cells to each tube.
3. Add 10 μl of KCM to each tube.
4. Incubate the cells on ice for 10 mins.
5. Add 2-5 μL of plasmid and mix gently.
6. Incubate on ice for 30 mins.
7. Heat Shock: Heat-pulse for 15 sec, 42°C.
8. Incubate on ice for 2 mins.
9. Add 100 μl of per-heated TSB to each tube.
10. Incubate the cells in shaking incubator for 1 h at 37°C, 200 rpm and preheated plates with relevant antibiotic in
    the incubator.
11. Mix and plate it on the agar plate and spread it evenly.
12. Incubate overnight (12-17 h) at 37°C.

Protein expression

The target protein is expressed in *E. coli* BL21 cells.

---

###### Materials

* Transformed *E. coli* BL21
* TBS media supplemented with ampicillin
* IPTG (0.5 M)
* Lysis buffer

###### Procedure

###### *Pre-culture*

- Pick a colony and inoculate in 10 mL of TSB Media with 10 μL Amp in a 50 mL falcon tube overnight at 37°C in
  a shaking incubator.

###### *Main culture*

- Centrifuge cell culture at 4600 rpm for 10 min. Discard supernatant and resuspend pellet in 10 mL TSB + 10 μl Amp
  (1:1000).
- Inoculate 200 mL TSB + 200 ul Amp (1:1000) with 4 mL cell suspension (1:50).
- Incubation for 2-4 h at 37°C shaking (220 rpm) until OD600 is 0.3-0.6 (then take a sample - 1mL - to be
  processed!)
- ((Glycerol stock: 900 μl of the preculture and 300 μl of 100% glycerol store at -80°C))
- Add IPTG (final concentration:1 mM)
- Incubate 3 h (37°C, 220 rpm) or overnight at 25°C.
- Take a sample (1 mL - to be processed)
- Transfer the rest of the sample to GSA tubes or fresh falcon tubes. Centrifuge at 4600 rpm (2800 g) and 4°C for 15 min.
- (Discard supernatant and store pellet at -80°C or) continue with cell lysis

###### *Cell lysis by sonication*

- Resuspend the pellet in 10 mL (batch purification) equilibration buffer and transfer to SS34 tubes (50 mL)
- Disrupted by sonication on ice, repeat twice:
  1. First round 1:30 min, interval 1 s, 23% amplitude
  2. Second round 3:00 min, interval 1 s, 23% amplitude
- Make sure that the sample is not over-heated. To ensure proper lysis, sonicate all samples
  once (storing them on ice at all times to cool before and after sonication), and then sonicate each sample
  again. Wash sonicator with EtOH in between each sample-run. Clean sonicator gently with fine sand paper
  afterwards.
- Spin at 25 000xg, 15 min, 4°C (keep the supernatant!)
- Filter the supernatant through a 0.45μm filter.
- Take a 30 μl aliquot of the filtered supernatant for SDS-gel analysis, and store at -20°C. Also dip
  a tip in the pellet and dissolve in 30 μL MilliQ, as a sample for SDS-PAGE of the pellet fraction
  post-lysis.
- Run SDS-PAGE and western blot to confirm expression.

Batch purification

Batch purification of our His6-tagged proteins was carried out. This is protocol is for 200 ml cultures and
should be scaled for other volumes.

---

###### Materials

* Pellet of 100-mL production, in SS34 tube
* Equilibration buffer (optional)
  + 20 mM Tris-HCl, 300 mM NaCl, 10 mM Imidazole, pH 8.0. Filtered 0.2 μm.
  + 40-60 ml per construct (20ml if washing once for equilibration, 60ml if twice)
* Wash buffer (it can be used also for equilibration)
  + 20 mM Tris-HCl, 300 mM NaCl, 30 mM Imidazole, pH 8.0. Filtered 0.2 μm.
  + 60 ml per construct
* Elution buffer
  + 20 mM Tris-HCl, 300 mM NaCl, 500 mM Imidazole, pH 8.0. Filtered 0.2 μm.
  + 2.5 ml per construct
* PD10 columns
* Goal buffer: ca. 30ml per construct

###### Procedure

###### *Buffer preparation:*

- Equilibration buffer:

| Component | Concentration (M) | Mw (g/mol) | Volume (l) | Mass (g) |
| --- | --- | --- | --- | --- |
| Tris-HCl | 0.02 | 157.60 | 0.5 | 1.576 |
| NaCl | 0.3 | 58.44 | 0.5 | 8.766 |
| Imidazole | 0.01 | 68.08 | 0.5 | 0.3404 |

- Wash buffer:

| Component | Concentration (M) | Mw (g/mol) | Volume (l) | Mass (g) |
| --- | --- | --- | --- | --- |
| Tris-HCl | 0.02 | 157.60 | 1 | 3.152 |
| NaCl | 0.3 | 58.44 | 1 | 17.532 |
| Imidazole | 0.03 | 68.08 | 1 | 2.0424 |

- Elution buffer:

| Component | Concentration (M) | Mw (g/mol) | Volume (l) | Mass (g) |
| --- | --- | --- | --- | --- |
| Tris-HCl | 0.02 | 157.60 | 0.5 | 1.576 |
| NaCl | 0.3 | 58.44 | 0.5 | 8.766 |
| Imidazole | 0.5 | 68.08 | 0.5 | 17.2 |

###### *Equilibration of cobalt resin*

- Thoroughly resuspend the Cobalt Resin.
- Leave at room temperature to equilibrate for 15 minutes, followed by thorough resuspension, to ensure a
  homogeneous solution. This keeps the ratio of resin to slurry at 1:2.
- Immediately transfer 4 ml slurry to a 50 ml Falcon tube. Centrifuge at 1000 x g for 1 min and remove
  supernatant. This corresponds to a CV of 2 mL resin. Supernatant removal is best done by decisive decantation
  (i.e. quick turning of the tube, with one extra flick).
- Wash with 10 CV equilibration Buffer (or washing buffer)

###### *Sample application*

- Resuspend the Cobalt Resin in the pulp free *E. coli* juice.
- Gently agitate in cold room for 30 min on a rotamixer to allow the his-tagged protein to bind the resin.
- Centrifuge at 1000 x g for 1 min.
- Carefully remove as much supernatant as possible without disturbing the resin pellet. Note: take a 30 uL sample as flow-through for SDS-PAGE analysis.

###### *Washing*

- Wash the resin by adding 10 CV of Wash Buffer. Gently agitate the suspension in cold room for 5 min on a
  rotamixer for thorough washing.
- Centrifuge at 1000 x g for 1 min. Remove and discard the supernatant. Take a 30 uL sample of the supernatant and save for SDS-PAGE analysis.
- Repeat 12-13 for a total of three washes.

###### *Elution*

- Elute the his-tagged protein by adding 2.5 ml elution buffer to the resin and incubate for 10 min in the cold
  room.
- Centrifuge at 1000 x g for 1 min. Add the 2.5 ml supernatant to PD10 for buffer exchange.

###### *PD10 column buffer exchange*

- Remove the bottom and cap off new columns.
- Let the buffer flow through.
- Equilibrate the column with 25 ml of the goal buffer.
- Add exactly 2.5 ml of the sample. Do not collect the first 2.5 ml flow through as the sample enters the column.
- When the sample has completely entered the column, switch to 15 ml collection tubes at the bottom of the
  column.
- Add exactly 3.5 ml goal buffer, collect the flow through, which contains the sample exchanged into the new
  buffer.
- Measure A280 and run SDS-PAGE analysis.

Äkta purification

Protocol for purification of His6-tagged constructs.

---

###### Materials

* 20ml of clarified cell lysate
* Wash buffer
  + 20 mM Tris-HCl, 300 mM NaCl, 30 mM Imidazole, pH 8.0. Filtered 0.2 μm.
* Elution buffer
  + 20 mM Tris-HCl, 300 mM NaCl, 500 mM Imidazole, pH 8.0. Filtered 0.2 μm.
* MilliQ
* 70% ethanol
* ÄKTA:Start
* Frac30 Fraction Collector
* Cytiva HisTrap FF Crude (1ml) (or other appropriate ÄKTA column)

###### Procedure

###### *Buffer preparation:*

- Wash buffer:

  Concentration of imidazole in wash buffer must be empirically determined from construct to construct to ensure high purity and high yield.

| Component | Concentration (M) | Mw (g/mol) | Volume (l) | Mass (g) |
| --- | --- | --- | --- | --- |
| Tris-HCl | 0.02 | 157.60 | 1 | 3.152 |
| NaCl | 0.3 | 58.44 | 1 | 17.532 |
| Imidazole | 0.03 | 68.08 | 1 | 2.0424 |

- Elution buffer:

| Component | Concentration (M) | Mw (g/mol) | Volume (l) | Mass (g) |
| --- | --- | --- | --- | --- |
| Tris-HCl | 0.02 | 157.60 | 0.5 | 1.576 |
| NaCl | 0.3 | 58.44 | 0.5 | 8.766 |
| Imidazole | 0.5 | 68.08 | 0.5 | 17.2 |

###### *Programme set-up and column connection*

- Power on the ÄKTA:start system and open the UNICORN software on the connected computer.
- Connect the UNICORN software to the ÄKTA:start.
- Place the wash buffer, elution buffer, MilliQ water, and 70% Ethanol on top of the machine and immerse inlet tubes A and B into the Ethanol flask.
- Place a waste collection flask next to the machine and place all waste outlet tubes into it.
- Initiate a manual run with a slow flow rate (0.5ml/min).
- Disconnect the G5 connector from the coupling, and connect the tubing to the HisTrap column in a drop-to-drop manner, ensuring no airbubbles are introduced.
- Disconnect the G6 connector from the coupling and connect the HisTrap column in the same drop-to-drop manner to it, ensuring no airbubbles are introduced.
- End the run.

###### *Equilibriation of the ÄKTA:Start*

- Move inlet tubes A and B from the ethanol flask to the MilliQ water flask.
- Initiate a manual run with a flow rate of 2ml/min in the software.
- Wash all tubing with 10CV of MilliQ by changing the flow path to include all waste tubes as well as the sample inlet and coupling tubes.
- End the run once all tubing has been washed.
- Move the inlet tubings A and B into the wash buffer and elution buffer respectively.
- Repeat steps 14-16 with the wash buffer only.

###### *Set-up before starting method*

- Create a purification method in UNICORN
- Insert the sample inlet tube into the clarified lysate.
- Move the fractionation tubing and insert it into the holder on the Frac30 system.
- Insert sufficient eppendorf tubes into the Frac30 system.

###### *Running the method*

- Run the method keeping a close eye on the system readings (UV, Conductivity, Pressure).
- Once the method has run, save the chromatogram and equilibriate the entire system as in steps 14-16 with MilliQ water, then with 70% ethanol.
- Buffer exchange can be done on the ÄKTA system as well, though the volume is limited to 1.5ml of sample. Therefore it is recommended to do a buffer exchange using PD10 Columns instead.

SDS-PAGE

To seperate proteins by size via electrophoresis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was used.
Molecular weight standard (SM) was applied in parallel to the samples to determine the protein size of the unknown sample.

---

###### Materials

* Protein sample
* Precast SDS-PAGE gel
* PageRuler Prestained Protein Ladder
* 3X sample loading buffer
* 1X SDS running buffer (1x MES)
* Gelcode Blue Safe Stain

###### Procedure

###### *Running the gel*

- Add 3X sample loading buffer in a ratio of 1:3 to the protein sample (typically load around 2 μg of protein per
  sample). The final volume is dependent on the well size of the gel.
- Incubate samples for 5 minutes in a heating block at 95°C.
- Centrifuge samples at 13,000 rpm for 60 seconds.
- Remove the tape at the bottom of the precast gel cassette and assemble the cassette into the SDS-PAGE
  chamber.
- Remove the comb from the gel.
- Add running buffer to the inner and outer chambers.
- Load the samples (volume depending on well size) and protein ladder (5 μL) into the wells.
- Run the gel at 200V for 25 to 35 minutes in a cold room at 4°C, until the dye front reaches the reference line.
- Remove cassette from the chamber and remove the gel from the cassette using opening levers. Place the gel
  directly into a staining container with dH2O (see below).

###### *Staining the gel*

- Place the gel in a staining container and add dH2O.
- Incubate for 3x 5 min on an orbital shaker, to wash the gel. Exchange dH2O in between the incubations.
- Remove the dH2O and add Gelcode Blue Safe Stain solution until it covers the gel.
- Incubate for 1 hour on an orbital shaker.
- Destain the gel in dH2O on an orbital shaker over night.
- Next day, take a picture of your gel using the imager.

Western blot

For use on the gel obtained by SDS-PAGE.

---

###### Materials

* Prepackaged turbo transblot PDVF membrane
* Blocking buffer (PBS buffer with 5% milk poweder and 0.5% v/v Tween 20)
* PBS-Tween (PBS buffer with 0.1% v/v Tween 20)
* Anti-His-tag Antibody HRP conjugated

###### Procedure

1. *Note: to be started directly after removinf gel from the chamber.*
2. Transfer gel to a turbo transblot PDVF membrane with prepackaged westernblot membrane for
   tranturbo system
3. Transfer of proteins from gel to membrane will be carried out via electroblotting for 10 min at 1.3A and 25V
4. Post transfer take the membrane and put it in a 50 mL falcon tube.
5. Add 5 ml blocking buffer to your tube.
6. Place the tube on a rocking table for 1h.
7. Wash the membrane by filling the tube with 20 mL PBS-Tween and put it
   on a rocking table for 5 min. Empty the tube of buffer.
8. Repeat the washing two times (a total of three washes).
9. Add Anti-His-tag Antibody HRP conjugate diluted 1:2500 in 5ml blocking buffer to the membrane. Incubate on a
   rocking table for 1 h.
10. Wash the membrane as described above 3 times.
11. Put the membrane in a plastic box with PBS-Tween for 5 min.
12. Pour out the buffer.
13. Dip the membrane in HRP substrate.
14. Put the membrane in a plastic folder; mark the bands on the protein marker on the plastic film with a marker
    pen.
15. Run the camera for blot development.

Analysis of Protein Binding

Protocol for analysis of NLuc-anchor peptide construct binding to 96-well plates.
Different plate colours were used due to accessibility reasons.

---

###### Materials

* Greiner Bio-One Polystyrene 96-well F-Bottom Microplates (White)
* Greiner Bio-One Polypropylene 96-well F-Bottom Microplates (Black)
* CLARIOstar Plus Microplate Reader
* Promega NanoGlo Substrate
* Purified, buffer exchanged NLuc-anchor peptide construct
* 1x PBS

###### Procedure

1. Make a dilution series using the NLuc construct to get 7 desired concentrations:- 16µg/µl, 8µg/µl, 4µg/µl, 2µg/µl, 1µg/µl, 0.5µg/µl, and 0.25µg/µl were used in our testing.
2. Mix the NanoGlo buffer with the NanoGlo substrate at a 50:1 ratio:- Prepare 2x the volume required and an extra 1ml to ensure enough substrate for subsequent steps.
3. Add 50µl of 1x PBS into the blank wells
4. Add 50µl of the protein sample to the bottom of the sample wells.
5. Add a subsequent 50µl of the NanoGlo substrate mix into the bottom of all the used wells (both blank and sample).
6. Incubate the plates at room temperature without shaking for 5 minutes.
7. Measure the luminescence using the plate reader and save the data for analysis.
8. Discard all the liquid in the wells by quickly and decisively invert the well plate, over a sink/waste container, with force.
9. Wash the used wells with 200µl of 1x PBS and wait 10-30 seconds and once again emptying the plate as in step 7.
10. Repeat steps 7-8 2 more times.
11. Add 50µl of 1x PBS into the bottom of all the used wells.
12. Add a subsequent 50µl of the NanoGlo substrate mix into the bottom of all the used wells.
13. Measure the Luminescense using the plate reader and save the data for analysis.

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{% block title %}Results{% endblock %}
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![](https://static.igem.wiki/teams/4658/wiki/header-bolder.png)

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{% block header %}The detection and quantification of microplastics is crucial for understanding their impact and developing effective mitigation strategies.
To overcome this problem of detection and quantification of microplastics in water bodies, we devised an assay where an anchor peptide, which can easily attach
to a plastic surface, along with luciferase helps with the detection of the microplastic by bioluminesce. The assay essentially consists of three parts. First,
is the biotinylated anchor peptide which is attached to a streptavidin bead. Second, is the microplastic which gets captured by the anchor peptide and third, is
the luciferase which is attached to the anchor peptide in our fusion protein. The oxidation of furimazine by our luciferase gives off a luminescent signal that
can be detected with smartphone technology. Below we describe the results we obtained from the development of our assy.
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{% block page\_content %}

[Cloning and transformation](#cloning-and-transformation)
[Protein expression and purification](#protein expression-and-purification)
[Assay development](#assay-development)
[References](#references)

### Cloning and transformation

We went forward with the cloning of two insert constructs (Fig. 1), consisting of an anchor peptide fused with NanoLuc luciferase (NLuc) and another anchor peptide fused with eGFP.
The eGFP construct was included as a control for the design of our NLuc construct.
Our inspriration was taken from the eGFP construct developed by Ji et al. which has been conifirmed to bind polypropolene for microplastic detection. (Ji et al., 2021)
The cloning of our inserts into the pET45b expression vector was initially met with challenges. In the early attempts, transformation of the gene of
interest into Top10 *E. coli* cells resulted in either the absence of colonies or an excessive number of colonies in
the negative control. These unexpected outcomes prompted us to critically evaluate and revise our cloning and transformation
protocols.

![](https://static.igem.wiki/teams/4658/wiki/project/inserts.png)

Figure 1: The sequence inserts for our protein constructs visualised in snapgene. A) shows our NLuc construct of 828 bp.
B) shows our eGFP construct of 1032 bp. In both you can see the details of the polypropolene binding anchor peptide and the linker
sequence.

One of the key troubleshooting steps involved the analysis of restriction digestion. However, our initial efforts to verify
successful restriction digestion using HindIII HF and SacI HF enzymes were unsuccessful. Despite conducting several gel
electrophoresis experiments, we could not obtain conclusive evidence of successful restriction digestion. After weeks of careful
consideration and brainstorming, we identified critical errors in the construct that had been ordered for this cloning project.
These errors necessitated a revision of the construct, correcting the issues that had been identified.

With the revised construct in hand, we proceeded with the cloning process into the pET45b expression vector. This time, the
cloning procedure was successful, resulting in the generation of colonies. To confirm the presence of the desired insert,
colony PCR was performed, and subsequently, mini-prep was carried out on selected colonies. These purified plasmids were
then sent for sequencing to validate the accuracy of the insert.

Upon receipt of the verified plasmid sequences, the constructs were transformed into Top10 *E. coli* cells. This set of
transformed cells was employed for protein expression and subsequent purification, marking the successful completion of the
cloning process.

### Protein expression and purification

Once the sequencing results were validated, we started transformation in the BL21 cells which again gave good colonies as a
result and then followed by pre-culturing and making glycerol stocks. This was followed by the main culturing and with an overnight
IPTG induction for protein expression which was done at two different temperatures, 25°C and 37°C, to ensure and check which
temperature is the best for cells to grow and express the protein. 25°C had the best results in terms of growth and expression.
This was confirmed by doing cell lysis and performing an SDS page for the lysis product. The protein purification was done by
batch purification method, as well as using ÄKTA columns to get more concentrated purified protein without contamination through
other proteins (Fig. 2).

![](https://static.igem.wiki/teams/4658/wiki/project/pre-post5.png)

Figure 2: The SDSPAGE results obtained form batch purification for both the eGFP (A) and NLuc (B) fusion proteins. The gel
images confirm the successful purification of our protein.

### Assay development

The purified protein construct would then need to be thoroughly tested to ascertain binding specificity as well as functionality of
the luciferase domain. This was done by analysing the luminescence of bound proteins on a 96-well plate with the help of a plate reader.
A concentration gradient was tested alongside a blank containing no protein (Fig. 3).

![](https://static.igem.wiki/teams/4658/wiki/project/pre-post4.png)

Figure 3: The luminescence intensity was measured at different concentrations (µg/µl) pre-wash (A) and post-wash (B). Pre-wash luminescence
values are significantly higher than that of the post wash, with an unusual decrease in luminescence intensity for the pre-wash samples
at the two highest concentrations. Post-wash luminescent signal increases following a threshold protein concentration of 1µg/µl. This indicates the
successful binding of our fusion protein. However, the optimal protein concentration is yet to be determined.

Pre-wash luminescence values are observed to be significantly higher than that of the post wash as a result of the high concentration
of unbound protein. When analysing the post-wash samples independently of the pre-wash samples, it can be seen that binding does seem to
occur, as the luminescent signal remains (though substantially weaker than that of the pre-wash). What can also be seen is that the binding
capacity of the 96-well plate was not reached at a concentration of 8µg/µl, and significantly higher concentrations must be tested to
obtain a standard curve based on our fusion protein.

An initial test was done to ascertain the binding of the protein construct to polystyrene. No luminescent signal was detected post-wash,
supporting the specificity of our construct. However, due to time constraints, a full binding analysis with the appropriate controls, comparable
to the one done with polypropolene plates, was not carried out. These results would enable us confirm our binding specificity.

Read more about our [proof of concept](https://2023.igem.wiki/stockholm/proof-of-concept) here.

### References

Bauten, W., Nöth, M., Kurkina, T., Contreras, F., Ji, Y., Desmet, C., ... & Schwaneberg, U. (2023). Plastibodies for multiplexed detection and sorting of microplastic particles in high-throughput. Science of The Total Environment, 860, 160450.

Ji, Y., Lu, Y., Puetz, H., & Schwaneberg, U. (2021). Anchor peptides promote degradation of mixed plastics for recycling. In Methods in Enzymology (Vol. 648, pp. 271-292). Academic Press.

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