iGEM 2020 — Team Tuebingen

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# Team:Tuebingen/Notebook

PacMn

# Notebook

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[##### PacMn](https://2020.igem.org/Team%3ATuebingen)

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  [Notebook](/Team%3ATuebingen/Notebook)
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  Video](/Team%3ATuebingen/Promo_Video)

![Wetlab Overview](https://static.igem.org/mediawiki/2020/6/62/T--Tuebingen--Overview_wetlab.png)

##### Week 1 (27 Jul - 31 Jul)

**Objectives:**

* Moving to lab and organizing materials
* Resuspending of primers and DNA fragments
* PCR removal of extra base pairs (bp) of fragments **B** and **E**
  (**Table 1**)

**Resuspending primers and DNA fragments (Table 1):**

*(Lea, Florian)*

* Resuspensions performed with nuclease-free water (H2ONF)
* Primer stock concentration: Set to **100 µM**
* Primer working solution concentration: Set to **10 µM**
* Fragment stock concentration: Set to **10 ng/µl**

| Label | Fragment | Found in Part ID(s): |
| --- | --- | --- |
| A | Anderson Promoter - B12-Riboswitch - ssRA Tag | BBa\_K3510006, BBa\_K351007 |
| B | Tet-Inverter System | BBa\_K3510006, BBa\_K351007 |
| C | GFP - Terminator | BBa\_K3510006 |
| D | mRFP - Terminator | BBa\_K351007 |
| E | Mn Promoter - Mn Riboswitch | BBa\_K3510002, BBa\_K3510003 |
| F | FAST - Chromoprotein - Terminator | BBa\_K3510002, BBa\_K3510004 |
| G | FAST - Phytochelatine-Terminator | BBa\_K3510003, BBa\_K3510005 |
| H | Anderson Promoter - Mn Riboswitch | BBa\_K3510004, BBa\_K3510005 |
|  |  |  |
| Label | Primer | Sequence (5' --> 3') |
| --- | --- | --- |
| 1 | GFP-Terminator (for) | gtgatagagatactgagcacatgcgtaaaggcgaagagctg |
| 2 | GFP-Terminator (rev) | gggttttcccagtcacgacgttgtaaaacgacggccagtgaattctataaacgcagaaaggcccaccc |
| 3 | mRFP-Terminator (for) | gtgatagagatactgagcacatggcttcctccgaagacg |
| 4 | Tet-Inverter system with GFP (for) | cgctctggctgcttaataaaaagaggagaaatactagatgtccagattagataaaagtaaag |
| 5 | Tet-Inverter system with GFP-Terminator (rev) | gctcttcgcctttacgcatgtgctcagtatctctatcactgataggga |
| 6 | Tet-Inverter system with mRFP-Terminator (rev) | cgtcttcggaggaagccatgtgctcagtatctctatcactgataggg |
| 7 | Anderson Promoter - B12-Riboswitch - ssRA Tag (for) | cagctatgaccatgattacgccaagcttgcatgcctgcagttgacggctagctcagtcctagg |
| 8 | Anderson Promoter - B12-Riboswitch - ssRA Tag (rev) | ctagtatttctcctctttttattaagcagccagagcgtagttttcg |
| 9 | mRFP-Terminator (rev) | gggttttcccagtcacgacgttgtaaaacgacggccagtgaattctataaacgcagaaaggcccaccc |
| 10 | Tet-Inverter system with -mRFP-Terminator (for) | cgctctggctgcttaataaaaagaggagaaatactagatgtccagattagataaaag |
| 11 | Anderson Promoter-Mn Riboswitch (for) | gggttttcccagtcacgacgttgtaaaacgacggccagtgaattcttgacagctagctcagtcctaggtac |
| 12 | Anderson Promoter-Mn Riboswitch (rev) | ccaaaggcaacatgctccatgacaatgtcctgaccgggg |
| 13 | Mn Promoter-Mn Riboswitch (for) | gggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgtttcaactcaataagttatgaatttagccaaagctatg |
| 14 | Mn Promoter-Mn Riboswitch rev) | ccaaaggcaacatgctccatgacaatgtcctgaccgggg |
| 15 | FAST-Phytochelatine - Terminator (for) | ccccggtcaggacattgtcatggagcatgttgcctttggc |
| 16 | FAST-Phytochelatine-Terminator (rev) | caatttcacacaggaaacagctatgaccatgattacgccaagctttataaacgcagaaaggcccaccc |
| 17 | puc19 M13 fwd (colony PCR) | cccagtcacgacgttgtaaaacg |
| 18 | puc19 M13 rev (colony PCR) | agcggataacaatttcacacagg |
| 19 | Mn Promoter-Mn Riboswitch (for) (PCR IDT bp removal) | gtttcaactcaataagttatgaatttagccaaagct |
| 20 | Mn Promoter-Mn Riboswitch (rev) (PCR IDT bp removal) | gacaatgtcctgaccggggt |
| 21 | Tet-Inverter system (for) (PCR IDT bp removal) | aaagaggagaaatactagatgtccagattagataaaagtaaag |
| 22 | Tet-Inverter system (rev) (PCR IDT bp removal) | gtgctcagtatctctatcactgatagggat |

**PCR removal of extra base pairs (bps) of fragments B and E**:

*(Lea, Benedikt, Anirudh)*

To order fragments **B** and **E** (Table 1) from
Integrated DNA
Technologies (IDT), we had
to reduce their complexity score by adding additional bps to both ends. To get
rid
of these additions, we designed primers to exclude them during amplification via
PCR
(**Protocol 1**).

PCR was analyzed by gel electrophoresis (**Protocol 2**): Since the overhangs
of the
ordered fragments were only 10 bp for each fragment, this cannot determine
whether
the PCR successfully eliminated them. However, with bands at desired positions
(**Fig. 1**: ~1000 bp for fragment B / 500 bp for fragment E) it shows that
the PCR worked
in
general. The PCR products from this step were then called Bstock (Bs) and Estock
(Es).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/2/20/T--Tuebingen--Gel_Fig1.jpg)

**Figure 1: Gel
electrophoresis of IDT-overhang removal PCR.** Gel loaded (from left
to right): GeneRuler 1kb ladder - Fragment Bs - Fragment Es.

##### Week 2 (03 Aug - 07 Aug)

**Objectives:**

* Creation of Gibson overhangs for fragments E, F, and H

**Creation of Gibson overhangs for fragment E, F, and H:**:

*(Anirudh, Sophia)*

Creation of Gibson overhangs for fragments E, F, and H
was attempted with 25µl PCR reactions according to **Protocol 1** and the PCR
products were analyzed according to **Protocol 2**. Fragments H and E had a
gel band
at the correct size, but for fragment E, unpurified Es was used as a template,
so it was decided to repeat that PCR. Fragment H was directly purified and
eluted in 10 µl elution buffer (**Protocol 3**). Fragment F did not show a
band at
the desired size (**Fig. 2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/4/4e/T--Tuebingen--Gel_Fig2.png)

**Figure 2: Gel
electrophoresis of Gibson-Overhang PCR.** Gel loaded (from left to
right):
GeneRuler 1kb ladder - Fragment E - Fragment F - Fragment H.

##### Week 3 (10 Aug - 14 Aug)

**Objectives:**

* Creation of Gibson overhangs for fragment F:

+ Increase in extension time + variation of template DNA
+ Gradient PCR

* Ampicillin working solutions

**Creation of Gibson overhangs for fragment F:**

*(Andreas, Luise)*

PCR for creation of Gibson overhangs for fragment F was
repeated (**Protocol 1**) with some variations. The extension time was
prolonged to
30s following the kit’s suggestion of 15-30s/kb for fragments larger than 1 kb
(F = 1.3 kb). For template DNA, three different types were used:

* The PCR product of the first Gibson overhang PCR for F
* DNA of the IDT stock
* H2ONF as a negative control

Analysis of the PCR (**Protocol 2**) showed no desired
band (**Fig. 3**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/8/8d/T--Tuebingen--Gel_Fig3.png)

**Figure 3: Gel
electrophoresis of Gibson-Overhang PCR of fragment F.** Gel loaded
(from left
to right): Benchtop 1kb ladder - negative control - Fragment F (template IDT
Stock) - Fragment F (template F from previous PCR).

Additionally, a gradient PCR with the following
temperatures was conducted: 57.8 / 59.1 / 60.5 / 61.8 / 63.1 / 64.2 °C
The PCR (**Protocol 1**) was performed again with 30s extension time, but in
the
subsequent gel electrophoresis (**Protocol 2**) no DNA was visible at all,
except
for the ladders.

**Ampicillin working solutions and plates:** :

*(Andreas)*

* 500 µl aliquots of 100 mg/ml (w/v) working solutions were stored at -20 °C
* Selective agar plates with a final ampicillin concentration of 100 µg/mL
  were poured with approximately 25 ml each and stored at 8 °C
* Note: Due to rapid degradation of ampicillin, selective agar plates were
  always prepared fresh for new experiments

##### Week 4 (17 Aug - 21 Aug)

**Objectives:**

* Creation of Gibson overhangs for fragments A, B, C, and D
* Creation of Bs and Es
* Gradient PCRs for fragment F
* Different PCR template and primer parameters for fragment H
* Isolation of plasmid pUC19
* Creating a larger stock of pUC19

**Creation of Gibson overhangs for fragment A, B, C, and D:**

*(David, Aaron, Flo)*

Since B is contained in two different Gibson Assemblies,
two versions with different overhangs were required: B1 and B2. The PCRs were
done according to **Protocol 1** and analyzed according to **Protocol 2**.
The desired
specific bands were visible for A, B2, C, and D, but strong primer clouds were
also present (**Fig. 4 A**). These primer clouds were still visible after
purification (**Fig. 4 B**) (**Protocol 3**). B1 had the wrong size (below
500 bp
instead of 940 bp). In addition, both B1 and B2 were amplified with unpurified
Bs as template DNA, and were therefore repeated later.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/8/8e/T--Tuebingen--Gel_Fig4.png)

**Figure 4: A: Gel
electrophoresis of Gibson-Overhang PCR of fragment A, B1, B2, C, D.**
Gel
loaded (from left to right): Benchtop 1kb ladder - Fragment A - Fragment B1
- Fragment B2 - Fragment C - Fragment D
**B: Gel electrophoresis of purified Gibson-Overhang PCR of fragment A, C,
D,
and digested pUC19 DNA with EcoRI.** Gel loaded (from left to right):
Benchtop
1kb ladder - Fragment A - C - D - test digest culture 1 - culture 2 -
culture 3 - culture 4.

After amplification (**Protocol 1**) and purification
(**Protocol 3**) of new Bs and Es, the Gibson-Overhang PCR for fragments B1
and B2
were repeated (**Protocol 1**). This time, both PCRs yielded the desired gel
bands.
An additional unspecific band was detected at around 750 bp in both cases. E was
also amplified for Gibson overhangs, and a band was visible at the correct size
(**Fig. 5 A**).

**Different PCR template and primer parameters for fragment H:**

*(Andreas, Aaron, David)*

Since the PCR for fragment H was successful, we used it to
try different PCR parameters (primer and template concentrations) to potentially
optimize the protocol. **Protocol 1** was used with the following
combinations:

* H1: 10 ng DNA + 0.2 µM per primer
* H2: 5 ngl DNA + 0.2 µM per primer
* H3: 10 ng DNA + 0.25 µM per primer
* H4: 5 ng DNA + 0.25 µM per primer

No pattern was apparent (**Fig. 5 B**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/2/25/T--Tuebingen--Gel_Fig5.png)

**Figure 5: A: Gel
electrophoresis of Gibson-Overhang PCR of fragments B1, B2, and E.**
Gel
loaded (from left to right): GeneRuler 1kb ladder - Fragment B1 -
Fragment B2 - Fragment E.
 **B: Gel electrophoresis (**Protocol 2**)
of
Gibson-Overhang PCR of fragment H with different template and primer
concentrations.** Gel loaded (from left to right): H1 - H2 - H3 - H4 -
nothing - GeneRuler 1kb ladder.

**Gradient PCRs fragment F:**

*(David, Aaron, Florian)*

Second gradient PCR (**Protocol 1**, 30s extension
time) for
fragment F with temperatures 63.5 / 64.9 / 66.1 / 67.2 / 68.0 / 68.5 °C. A band
approximately at the correct size (1.3 kb) is observable for the 68.5 °C sample
(**Fig. 6**) (**Protocol 2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/0/03/T--Tuebingen--Gel_Fig6.png)

**Figure 6: Gel
electrophoresis of Gradient PCR fragment F.** Gel loaded (from left to
right): GeneRuler 1kb ladder - Fragment F 63.5 - 64.9 - 66.1 - 67.2 - 68.0 -
68.5 °C - ladder.

Because of the detected band between 1-1.5 kb at 68.5 °C,
a third gradient PCR (**Protocol 1**, 30s extension time) for fragment F was
set up
with higher temperatures (68-72 °C). Additionally, duplicates were performed
with 5 µl GC-Enhancer per 25 µl reaction (5 µl H2ONF were substituted). The gel
image (**Fig. 7**) (**Protocol 2**) suggests an increase in DNA yield with
GC enhancer,
but the specific band is not visible in either case.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/6/6c/T--Tuebingen--Gel_Fig7.png)

**Figure 7: Gel
electrophoresis of Gradient PCR fragment F.** Gels loaded (from left
to
right): **A**: Fragment F without GC enhancer: GeneRuler 1kb ladder -
68.0 -
69.1 - 69.9 - 71.2 - 72.0 °C - ladder.
**B**: Fragment F with GC
enhancer:
GeneRuler 1kb ladder - 68.0 - 69.1 - 69.9 - 71.1 - 72°C

**Isolation of plasmid pUC19:**

*(Aaron, David, Lea)*

To obtain pUC19 plasmid DNA to use as a vector backbone
for the Gibson Assemblies, NEB® 5-alpha Competent E. coli (High Efficiency)
cells were transformed (**Protocol 4**) with 1 ng pUC19 plasmid DNA. Four
colonies
were picked the next day, inoculated in selective LB media (100 µg/ml (w/v), and
grown overnight. The overnight cultures were used for isolation of plasmid DNA
according to **Protocol 5**. To screen for correct pUC19 transformants, the
isolated
plasmid DNA was subjected to restriction digestion with EcoRI for linearization
(**Protocol 6**). The gel electrophoresis (**Protocol 2**) showed the
expected size
(2686 bp) for three out of four samples (**Fig. 4 B**).

**Creating a larger stock of pUC19:**

*(Andreas, Lea)*

A 100 ml selective culture (100 µg/ml (w/v) ampicillin)
was inoculated with one of the positive pUC19 transformants. The next day, the
culture was splitted into two 50 ml falcon tubes and plasmid DNA was isolated
according to **Protocol 7**. To confirm the presence of pUC19, the isolated
plasmid
DNA was digested with EcoRI and HindIII separately (**Protocol 6**). Gel
electrophoresis (**Protocol 2**) resulted in faint bands that can be seen at
the
right size for both digests (**Fig. 8**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/6/6a/T--Tuebingen--Gel_Fig8.png)

**Figure 8: Gel
electrophoresis of analytical restriction digestion of pUC19 with EcoRI
and HindIII.** Gel loaded (from left to right): Benchtop 1 kb ladder -
Analytical digest with EcoRI - Analytical digest with HindIII.

##### Week 5 (24 Aug - 29 Aug)

**Objectives:**

* Creation of Gibson overhangs for fragment A, B, C, and D with reduced primer
  concentrations
* Gradient PCR for C and D with lower primer concentrations and varied PCR
  cycles
* Creation of Gibson overhangs for fragment C with reduced primer
  concentrations, but more cycles and template DNA
* Creation of Gibson overhangs for fragment F with a robust PCR kit

**Creation of Gibson overhangs for fragment A, C, and D with reduced primer
concentrations:**

*(David, Lea)*

To eliminate or reduce primer clouds, the PCRs for
creation of Gibson overhangs for fragments A, C, and D were repeated. This time,
primer concentrations were reduced to ~50 % (0.13 µM) and to ~25 % (0.065 µM)
compared to the recommended concentration (0.25 µM) (**Protocol 1**).
On the one hand, PCR analysis (**Fig. 9**) (**Protocol 2**) revealed that
for fragment A, a lower primer concentration seems to reduce unspecific bands.
On the other hand, for C and D, both primer clouds and the desired bands were
reduced with lower primer concentrations.
*Note: The PCR should have been repeated with the normal primer concentrations
to be able to compare the new conditions to the standard one.*

![A generic square placeholder](https://static.igem.org/mediawiki/2020/9/92/T--Tuebingen--Gel_Fig9.png)

**Figure 9: Gel
electrophoresis of Gibson-Overhang PCR of fragment A, C, and D with
different primer concentrations.** Gel loaded (from left to right):
BenchTop 1kb ladder - A (50% primer) - C (50% primer) - D (50% primer) - A
(25% primer) - C (25% primer) - D (25% primer).

**Gradient PCR C and D with lower primer concentrations:**

*(Andreas)*

Considering the results of the PCR before, we decided to
run a gradient PCR with 25% of the standard primer concentration. The gradient
comprised the following annealing temperatures: 63, 64, 65, and 66 °C.
Additionally, denaturation time was increased by 10 s, extension time by 15 s,
and template DNA was increased to 20 ng, compared to **Protocol 1**. For
fragments C
and D, increasing the annealing temperature reduced the intensity of the primer
clouds but also of the desired bands (**Fig. 10**) (**Protocol 2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/b/b1/T--Tuebingen--Gel_Fig10.png)

**Figure 10: Gel
electrophoresis of Gibson-Overhang PCR of fragment A, C, and D with
temperature gradient.** Gel loaded (from left to right): BenchTop 1kb
ladder - C (63 °C - 66 °C, increasing by 1 °C) - D (63 °C - 66 °C,
increasing by 1 °C).

**Creation of Gibson overhangs for fragment C with reduced primer
concentrations, but more cycles and template:**

*(Lea, Andreas)*

With fragment C, further PCR optimization was attempted
with two different cycle numbers (35 and 40), a template DNA increase to 3 µl (=
30 ng), and primer reduction to 20% (~0.05 µM). These conditions were tested for
two annealing temperatures (66 °C and 72 °C). Everything else was done according
to **Protocol 1** and **Protocol 2**. No interesting difference was
recognized (**Fig. 11**
A), so optimization was abandoned.

**Creation of Gibson overhangs for fragment F with a robust PCR kit:**

*(Lea)*

Since we had the most issues with fragment F, we decided
to try the Gibson-Overhang PCR with a robust PCR kit (**Protocol 8**), which
is
designed to help amplify difficult fragments by facilitating primer binding.
Although the robust kit is supposed to work independent of PCR optimization, we
immediately set up a gradient PCR for the annealing temperature (60, 61.1, 62.2,
and 63.3 °C) as a precautionary measure.
For all temperatures, a band is
visible at the right size of ~ 1300 bp, which gets stronger with higher
temperatures (**Fig. 11 B**). As expected, this kit produced many unspecific
amplicons, but it allowed us to continue our work with fragment F without having
to re-design the primers (**Protocol 2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/a/a8/T--Tuebingen--Gel_Fig11.png)

**Figure 11: A: Gel
electrophoresis of Gibson-Overhang PCR of fragment C with increased
template and cycles at two temperatures.** Gel loaded (from left to
right): BenchTop 1kb ladder - C (66 °C, 35 cycles) - C (72 °C, 35 cycles) -
C (66 °C, 40 cycles).
**B: Gel electrophoresis of Gibson-Overhang PCR of fragment F with Protocol
8
and increasing annealing temperatures.** Gel loaded (from left to
right):
BenchTop 1kb ladder - F (60 °C) - F (61.1 °C) - F (62.2 °C) - F (63.3 °C).

##### Week 6 (31 Aug - 04 Sep)

**Objectives:**

* Creation of Gibson overhangs for fragment H with reduced primer
  concentrations
* Gradient PCR for fragments F and D with the robust PCR kit
* Restriction digestion and CIP treatment of pUC19 plasmid
* Creation of Gibson overhangs for fragment B, C, and D with more template,
  more cycles, or both
* Creation of Gibson overhangs for fragments C and F with greater PCR volumes
  for subsequent gel extraction

**Creation of Gibson overhangs for fragment H with reduced primer
concentrations:**

*(Anirudh, Sophia)*

A reduction of the primer concentration to 50% was
attempted for fragment H (**Protocol 1**), but it led to very faint bands
(**Protocol
2**). This was most likely explained by a procedural mistake with gel
electrophoresis, as fragment A, which was repeated with 25% primer
concentration, was also extremely faint (compared to **Fig. 11**).

**Gradient PCR for fragments F and D with the robust PCR Kit:**

*(Lea)*

After the PCR **Protocol 8** yielded the desired band
for
fragment F and a rising annealing temperature was accompanied by a rising band
intensity, the gradient PCR was continued with higher temperatures (64.9, 66.6,
68.2, and 69.7 °C). In addition, amplification of fragment D was also attempted
with **Protocol 8** for the temperatures 62, 63.3, 66.6, and 68.8 °C.

Gel electrophoresis (**Protocol 2**) showed that PCR
with
Protocol 8 worked for D as well, however, rising annealing temperatures lead to
fainter bands (**Fig. 12 A**). For fragment F, higher temperatures did not
improve
the PCR yield any further (**Fig. 12 B**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/b/b5/T--Tuebingen--Gel_Fig12.png)

**Figure 12: Gel
electrophoresis of Gibson-Overhang PCR of fragment D and F with the
AllTaq PCR Kit and increasing annealing temperatures. A:**  Gel loaded
(from left to right): BenchTop 1kb ladder - D (62 °C) - D (63.3 °C) - D
(66.6 °C) - D (68.8 °C).
**B:**Gel loaded (from left to right): GeneRuler 1kb ladder - F (64.9 °C)
- F (66.6 °C) - F (68.2 °C) - F (69.7 °C)

**Restriction digestion and CIP treatment of pUC19 plasmid:**

*(Lea, Anirudh)*

With progress in PCR optimization and getting closer to
Gibson Assembly, the next step was to prepare our vector backbone. Therefore,
pUC19 DNA (Maxiprep) was digested with both RE EcoRI and HindIII to linearize
the plasmid and produce the necessary overhangs for the assembly. Additionally,
it was treated with NEB Quick CIP (calf-intestinal alkaline phosphatase) to
prevent re-ligation of linearized plasmid DNA (**Protocol 9**).

**Creation of Gibson overhangs for fragments B, C, and D with more template,
more cycles, or both:**

*(Florian, David)*

We concluded that the unspecific amplicons would have to
be excluded through gel extraction of our specific amplicon DNA. To produce a
higher amplicon yield for gel extraction and in general for having more DNA for
Gibson Assembly reactions, we tried playing with other PCR parameters. We added
more template DNA (25 ng instead of 10 ng), ran PCR for more cycles (40 vs. 35),
or the combination of both. Everything else was done according to **Protocol
1**.
This was done for fragment B1, B2, C, and D. In the gel analysis (**Protocol
2**),
we could not really compare the different conditions, as hardly any bands were
visible.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/8/85/T--Tuebingen--Gel_Fig13.png)

**Figure 13: B: Gel
electrophoresis (Protocol 2) of Gibson-Overhang PCR of fragment B1, B2,
C, and D with different template concentrations and number of
cycles.** Gel loaded (from left to right): BenchTop 1kb ladder - B1 -
B2 - C - D (25 ng template, 35 cycles) - B1 - B2 - C - D (10 ng, 40 cycles)
- B1 - B2 - C - D (25 ng, 40 cycles) - BenchTop 1kb ladder

**Creation of Gibson overhangs for fragment C and F with greater PCR volumes
for subsequent gel extraction:**

*(Aaron, David)*

Since we repeatedly observed an unspecific band at 750 bp
for many of our fragments, and primer clouds seemed to prevail, we decided to
upscale our PCRs and perform gel extraction of the specific DNA loaded on the
agarose gel. The same procedure was done with fragment F, since it only worked
with the robust PCR method (**Protocol 8**), which produces many unspecific
amplicons. This was first done with fragment C and fragment F, where the 200 µl
reaction mix was split into 2 x 100µl for a more precise temperature
transmission during PCR. **Fig. 14 A** (**Protocol 2**) shows that F1
didn’t work, but
unfortunately F1 and F2 were already pooled back together for gel extraction.
Gel extraction (**Protocol 10**) of F and C was very successful (Week 7),
decreasing
the unspecific bands substantially (**Fig. 14 B**). Note: for the gels of
**Fig. 14
A/B**, we had already changed the DNA stain from SYBR® Gold DNA Stain to
SYBR®
Safe DNA Stain, as the former one was old and ineffective. With SYBR® Safe, we
performed precast staining as opposed to incubating gels in stain baths as we
did for SYBR® Gold.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/1/1b/T--Tuebingen--Gel_Fig14.png)

**Figure 14: A: Gel
electrophoresis of Gibson-Overhang PCR of fragment C with Protocol 1 and
fragment F with Protocol 8.** Gel loaded (from left to right):
BenchTop
1kb ladder - C(1) - C(2) - F(1) - F(2)
**B: Gel electrophoresis of
Fragments C and F after Gel extraction and purification.** Gel loaded
(from left to right): GeneRuler 1kb ladder - F (extracted, purified) - C
(extracted, purified).

##### Week 7 (07 Sep - 11 Sep)

From this week forward, we used SYBR® Safe DNA Stain and only did precast
staining, unless specified otherwise. This change highly improved the quality of
our gel images and saved us the 30 minutes required for the bath staining with
SYBR® Gold DNA Stain, which was also old and therefore ineffective.

**Objectives:**

* Gel extraction of fragments C and F
* Creation of Gibson overhangs for fragments A and H
* Gel extraction of fragments B1, B2, and D
* Creation of Gibson overhangs for fragment E
* **Double restriction digestion and CIP treatment pUC19 plasmid**
* Gibson Assemblies GA1, GA1(1:4), GA3, GA5, GA5(1:4), and GA6
* Creation of Gibson overhangs for fragments A, B1, C, and F, and gel
  extraction

**Gel extraction of fragments C and F:**

*(Andreas, Lea)*

For gel extraction (**Protocol 10**), both 100 µl PCR
set ups
were pooled back together and were then purified and eluted in only 15 µl
(**Protocol 3**) to be able to load all of the DNA together. After
extraction, the
samples were additionally purified and eluted in 10 µl (**Protocol 3**). Both
fragments then showed clear, bright bands in the gel (**Fig. 14 B**, Week 6)
(**Protocol 2**).

**Creation of Gibson overhangs for fragments A and H:**

*(Andreas)*

Fragments A and H did not show any unspecific bands,
therefore, their total PCR reaction volumes were only increased to 50 µl (2 x 25
µl each) (**Protocol 1**) and then purified and eluted in 10 µl (**Protocol
3**). For
fragment A, only 25 % primer concentration was used (compared to standard), as
this modification had previously shown good results (**Fig. 1**2). Clear
specific
bands were visible for both fragments with no noticeable unspecific bands
(**Fig.
15**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/e/e8/T--Tuebingen--Gel_Fig15.png)

**Figure 15: Gel
electrophoresis of Gibson-Overhang PCR of fragments A and H after
purification.** Gel loaded (from left to right): GeneRuler 1kb ladder
- A(1) - A(2) - H(1) - H(2).

**Gel extraction of fragments B1, B2, and D:**

*(Florian, Lea, David)*

For fragments B1, B2, and D, 100 µl or 200 µl
Gibson-Overhang PCR reactions (**Protocol 1**) were concentrated to 15 µl
(**Protocol
3**) and then loaded onto a gel for gel extraction (**Protocol 10**).
Isolation of the
specific bands for B1, B2, and D was successful (**Fig. 16 A and B**)
(**Protocol 2**).

**Creation of Gibson overhangs for fragment E:**

*(Aaron, Lea)*

Fragment E didn’t show unspecific bands or big primer
clouds and therefore a 200 µl PCR reaction was performed (**Protocol 1**) and
the
product was purified into 10 µl (**Protocol 3**). Unexpectedly, an unspecific
band
for E appeared at 1000 bp (**Fig. 16 B**). This can be explained by the high
amount
of DNA resulting from the large PCR reaction volume. Since the unspecific band
was very faint, the sample was still used for the Gibson Assemblies.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/2/29/T--Tuebingen--Gel_Fig16.png)

**Figure 16: A: Gel
electrophoresis of Gibson-Overhang PCR of fragment B1 and D after gel
extraction and purification. Additionally, new Bs was created.** Gel
loaded (from left to right): BenchTop 1kb ladder - Bs - B1 - D.
**B: Gel electrophoresis of fragment E after purification and fragments B2
after Gel extraction and purification.** Gel loaded (from left to
right):
BenchTop 1kb ladder - B2 - E.

**Double restriction digestion and CIP treatment pUC19 plasmid:**

*(Lea, David)*

Unfortunately, concentration of the first double-digested
(i.e. with two restriction enzymes) and CIP treated pUC19 DNA was really low, so
the process was repeated (**Protocol 9**). Again, the yield was too low. To
achieve
a higher concentration, both samples were pooled together and concentrated into
10 µl (**Protocol 3**), resulting in a concentration of ~50 ng/µl (ideal for
a
Gibson Assembly reaction, where 50 ng of vector backbone DNA suffices).

**Gibson Assemblies GA1, GA1(1:4), GA3, GA5, GA5(1:4), and GA6:**

*(Lea, David)*

Having several fragments ready for Gibson Assembly, the
assemblies GA1, GA3, GA5, and GA6 were attempted (**Table 2**). Gibson
Assembly was
done according to **Protocol 11** and chemically competent cells were
transformed
with the resulting GA1, GA1(1:4), GA3, and GA6 reaction products (**Protocol
4**). A
25 µl aliquot of competent cells was transformed with half (5 µl) of the
assembly reaction. For the assemblies described as “(1:4)”, half (5 µl) of an
assembly reaction were diluted 1:4 to 20 µl, and 5 µl of this dilution were used
for transformation. Unfortunately, plates with GA3 did not show colonies the
next day.

**Table 2: Overview Gibson Assemblies**

| Assembly name | Fragments |  | Primers |
| --- | --- | --- | --- |
| GA1 | E | Mn-Promoter - Mn-Riboswitch | 13+14 |
|  | F | FAST - Chromoprotein - Terminator | 15+16 |
| GA2 | E | Mn-Promoter - Mn-Riboswitch | 13+14 |
|  | G | FAST - Phytochelatine- Terminator | 15+16 |
| GA3 | H | Anderson-Promoter - Mn Riboswitch | 11+12 |
|  | F | FAST - Chromoprotein - Terminator | 15+16 |
| GA4 | H | Anderson-Promoter - Mn Riboswitch | 11+12 |
|  | G | FAST - Phytochelatine- Terminator | 15+16 |
| GA5 | A | Anderson-Promoter - B12 Riboswitch | 7+8 |
|  | B1 | Tet-Inverter-System | 4+5 |
|  | C | GFP-Terminator | 1+2 |
| GA6 | A | Anderson-Promoter - B12 Riboswitch | 7+8 |
|  | B2 | Tet-Inverter-System | 10+6 |
|  | D | mRFP-Terminator | 3+9 |

**Creation of Gibson overhangs for fragments A, B1, C, and F, and gel
extraction:**

*(Aaron, Florian, David)*

To ensure continuous repetition of failed Gibson
Assemblies, more PCRs were performed. Two 100 µl PCRs for fragment F were done
according to **Protocol 8**. For fragments A, B1, and C, 100 µl (2 x 50 µl or
100
µl) PCRs were done according to Protocol 1, but with 3 µl DNA template (= 30 ng)
per 25 µl of reaction mix to try to increase yield. After verifying that the
PCRs had worked (**Fig. 17**) (**Protocol 2**), the PCR reactions were
pooled
respectively and then purified (**Protocol 3**). As A(1) and A(2) only showed
the
desired specific band on the gel, the DNA was eluted in 30 µl and then stored.
The other PCR products displayed unspecific bands on the gel, so gel extraction
was necessary. After DNA elution in 15 µl, this was performed according to
Protocol 10.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/4/46/T--Tuebingen--Gel_Fig17.png)

**Figure 17: Gel
electrophoresis of Gibson-Overhang PCR of fragments A and H after
purification.** Gel loaded (from left to right): BenchTop 1kb ladder -
A(1) - A(2) - B1 - C(1) - C(2) - F(1) - F(2).

##### Week 8 (14 Sep - 18 Sep)

**Objectives:**

* Colony PCR of GA1, GA1(1:4), and GA6
* Double restriction digestion and CIP treatment of pUC19 plasmid DNA
* Plasmid DNA isolation of promising GA1 and GA1(1:4) transformants and
  subsequent analytical restriction digestion
* Isolation of pUC19 plasmid DNA and subsequent analytical restriction
  digestion
* Transformation with GA5 and GA5(1:4) and colony PCR
* Creation of Gibson overhangs for fragment H and new Bs

**Colony PCR of GA1, GA1(1:4), and GA6:**

*(Benedikt, Luise)*

Colony PCR was performed to assess which Gibson Assemblies
and transformations worked (**Protocol 12**). PCR analysis of GA1 (**Fig.
18** and **Fig.
19 A**) (**Protocol 2**) revealed that colonies 12 and 13 from GA1
contained an
insert, and the insert size of colony 13 corresponded to ca. 1800 bp, the
desired size. Unfortunately, GA6 didn’t show any specific bands (**Fig. 19
B**). For
GA1(1:4), colonies 12, 13, 14, 16, 18, and 19 showed correct insert sizes
(**Fig.
20**), indicating that dilution of the reaction before transformation had
had an
effect in the efficiency, presumably due to the decrease in reaction components.
The colonies with bands below 250 bp are presumably transformants of undigested
pUC19, as digestion is never 100 % efficient.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/d/da/T--Tuebingen--Gel_Fig18.png)

**Figure 18: Gel
electrophoresis of colony PCR from GA1.** pUC19 is a negative control,
where only the empty digested plasmid was transformed. Samples 12 and 13
are labelled, as these were the only ones showing insert amplification.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/1/13/T--Tuebingen--Gel_Fig19.png)

**Figure 19: Gel
electrophoresis of colony PCR from GA1 (A) and GA6 (B).** pUC19 is a
negative control, where only the empty plasmid was transformed.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/e/ef/T--Tuebingen--Gel_Fig20.png)

**Figure 20: Gel
electrophoresis of colony PCR from GA1(1:4).** Only the colonies with
the
right insert size are labelled.

**Double restriction digestion and CIP treatment of pUC19 plasmid DNA:**

*(Aaron)*

With previous low concentrations for double-digested and
CIP treated pUC19, we were in need of more and repeated this step with 2.6 µg
DNA (**Protocol 9**).

**Plasmid DNA isolation of promising GA1 and GA1(1:4) transformants and
subsequent analytical restriction digestion:**

*(Lea)*

Colonies from the corresponding samples were inoculated in
selective media (100 µg/ml ampicillin sodium salt) for overnight growth. The
next day, plasmid DNA was isolated (**Protocol 5**). Concentrations are given
in
**Table 3**.

- GA1: samples 12 and 13
- GA1(1:4): samples 12, 13, 14, 16, 18, and 19

**Table 3: Plasmid DNA concentrations of promising GA1 and GA1 (1:4)
transformants.**

| Sample | Concentration (ng/µl) |
| --- | --- |
| GA1 (1:4) 12 | 373.6 |
| GA1 (1:4) 13 | 477.1 |
| GA1 (1:4) 14 | 307.9 |
| GA1 (1:4) 16 | 341.3 |
| GA1 (1:4) 18 | 408.7 |
| GA1 (1:4) 19 | 391.3 |
| GA1 12 | 423.4 |
| GA1 13 | 466.2 |

Isolated plasmid DNA was digested once with EcoRI and once
with HindIII (**Protocol 6**) and the linearized plasmid DNA was then
analyzed via
gel electrophoresis (**Protocol 2**). Plasmid DNA was observed at the correct
size
(ca. 4300 bp). However, HindIII seemed to produce an extra gel band, presumably
nicked DNA since the enzyme was old and the extra band is positioned above the
specific band (**Fig. 21 A and B**) (**Protocol 2**).

**Isolation of pUC19 plasmid DNA and subsequent analytical restriction
digestion:**

*(Lea, Andreas)*

After inoculation of pUC19-containing E. coli in 5 x 20 ml
selective medium (100 µg/ml ampicillin sodium salt) and overnight growth,
plasmid DNA was isolated (**Protocol 7**). This time, a high concentration of
373.1
ng/µl was achieved for a total of 200 µl. The plasmid DNA was digested with
EcoRI and HindIII separately (**Protocol 6**) and the gel analysis (**Fig.
21 B**)
(**Protocol 2**) confirmed pUC19 with bands at 2686 bp.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/6/6d/T--Tuebingen--Gel_Fig21.png)

**Figure 21: Gel
electrophoresis restrictions digest with EcoRI and HindIII for GA1
sample 12 and 13 and GA1(1:4) samples 12, 13, 14, 16, 18, 19 and pUC19
plasmid from Maxi-Prep. A:** Gel loaded (from left to right): BenchTop
1kb
ladder - GA1 sample 13, EcoRI - GA1(1:4) sample 13, 14, 16, 18, 19 with
EcoRI - BenchTop 1kb ladder - GA1 sample 13, HindIII - GA1(1:4) sample
13, 14, 16, 18, 19 with HindIII
**B:** Gel loaded (from left to
right):
BenchTop 1kb ladder - GA1 sample 12, EcoRI - GA1 sample 12, HindIII -
GA1(1:4) sample 12, EcoRI - GA1(1:4) sample 12, HindIII - pUC19 EcoRÎ -
pUC19 HindIII.

**Transformation with GA5 and GA5(1:4) and colony PCR:**

*(Andreas, Lea)*

**GA5 and GA5(1:4)** were transformed into *E.
coli* (**Protocol
4**) and subjected to colony PCR the next day (**Protocol 12**). Gel
analysis (**Fig.
22**) (**Protocol 2**) revealed insertion of fragment A (530 bp
including overhangs)
in many samples. In later Gibson Assemblies, the molar ratio of fragment A was
relatively reduced.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/f/f6/T--Tuebingen--Gel_Fig22.png)

**Figure 22: Gel
electrophoresis of colony PCR from GA5 and GA5 (1:4).** Bands right
above
500 bp represent fragment A insertions.

**Creation of Gibson overhangs for fragment H and new Bs:**

*(Aaron, Lea)*

PCR for Gibson overhangs was performed for fragment H
along with a PCR to remove IDT base pairs from fragment B (**Protocol 1**).
Afterwards, the samples were purified and concentrated to 20µl (Bs) and 15µl (H)
(**Protocol 3**).

##### Week 9 (21 Sep - 25 Sep)

**Objectives:**

* Resuspension of fragment G
* Creation of Gibson overhangs for fragments G and B2
* Creation of Gibson overhangs for fragment F
* Gel extraction of fragments B2, G, and F
* Gibson Assemblies GA2, GA3, and GA4

**Resuspension of fragment G:**

*(Florian)*

Fragment G finally arrived and was dissolved with
nuclease-free water to a concentration of 10 ng/µl.

**Creation of Gibson overhangs for fragments G and B2:**

*(Florian)*

The newly arrived fragment G was tested with both PCR
protocols (**Protocol 1** and **Protocol 8**). Additionally, a 100 µl
Gibson overhang
PCR for fragment B2 was done (**Protocol 1**). PCR analysis (**Protocol
2**) revealed
that fragment G could be amplified correctly with both protocols (**Fig. 23
A**).
Since the DNA polymerase used in **Protocol 1** has a higher fidelity, the
**Protocol
1** was then used for a 100µl PCR of fragment G.

**Creation of Gibson overhangs for fragment F:**

*(Florian)*

Two 100µl PCRs for Gibson overhangs on fragment F were
done according to **Protocol 8** and gel analysis (**Protocol 2**)
confirmed it worked
(**Fig. 23 B**).

**Gel extraction of fragments B2, G, and F:**

*(Andreas)*

All PCR products were concentrated first to 15 µl (B2, G)
or 20 µl (combined F samples) (**Protocol 3**) and then gel extracted
(**Protocol 10**).
Through gel extraction, the unspecific bands of fragment F were excluded. For
fragment G, an unspecific but very faint band is visible at around 1400 bp
(**Fig.
23 C**) (**Protocol 2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/9/9d/T--Tuebingen--Gel_Fig23.png)

**Figure 23: A: Gel
electrophoresis of Gibson-Overhang PCR of fragments G and B2.** Gel
loaded
(from left to right): BenchTop 1kb ladder - B2(Protocol 1) - G(Protocol
1) - G(Protocol 8) - BenchTop 1kb ladder
**B: Gel electrophoresis of
Gibson-Overhang PCR of fragment F.** Gel loaded (from left to right):
BenchTop 1kb ladder - F(1) - F(2)
**C: Gel electrophoresis of fragment
B2,
G, and F after gel extraction and purification.** Gel loaded (from
left to
right): BenchTop 1kb ladder - B2 (extracted, purified) - F (extracted,
purified) - G (extracted, purified)

**Gibson Assemblies GA2, GA3, and GA4:**

*(Florian)*

GA2 and GA4 were performed for the first time and GA3 was
repeated (**Protocol 11**). Since GA1 had worked (F+E) and GA3 (F+H) had not,
the
molar ratio of H was increased to 5:1 (insert:backbone) as the common
denominator was F. E. coli was subsequently transformed (**Protocol 4**).
After
overnight growth, all plates showed colonies.

##### Week 10 (28 Sep - 02 Oct)

**Objectives:**

* Colony PCR of GA2, GA3, and GA4
* Sequencing of positive clones from GA1, GA2, GA3, and GA4

**Colony PCR of GA2, GA3, and GA4:**

*(Luise, David, Andreas)*

To assess which Gibson Assemblies and transformations
worked, colony PCR was performed for ca. 20 colonies per GA (**Protocol
12**).
Analysis of PCR products by gel electrophoresis (**Protocol 2**) revealed
multiple
potential clones for all assemblies (**Fig. 24**). Approximately five clones
were
inoculated for isolation of their plasmid DNA (**Protocol 5**) and subsequent
Sanger
sequencing (Microsynth service).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/c/c7/T--Tuebingen--Gel_Fig24.png)

**Figure 24: Gel
electrophoresis of colony PCR from GA2, GA3, and GA4.** Positive
samples
for GA3 are marked with arrows. For all other GA there were several
positive samples. Approximate desired band/insert sizes: 1200 bp for
GA2, 1600 bp for GA3, and 1000 bp for GA4.

**Sequencing of positive clones from GA1, GA2, GA3, and GA4:**

*(Andreas, Aaron, Luise)*

To verify the sequence of the inserts, isolated plasmid
DNA was prepared for the Sanger sequencing service by Microsynth (Balgach). For
this, specific M13 primers offered by Microsynth were selected to amplify the
insert region within the pUC19 vector. After alignment analysis with SnapGene
(GSL Biotech, California), one clone containing an intact insert sequence was
chosen for the measurement experiments for each GA (four clones in total). We
quickly noticed a mistake in our sequence: A PstI restriction site which is
incompatible with the iGEM standards BioBrick RFC[10] and Type IIS RFC[1000]
(**Fig. 25**). We had ordered the wrong sequences, so we decided to design
and order
primers for site-directed mutagenesis (SDM) (**Table 4**) as well as the
initially
designed FAST2 sequences (of F and G) that did not contain the PstI (silent
mutation of leucine codon from CTG to CTT) for repetition of Gibson Assemblies
(**Fig. 26**). We placed different orders for the SDM primers and the
corrected
fragments F and G and decided to execute the first available method.
Furthermore, we noticed that due to the larger insert sizes of GA1 and GA3, the
first few dozen base pairs downstream of the sequencing primers were not covered
by the sequencing. To complete sequencing of the insert borders, we designed
sequencing primers annealing in the FAST2 sequence according to the Microsynth
requirements (**Fig. 26**) (**Table 4**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/5/5a/T--Tuebingen--Gel_Fig25.png)

**Figure 25: Alignment of
Sanger sequencing results of GA1 with hypothetical reference sequence
(SnapGene).** The sequence aligns perfectly with our old sequence
containing the illegal PstI restriction site.

![A generic square placeholder](https://static.igem.org/mediawiki/2020/9/93/T--Tuebingen--Gel_Fig26.png)

**Figure 26: FAST2
sequence indicating location of SDM primers Pst\_fix\_RV/FW and sequencing
primers FAST\_mid\_RV/FW.** The SDM primers partially overlap, creating
nicked plasmid DNA during PCR which is re-ligated in the host cell after
transformation. The sequencing primers completely overlap with each other,
but are used in separate reactions for sequencing.

**Table 4: Primers for SDM and for complete sequencing of GA1 and
GA3**

| Primer name | Sequence (5' --> 3') |
| --- | --- |
| Pst\_fix\_RV | ttgtactgaaggatattcccgtcaccatcgag |
| Pst\_fix\_FW | ggaatatccttcagtacaatgctgctgaaggagac |
| FAST\_mid\_RV | ctgcttgtcggtatcatcc |
| FAST\_mid\_FW | ggatgataccgacaagcag |

##### Week 11 (05 Oct - 09 Oct)

**Objectives:**

* GA5 and GA6 with increased B1/2 and C concentrations and subsequent colony
  PCR
* Plasmid DNA isolation and analytical restriction digestion of interesting
  GA5 and GA6 samples
* Streak plates for all four GA1-4 clones and a pUC19 clone for
  sub-cultivation

**GA5 and GA6 with increased B1/2 and C concentrations and subsequent colony
PCR:**

*(Andreas)*

Since only insertion of fragment A was observed in GA5 and
GA6 (**Fig. 22**), the assemblies were repeated (**Table 2**) (**Protocol
11**). This time,
fragments B1/2 and C were added to a molar ratio of 5:1 (insert : backbone). The
finished reactions were used for transformation of *E. coli* (**Protocol
4**). The
next day, colonies were picked for colony PCR (**Protocol 12**), however, no
correct
bands were observed on the gel (**Protocol 2**). The expected insert size is
approximately 2300 bp for both GAs, and only unspecific bands were visible
(**Fig.
27**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/5/5d/T--Tuebingen--Gel_Fig27.png)

**Figure 27: Colony PCR
of GA5 and GA6.** Only unspecific bands are visible between 2500-3000
bp, 1500-2000 bp (presumably B + C insertion), and 500-750 bp (presumably A
insertion). The red arrows point at samples that were analyzed further.

**Plasmid DNA isolation and analytical restriction digestion of interesting GA5
and GA6 samples:**

*(Andreas)*

Eight interesting samples of GA5 and GA6 (**Fig. 27**)
were
analyzed further. For this, plasmid DNA was isolated (**Protocol 5**) and
subsequently digested with EcoRI (**Protocol 6**). As a control, plasmid DNA
of
GA2-4 sequenced clones was digested the same way. The plasmid DNA isolated from
the interesting GA5 and GA6 clones was smaller than the desired size of ca. 4900
bp (**Fig. 28**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/1/15/T--Tuebingen--Gel_Fig28.png)

**Figure 28: Analytical
restriction digestion of plasmid DNA isolated from interesting GA5 and
GA5 clones.** The plasmid DNA of GA5 and GA6 clones is under the
desired
size of ca. 4900 bp. The controls, GA2, GA3, and GA4, show the correct
band sizes.

**Streak plates for all four GA1-4 clones and a pUC19 clone for
sub-cultivation:**

*(Andreas)*

The chosen sequenced GA1-4 clones and a pUC19 clone were
streaked on non-selective LB agar plates to commence a sub-cultivation
experiment with non-selective media. This experiment was designed to assess
transgenerational propagation stability of our constructs without selective
pressure.

##### Week 12 (12 Oct - 18 Oct)

**Objectives:**

* Assembly of correct GA1, GA2, GA3, and GA4
* Colony PCR of correct GA1, GA2, GA3, and GA4
* Sequencing of correct GA1, GA2, GA3, and GA4 clones
* Continuing stability test

**Assembly of correct GA1, GA2, GA3, and GA4:**

*(Andreas, Florian, Luise)*

Since the site-directed mutagenesis primers arrived at the
same time as the correct fragments F and G, we chose to repeat Gibson Assembly
with the new fragments, the more reliable option considering we had optimized
the procedure. After PCR of F (**Protocol 8**) and G (**Protocol 1**) for
addition of
Gibson overhangs, the amplified DNA was concentrated (**Protocol 3**), loaded
on a
gel, and extracted (**Protocol 10**). After concentration measurements, the
Gibson
Assemblies of GA1, GA2, GA3, and GA4 were repeated with the correct versions of
F and G (no PstI restriction site) (**Protocol 11**). The assembly reactions
were
used to transform E. coli (**Protocol 4**).

**Colony PCR of correct GA1, GA2, GA3, and GA4:**

*(Anirudh, Isabel)*

All GA1-4 plates had hundreds of colonies on them (**Fig.
29**). Dozens of colonies were picked for each GA and subjected to colony
PCR
(**Protocol 12**). The efficiency was very high, with most colonies giving
positive
signals on the gel (**Fig. 29**) (**Protocol 2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/8/81/T--Tuebingen--Gel_Fig29.png)

**Figure 29: Colony PCR
of correct GA1, GA2, GA3, and GA4.** Correct bands: ca. 1800 bp (GA1),
ca. 1200 bp (GA2), ca. 1600 bp (GA3), and ca. 1000 bp (GA4). Cell material
of the old GA1-4 sequenced clones was used for PCR and gel electrophoresis
control (positive). The negative control was the PCR master mix without
template DNA. Only 9 out of 76 test samples were incorrect.

**Sequencing of correct GA1, GA2, GA3, and GA4 clones:**

*(Luise, Anirudh, Andreas, Isabel)*

After confirmation of successful assembly and
transformation (**Fig. 29**), plasmid DNA was isolated (**Protocol 5**)
from multiple
clones for each GA and sent for Sanger sequencing by Microsynth to inspect the
insert sequences. Mutation-less clones were found for each GA (**Fig. 30**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/3/3b/T--Tuebingen--Gel_Fig30.png)

**Figure 30: Alignment of
Sanger sequencing results of GA1 wth hypothetical reference sequence as
an example of an intact insert sequence (SnapGene).** Microsynth
standard
primers M13-40 (forward), M13-r (reverse), and in-house primers
FAST\_mid\_FW and FAST\_mid\_RV were used for the Sanger sequencing reaction
of GA1 and GA3. For GA2 and GA4, only the Microsynth M13 primers were
used due to the smaller size of the insert sequence. A point mutation is
visible in the PstI restriction site, indicating that the restriction
site is no longer present in the new FAST2 sequences.

**Fluorescence measurement:**

*(Luise, Isabel, Anirudh, Andreas)*

To test the functionality of our constructs, fluorescence
was measured (3 experimental replications, 3 sample replications for each
experiment) (**Protocol 13**). For this, we tested different manganese(II)
chloride
concentrations and used sequenced clones from the old GA1-4 and the new
(corrected) GA1-4, as well as a pUC19 clone as a control. Unfortunately, the
2020 iGEM measurement kit was not available, and our 2018 version was
non-functional. Therefore, our plate reader was not calibrated. Furthermore,
fluorescence values, although measured at similar cell densities, were random
and independent of manganese concentrations (**Fig. 31**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/5/58/T--Tuebingen--Gel_Fig31.png)

**Figure 31: Fluorescence
intensity (y-axis) measured at an OD600 of ca. 0.9 for different
manganese concentrations (A, B, and C) and constructs (x-axis).** The
values are random and no pattern is identifiable.

**Continuing stability test:**

*(Anirudh)*

The propagation stability test for the old constructs
GA1-4 was continued throughout the week by transferring cell material to new
non-selective LB agar plates every other day.

##### Week 13 (19 Oct - 23 Oct)

**Objectives:**

* Chromoprotein experiment
* Fluorescence microscopy
* mRNA experiment
* Analyzing stability test

**Chromoprotein experiment:**

*(Anirudh, Andreas)*

Since the results obtained from fluorescence measurement
with the plate reader were inconclusive (**Fig. 31**), we decided to
qualitatively
test the expression of the blue chromoprotein of GA1 and GA3. For this,
selective LB agar plates (100 µg/ml ampicillin sodium salt) were prepared with 3
different manganese(II) chloride concentrations (0, 10, and 20 µM). A sequenced
clone for each GA (old and new versions) and a pUC19 clone were streaked on the
plate and grown overnight. No color changes (i.e. no blue colonies) were
observed (**Fig. 32**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/f/fb/T--Tuebingen--results_fig14.png)

**Figure 32:
Chromoprotein experiment plates.** Three selective LB agar plates (100
µg/ml ampicillin sodium salt) containing three different manganese(II)
chloride concentrations - 0 µM (A), 10 µM (B), and 20 µM (C) - were
prepared and a clone for GA1 and GA3 (old and new/corrected versions)
was streaked. No blue color is visible on the cells.

**Fluorescence microscopy:**

*(Isabel, Luise)*

As a final qualitative test for the functionality of our
constructs, GA2 (new/corrected version) and pUC19 (control) cells were
visualized under the fluorescence microscope after growth in selective media
(100 µg/ml ampicillin sodium salt) with 10 µM manganese(II) chloride and
incubation in a fluorogen-labeling solution (see **Protocol 13** for details
on
labeling). The same autofluorescence was observed for the pUC19 and GA2 clones
(**Fig. 33**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/9/94/T--Tuebingen--results_fig13.png)

**Figure 33: Live cell
imaging of GA2 (corrected version) and pUC19 cells under the
fluorescence microscope.** Laser settings: Wavelengths 485 nm
(absorbance)
and 535 nm (emission). 1.5 x 630 zoom. (A) pUC19 control, growth in 10
µM Manganese. (B) GA2 (corrected version), growth in 10 µM Manganese.

**mRNA experiment:**

*(Luise, Anirudh, Andreas)*

The failure to produce fluorescence (**Fig. 31** and
**Fig. 33**)
or a visible blue color (**Fig. 32**) with our GA1-4 clones (old and
corrected
versions) made us take a closer look at our sequences again. We noticed the
absence of a ribosome binding site (RBS) between our riboswitch sequences and
the phytochelatin/chromoprotein gene. The mistake in our design was caused by
the misconception that the riboswitch sequence available in the parts registry
would include an RBS downstream of the riboswitch. With only two days left for
wet lab work, we quickly designed a Reverse Transcriptase (RT)-PCR experiment
(**Protocol 14**) to investigate mRNA synthesis and validate our promoters,
which
function independently of an RBS.

Unfortunately, the experiment was inconclusive. We
expected an amplicon of ca. 320 bp and a difference in signal intensity between
growth with 0 µM and 10 µM manganese(II) chloride for constructs with
manganese-inducible promoters, however, the negative controls had the same band
sizes and different manganese concentrations did not produce different signal
intensities. Although the band sizes of the negative controls seem to be
slightly larger, this could be explained by a variable gel morphology.
Interestingly, all negative controls have similar bands to the AllTaq PCR Master
Mix negative control, which did not contain template cDNA (**Fig. 34**)
(**Protocol
2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/5/57/T--Tuebingen--results_fig18.png)

**Figure 34: Gel
electrophoresis of cDNA amplification via PCR.** The desired band size
was ca. 320 bp. Test samples include the old GA1-4 and the corrected GA2 and
GA3 clones. Negative controls include the pUC19 samples, the reverse
transcription (RT) reaction mix, and the AllTaq PCR master mix without
template cDNA.

**Analyzing stability test:**

*(Anirudh, Andreas)*

To finalize the wet lab experience, we analyzed our
plasmid stability test by transferring cell material from each clone to a
selective lb agar plate (100 µg/ml ampicillin sodium salt) and performing colony
PCR (**Protocol 12**) after overnight growth. In total, nine transfers were
made
(**Fig. 35**). All clones grew when transferred back to selective media
(**Fig. 36**).
Correct signals were obtained for all tested clones in colony PCR (**Fig.
37**)
(**Protocol 2**).

![A generic square placeholder](https://static.igem.org/mediawiki/2020/8/83/T--Tuebingen--results_fig15.png)

**Figure 35:
Non-selective LB agar plates containing the old GA1, GA2, GA3, and GA4
clones, and a pUC19 clone, used for the stability test.**

![A generic square placeholder](https://static.igem.org/mediawiki/2020/0/00/T--Tuebingen--results_fig16.png)

**Figure 36: Final
selective plate from which colonies of the old GA1, GA2, GA3, and GA4
constructs were picked for colony PCR.**

![A generic square placeholder](https://static.igem.org/mediawiki/2020/4/40/T--Tuebingen--results_fig17.png)

**Figure 37: Colony PCR
of stability test.** A correct band is observable for all GA clones
(very faint for GA1 and GA3, marked with red arrows). The controls show the
expected band sizes.

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# Team:Tuebingen/Engineering

PacMn

# Engineering

[![logo](https://static.igem.org/mediawiki/2020/a/ab/T--Tuebingen--logo.png)](https://igem-tuebingen.com/index)
[##### PacMn](https://2020.igem.org/Team%3ATuebingen)

* Team & Supporters

  [Team](/Team%3ATuebingen/Team)
  [Team Statistics](/Team%3ATuebingen/Team_Statistics)
  [Attributions](/Team%3ATuebingen/Attributions)
  [Excellence](/Team%3ATuebingen/Excellence)
  [Sponsors](/Team%3ATuebingen/Sponsors)
* Project

  [Description](/Team%3ATuebingen/Description)
  [Design](/Team%3ATuebingen/Design)
  [Parts](/Team%3ATuebingen/Parts)
  [Engineering](/Team%3ATuebingen/Engineering)
  [Model](/Team%3ATuebingen/Model)
  [Experiments](/Team%3ATuebingen/Experiments)
  [Results](/Team%3ATuebingen/Results)
  [Contribution](/Team%3ATuebingen/Contribution)
  [Implementation](/Team%3ATuebingen/Implementation)
  [Notebook](/Team%3ATuebingen/Notebook)
  [Protocols](/Team%3ATuebingen/Notebook)
  [Safety](/Team%3ATuebingen/Safety)
* Outreach

  [Human Practices](/Team%3ATuebingen/Human_Practices)
  [Education](/Team%3ATuebingen/Education)
  [Collaborationss](/Team%3ATuebingen/Collaborations)
* Deliverables

  [Poster](/Team%3ATuebingen/Poster)
  [Judging
  Form](https://igem.org/2020_Judging_Form?team=Tuebingen)
  [Presentation Video](/Team%3ATuebingen/Presentation)
  [Project Promotion
  Video](/Team%3ATuebingen/Promo_Video)

### Wetlab

![Figure 1: Engineering-Design-Cycle with details how each step was or should be tackled within                         the wet lab work.](https://static.igem.org/mediawiki/2020/7/7a/T--Tuebingen--engi_fig1.png)

**Figure 1: Engineering-Design-Cycle with details how each step was or should be tackled within
the wet lab work.**

Biological engineering is a major part of iGEM’s objectives, already contained in the acronym itself.
Thus, this creative and systematic process, necessary for great projects and outcomes, is supposed to
receive greater attention this year. **Figure 1** gives an overview of the Engineering-Design-Cycle
and how
we fulfilled each step.

**Research**: During project finding and planning, we researched literature on heavy metal pollution
and on the possibilities of binding heavy metal ions. Environmental heavy metal contamination is often
man-made, stemming from our agricultural and industrial activities [1, 2]. This is not only the case in
developing countries [3, 4]; areas with heavy metal contaminated soil can be found in Europe [5].
Furthermore, it was shown that rising ground water temperatures are connected to higher manganese
concentrations in Germany [6]. Thus, rising temperatures caused by climate change could possibly enhance
problems with heavy metal pollution. This local connection and the fact that not many iGEM projects have
worked with this metal so far, led to our decision to focus on manganese. Plants, animals, and human
health can be affected by manganese exposure. In plants, exposure to manganese pollution causes biomass
loss. In humans, its inhalation causes neurotoxicity [7–10], and recent studies suggest that
manganese-contaminated drinking water poses health risks [11]. Interestingly, plants possess a
remarkable disposal mechanism for heavy metals; they produce cysteine-rich oligopeptides named
phytochelatins, which form complexes with heavy metal ions, especially cadmium. The complexes are then
stored in the vacuole, preventing interference with cellular biochemical processes [12]. To address
heavy metal contamination, an overexpression of synthetic phytochelatins on the surface of *E.
coli* was
attempted and resulted in more accumulation of cadmium in comparison to wild-type *E. coli* [13].

**Imagine**: This led us to our project PacMn: Phytochelatin-actuated complexation of Manganese. To
combat heavy metal contamination in water, methods for detection and capture of these ions are
crucial. Therefore, we want to create a bifunctional biosensor that fulfills both tasks using the
synthetic phytochelatin EC20 already included in the parts registry (BBa\_K1321005), which has been
shown to increase manganese adsorption when expressed on *C. metallidurans* (14). although to a
lower
extent than other metals. Thus, future adaptations of phytochelatin may be necessary to increase its
complexing efficiency for manganese.

One of our primary aims is to have a bifunctional biosensor with a high specificity for manganese ions.
Accordingly, we hypothesize that by using a riboswitch-based sensing mechanism, we will gain stronger
specificity than via standard repressor / activator proteins of bacterial operons [15]. The effects that
manganese-complexing will have on the functionality of the biosensor are unknown. At this point, we can
only assume that complexed manganese ions in the phytochelatin will not be sensed by the riboswitch due
to its high specificity for free dissolved ions [16], creating a self-regulated system in the form of a
negative feedback loop. In contrast, if riboswitches could be stimulated by complexed manganese ions, an
intracellular accumulation of manganese would lead to a positive feedback loop and, ultimately, a false
positive signal for extracellular manganese concentrations.

Because *E. coli* is a model organism for cloning work, we chose it as our biosensor host. However, other
microbial organisms might be more suitable for distinct extreme conditions. Thus, at a later stage, we
may consider switching hosts. Naturally, we wish to create a product that adheres to biosafety
regulations. Considering our work will entail the usage of genetically modified organisms (GMOs), a
potential safety measurement for the application of our product would be the implementation of a filter
system permeable to manganese ions and impermeable to cells.

**Design**: To address our questions and reduce unknown variables which would arise in a more complex
system, we decided to simplify our design as much as possible. In order to study the individual
components of each construct, we designed four similar constructs for comparative analysis (Table 1).
Although only one type of phytochelatin was included in our constructs, other variants were also
modelled by our dry lab.

**Table 1: Description of all components (BioBricks) of our PacMn constructs.**

| Name | Part ID | Function |
| --- | --- | --- |
| Manganese- promoter | BBa\_K902073 | A manganese-inducible promoter for transcriptional regulation |
| Anderson-promoter | BBa\_J23102 | A constitutive promoter to evaluate the effect of having and not having transcriptional regulation |
| Manganese- riboswitch | BBa\_K902074 | A manganese-inducible riboswitch for translational regulation |
| FAST2-tag | BBa\_K3510000 | A fluorogen-activating protein for interchangeable fluorescence |
| Phytochelatin | BBa\_K1321005 | A chelator for manganese-decontamination |
| Chromoprotein | BBa\_K864401 | A blue chromoprotein to evaluate the effect of phytochelatin on the sensing mechanism of the construct and to provide a direct color change for the naked eye |
| Double terminator | BBa\_B0015 | A reliable double terminator |

Different combinations of the seven components (**Table 1**) resulted in four PacMn constructs (**Fig.
2**). For
details regarding  [design](https://2020.igem.org/Team%3ATuebingen/Design) and function of these parts please visit the
corresponding wiki page.

![Figure 2: Schematic overview of the PacMn constructs. Each Gibson Assembly consisted of two fragments and a pUC19 vector backbone. The dotted line indicates the ends of the two insert fragments. Here they are going to get connected by Gibson Assembly.](https://static.igem.org/mediawiki/2020/7/75/T--Tuebingen--engi-fig2.jpg)

**Figure 2: Schematic overview of the PacMn constructs.** Each Gibson Assembly consisted of two
fragments and a pUC19 vector backbone. The dotted line indicates the ends of the two insert
fragments. Here they are going to get connected by Gibson Assembly.

**Build:** To assemble our constructs, we chose the Gibson Assembly® method, which is an efficient
and seamless method for assembly of multiple linear DNA fragments [17]. This method relies on the
addition of complementary overhangs to each DNA fragment by PCR. The overhangs of a fragment must be
complementary to its adjacent fragments, and careful primer design is required, in which we succeeded
with the support of the SnapGene software. The extended fragments were assembled together along with
pUC19 as the vector backbone. Upon successful cloning, our constructs were subjected to measurement
experiments and others.

As neither the initial PCRs nor the Gibson Assembly had worked on the first try, we had to iterate
through the **Build-Test-Learn-Improve** cycle multiple times, before we finally obtained the correct
constructs.

**Test**: Initial issues occurred with the PCRs to create the overhangs for Gibson Assembly. When
analyzing PCR products by gel electrophoresis, we encountered several problematic results ranging from
no bands, over faint bands, to many unspecific bands.

**Learn**: Therefore, we had to investigate ways to troubleshoot and optimize PCR. A summary can be
found
in [Contributions](https://2020.igem.org/Team%3ATuebingen/Contribution).

**Improve**: With this knowledge we then edited the PCR protocol for each fragment and decided to
include
gel-extraction in our process.

**Build**: Once all PCRs worked and we had all fragments with the necessary overhangs, we approached
Gibson Assembly.

**Test**: To assess successful assembly and transformation, we performed colony-PCR as an initial
detection
method. Plasmid DNA was then isolated from potential candidates and subjected to analytical restriction
digestion with the restriction enzymes EcoRI and HindIII. Only the GA1 construct (**Fig. 1**) was
successfully cloned on the first try.

**Learn & Improve**: Following the failed attempt to clone the other three constructs of the
manganese
project, we adjusted the vector-insert molar ratios based on the analysis of the gel images from
colony-PCR.

**Build & Test**: With this adjustment, we were able to clone the other three main constructs (GA2-4)
the
second time we attempted Gibson Assembly. All positive clones were then sequenced to check for
mutations. Unfortunately, we noticed that an ordering mistake was made for the FAST2I-tag. The
original FAST2-tag sequence contains the restriction site for PstI, which is incompatible with the
accepted iGEM standards BioBrick RFC[10] and Type IIS RFC[1000]. This issue was initially excluded
with a silent mutation in the restriction site. However, the original FAST2-tag sequence was
accidentally ordered instead of our modified version.

**Learn**: To solve the compatibility issue, we considered two options: Ordering the correct
sequences
and repeating the cloning work with our reliable protocols, or ordering primers for site-directed
mutagenesis. Due to the late detection of the problem and the differing shipping times of primers
and longer DNA fragments, we decided to order both and execute the method that was more readily
available.

**Build**: To limit invested time and money, and considering we already had a polymerase appropriate
for site-directed mutagenesis, we did not order a special kit for this. Since both orders arrived on the
same day, we chose to repeat PCR and Gibson Assembly with the new FAST2-tag sequences, which was the
more familiar and therefore reliable cloning method for us.

**Test**: To assess successful assembly and transformation, we applied the same test procedure as
previously: Colony PCR, analytical restriction digestion of isolated plasmid DNA, and sequencing.
Correct transformants were obtained on the first try for all four constructs. For assessment of the
site-directed mutagenesis, we had planned to digest isolated plasmid DNA of potential transformants
(after analysis of colony PCR) with PstI to verify if the restriction site was successfully changed
or not. The samples which were not cut would have then been sequenced for final confirmation of
successful mutagenesis. For our proposal on how to successfully assemble GA5 and GA6 from our
vitamin B12 side project, which are composed of one more fragment than GA1-4, see [Outlook](https://2020.igem.org/Team%3ATuebingen/Results).

Despite our encountered problem with the iGEM BioBrick compatibility, we continued experimenting with
the already older constructs to gain a first impression of what results to expect, and to optimize the
experimental set-up. By changing one nucleotide at a wobble position in the PstI restriction site, the
resulting amino acid sequence is identical. Of course, codon usage is irregular, but this only provides
more value by allowing comparative analysis of two constructs that are different in their nucleotide
sequence yet identical in their amino acid sequence. The information collected could then serve future
iGEM teams when deciding if a particular illegal sequence can be changed in a certain way that makes it
compatible with the standards and comparibly functional to its original form.

The experimental results obtained from the older and also from the newly modified constructs indicated
that there was a functional issue: No color change nor obvious fluorescence was observed in any case,
contrary to our anticipated results. The fluorescence intensity measured from *E. coli* containing FAST2
was similar to the negative control, as only autofluorescence was observed. Furthermore, no amount of
manganese was able to induce a color change in *E. coli* containing the chromoprotein.

**Research**: Therefore, we returned to literature research and took a closer look at our design to
figure out
where a problem might have occured. We quickly detected a misconception during our project
design that led to these results. Originally, we thought that due to the nature of the
relationship between a riboswitch and an RBS, they would have to be at a specific distance from
each other and, therefore, a RBS would be included in the riboswitch sequence from the parts
registry. In accordance with our presumption, we did not add an RBS in front of the FAST2
sequence, as this would have overridden the regulatory function of the riboswitch. This turned
out to be an expensive mistake leading to no expression of our genes of interest.

**Design & Test**: At the time of realization, we were a few days away from wiki freeze. Since there
was
no time to order new sequences, and repeat our mastered Gibson Assembly method, we came up with a
plan to at least test the functionality of our manganese-inducible promoter. Our plan was to analyze
transcriptional regulation, since this was independent of the RBS. The only method available to us
on such short notice was RT-PCR to analyze mRNA synthesis. We designed a qualitative experiment to
determine if mRNA synthesis was being promoted by the manganese-inducible promoter of GA1 and GA2 in
media containing 10 µM manganese(II) chloride. We even thought of a creative way of lysing our cells
for RNA extraction (see Troubleshooting). Unfortunately, the experiment was inconclusive (see
 [Results](https://2020.igem.org/Team%3ATuebingen/Results)).

**Research & Imagine**: While conducting our troubleshooting literature research, we also gathered
more knowledge about manganese homeostasis in *E. coli*. It was found that the cells reduce the expression
of the manganese importer MntH in the presence of high manganese levels [18, 19]. This may interfere
with our biosensor, as it would prevent a proportional response of the sensor towards extracellular
manganese concentrations. In contrast, it was shown that manganese import is increased under oxidative
stress [19]. Strains lacking catalase/peroxidase (Hpx-) cannot degrade hydrogen peroxide (H2O2) and
accumulate H2O2 when cultured in oxic media [20]. As presented, there are multiple options for
optimization of our system (see  [Outlook](https://2020.igem.org/Team%3ATuebingen/Results)).

**Conclusion**: Looking back at all the roadblocks that we were able to overcome, we consider our
engineering experience as successful. We experienced first-hand how unpredictable and sensitive
laboratory work can be, and how beautiful it is when different minds cooperate to find solutions for our
human mistakes. Like for many others, the SARS-CoV-2 pandemic was our largest roadblock. Its
consequences included difficulties finding a lab and consequently our late start there in the middle of
Summer, transferring to a second lab in Autumn, the limited time and space available in these labs to
ensure safety in the workplace, and not being able to meet in real life to discuss early design and
planning steps.

In spite of the difficult situation, we managed to clone our main composite parts by iterating through
the Engineering-Design-Cycle (**Fig. 1**). Using the extensive toolbox synthetic biology has to offer, we
overcame many issues occurring in the wet lab, especially for preparing our individual fragments for
Gibson Assembly, which took time to perfect. For cloning of our composite parts, we managed to
troubleshoot efficiently and can now provide reliable protocols to replicate this section of our work.
By applying the Build-Test-Learn-Improve cycle throughout the course of our work, we detected the
sequence mistake responsible for the shortcomings of our composite parts, and can now proceed to fix
this mistake and test them again. This proves how important it is to follow this procedure in a
meticulous way to implement a new system in synthetic biology.

Although we realized that our systems require a sequence adjustment to work as expected, and we lacked
the time to correct this before this year’s wiki freeze, all of us are satisfied with the development of
our project. Each and every one of us learned new skills and became a better scientist in a matter of
months. We could not have asked for a more realistic experience to teach us how complicated and fun
science can be.

### Drylab

![Figure 3: Engineering-Design-Cycle with details how each step was approached within the dry lab work.](https://static.igem.org/mediawiki/2020/6/60/T--Tuebingen--engi-fig3.png)

**Figure 3: Engineering-Design-Cycle with details how each step was approached within the dry lab
work.**

**Research**: In order to find a way to contribute to the project from a perspective of modelling,
the drylab members conducted literature and database research on their own, primarily about
riboswitches, phytochelatin, and their binding mechanisms to manganese and other ions. Based on this
research, we assumed that the amino acid cysteine, which appears often in our phytochelatin, binds to
the manganese ions through its thiol groups.

**Imagine**: We identified the great potential in the improvement of the binding of phytochelatin to
manganese, so we decided to tackle this challenge. Additionally, we realized that the three-dimensional
structure of the phytochelatin we are using is unknown. This led us to wonder how replacing cysteine
with other amino acids would affect the structure of our phytochelatin and its binding to manganese.

**Design**: We exchanged all cysteine codons in the phytochelatin sequence, once for histidine and
once
for aspartic acid codons. The decision was based on their higher electronegativity in comparison to
cysteine, which might strengthen the binding to manganese.

**Build**: The structures of the original sequence and the two modified sequences were predicted using a
mixture of homology and *ab initio* modelling (see  [Model](https://2020.igem.org/Team%3ATuebingen/Model)).

**Test**: To test the stability of our newfound structures, we needed to employ molecular dynamics
modelling over 50 ns.

**Learn**: While the structure of the original sequence and the histidine variant were stable over
the whole simulation time span, the aspartic acid variant was not. We then had to evaluate whether to
discard the structure or change the model parameters.
From our collaboration with Waterloo, we learned about the potential of quantum mechanical force fields
such as CHARMM for better estimation of sequence stability.

**Improve**: After changing the forcefield, the structure of the aspartic acid variant was stable for
a
relatively long time of 48 ns. Through this modification, we were able to validate this structure as
well.

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17. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO. Enzymatic
    assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009; 6(5):343–5.
18. Patzer SI, Hantke K. Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the
    mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli. J Bacteriol 2001;
    183(16):4806–13.
19. Anjem A, Varghese S, Imlay JA. Manganese import is a key element of the OxyR response to hydrogen
    peroxide in Escherichia coli. Mol Microbiol 2009; 72(4):844–58.
20. Seaver LC, Imlay JA. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia
    coli. J Bacteriol 2001; 183(24):7182–9.

[![](https://static.igem.org/mediawiki/2020/0/0a/T--Tuebingen--unibound.png)](https://uni-tuebingen.de/en/universitaet/profil/freunde-und-foerderer/universitaetsbund/)

[![](https://static.igem.org/mediawiki/2020/4/47/T--Tuebingen--vwr.png)](https://vwr.com/)

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[![](https://static.igem.org/mediawiki/2020/0/05/T--Tuebingen--moldevT.png)](https://www.moleculardevices.com/)

---

[![](https://static.igem.org/mediawiki/2019/0/0b/T--Tuebingen--4base_logo.png)](http://s466755640.online.de/4bl/)

[![](https://static.igem.org/mediawiki/2019/3/31/T--Tuebingen--JBS_logo.png)](https://www.jenabioscience.com/)

[![](https://static.igem.org/mediawiki/2019/1/18/T--Tuebingen--microsynth_logo.png)](https://www.microsynth.ch/home-ch.html)

[![](https://static.igem.org/mediawiki/2019/a/ae/T--Tuebingen--greiner_logo.png)](https://www.gbo.com/)

[![](https://static.igem.org/mediawiki/2019/f/f8/T--Tuebingen--promega_logo.png)](https://www.promega.de/en/)

[![](https://static.igem.org/mediawiki/2020/9/97/T--Tuebingen--tenbo.png)](https://tonbobio.com/)

---

[![](https://static.igem.org/mediawiki/2020/c/c4/T--Tuebingen--Quiagen.png)](https://www.qiagen.com/us/)

[![](https://static.igem.org/mediawiki/2020/2/2c/T--Tuebingen--Dieter.png)](https://www.dieter-schwarz-stiftung.de/homepage.html)

[![](https://static.igem.org/mediawiki/2020/2/2b/T--Tuebingen--absource.png)](https://absource.de/)

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[![](https://static.igem.org/mediawiki/2020/a/a2/T--Tuebingen--Roth.png)](https://www.carlroth.com/com/en/)

---

[![](https://static.igem.org/mediawiki/2020/9/98/T--Tuebingen--eppendorf.png)](https://www.eppendorf.com/OC-en/)

[![](https://static.igem.org/mediawiki/2020/0/04/T--Tuebingen--Zymo.png)](https://www.zymoresearch.de/)

[![](https://static.igem.org/mediawiki/2020/9/9e/T--Tuebingen--benchling.png)](https://www.benchling.com/)

[![](https://static.igem.org/mediawiki/2020/0/0d/T--Tuebingen--IDT.png)](https://eu.idtdna.com/)

[![](https://static.igem.org/mediawiki/2020/7/7e/T--Tuebingen--NEB.png)](https://www.neb-online.de/en/)

[![](https://static.igem.org/mediawiki/2019/3/3a/T--Tuebingen--snapgene_logo.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Tuebingen/Experiments

PacMn

# Experiments

[![logo](https://static.igem.org/mediawiki/2020/a/ab/T--Tuebingen--logo.png)](https://igem-tuebingen.com/index)
[##### PacMn](https://2020.igem.org/Team%3ATuebingen)

* Team & Supporters

  [Team](/Team%3ATuebingen/Team)
  [Team Statistics](/Team%3ATuebingen/Team_Statistics)
  [Attributions](/Team%3ATuebingen/Attributions)
  [Excellence](/Team%3ATuebingen/Excellence)
  [Sponsors](/Team%3ATuebingen/Sponsors)
* Project

  [Description](/Team%3ATuebingen/Description)
  [Design](/Team%3ATuebingen/Design)
  [Parts](/Team%3ATuebingen/Parts)
  [Engineering](/Team%3ATuebingen/Engineering)
  [Model](/Team%3ATuebingen/Model)
  [Experiments](/Team%3ATuebingen/Experiments)
  [Results](/Team%3ATuebingen/Results)
  [Contribution](/Team%3ATuebingen/Contribution)
  [Implementation](/Team%3ATuebingen/Implementation)
  [Notebook](/Team%3ATuebingen/Notebook)
  [Protocols](/Team%3ATuebingen/Protocols)[Safety](/Team%3ATuebingen/Safety)
* Outreach

  [Human Practices](/Team%3ATuebingen/Human_Practices)
  [Education](/Team%3ATuebingen/Education)
  [Collaborations](/Team%3ATuebingen/Collaborations)
* Deliverables

  [Poster](/Team%3ATuebingen/Poster)
  [Judging
  Form](https://igem.org/2020_Judging_Form?team=Tuebingen)
  [Presentation Video](/Team%3ATuebingen/Presentation)
  [Project Promotion
  Video](/Team%3ATuebingen/Promo_Video)

### Phase 1: Cloning

After designing our constructs and the primers for Gibson Assembly (GA), we ordered the DNA molecules
from Integrated DNA Technologies, Inc. (IDT). The first step in cloning was to prepare the individual
fragments for GA. This consisted in amplifying each fragment with the special primers designed to add
complementary overhangs, and linearizing the vector backbone through restriction digestion. The products
were qualitatively analyzed by gel electrophoresis and their concentrations (ng/µl) were measured with a
spectrophotometer (**Fig. 1**).

![Figure 1: Overview of fragment preparation for Gibson Assembly.](https://static.igem.org/mediawiki/2020/2/27/T--Tuebingen--experiments_fig1.png)

**Figure 1: Overview of fragment preparation for Gibson Assembly.**

With the ready-to-use fragments, the next step was to assemble them. The assembly itself only
takes about an hour, but the whole process from assembling to having a confirmed positive clone required
six main steps (**Fig. 2**):

1. **Gibson Assembly:** Assembling the fragments *in vitro*.
2. **Transformation:** Transforming chemically competent *E. coli* with DNA from the
   assembly
   reaction.
3. **Colony PCR:** Preliminary screening of potential positive transformants by amplifying
   insert DNA and analyzing its size.
4. **Plasmid isolation:** Extraction of plasmid DNA of potential positive transformants for
   further analysis.
5. **Restriction digestion:** Linearization of isolated plasmid DNA for a second size analysis
   (of the entire plasmid).
6. **Sequencing:** Sending the size-confirmed candidates for sequencing to rule out mutations
   within the insert DNA.

![Figure 2: Schematic overview of cloning workflow. The main steps are ordered from 1 to 6. ](https://static.igem.org/mediawiki/2020/a/a0/T--Tuebingen--experiments_fig2.png)

**Figure 2: Schematic overview of cloning workflow.**  The main steps
are ordered from 1 to 6.

### Phase 2: Testing

The most interesting test for our constructs consisted in measuring fluorescence in the presence of
different manganese(II) chloride concentrations (**Fig. 3**). The measurements for all
constructs were conducted simultaneously in a plate reader, including an empty pUC19 vector as a
control.

![Figure 3: Schematic overview of fluorescence measurement workflow. The main steps are ordered from 1 to 3.](https://static.igem.org/mediawiki/2020/9/9a/T--Tuebingen--experiments_fig3.png)

**Figure 3: Schematic overview of fluorescence measurement
workflow.** The main
steps are ordered from 1 to 3.

As a qualitative test for fluorescence, we analyzed our different clones via fluorescence microscopy. We
also observed the morphology of our cells containing the different constructs to search for stress
phenotypes (**Fig 4.**).

![Figure 4: Analysis of cell fluorescence and morphology via Microscopy.](https://static.igem.org/mediawiki/2020/1/19/T--Tuebingen--experiments_fig4.png)

**Figure 4: Analysis of cell fluorescence and morphology via
Microscopy.**

As a qualitative validation of our Mn-inducible components, we grew E. coli containing the chromoprotein
gene on selective solid and liquid LB medium with manganese(II) chloride (**Fig. 5**).

![Figure 5: Testing the effect of chromoprotein expression on cell color.](https://static.igem.org/mediawiki/2020/a/a9/T--Tuebingen--experiments_fig5.png)

**Figure 5: Testing the effect of chromoprotein expression on cell
color.**

To roughly test the transgenerational propagation stability of our constructs, we performed a
sub-cultivation experiment on non-selective solid LB medium (**Fig. 6**).

![Figure 6: Sub-cultivation experiment as a plasmid stability test.](https://static.igem.org/mediawiki/2020/6/60/T--Tuebingen--experiments_fig6.png)

**Figure 6: Sub-cultivation experiment as a plasmid stability test.**

To analyze the functionality of our manganese-inducible promoters in the absence of protein expression,
we performed Reverse Transcriptase (RT)-PCR. Here, we evaluated the transcriptional activation
presumably initiated by manganese(II) chloride (**Fig.
7**).

![Figure 7: Verification of mRNA synthesis via RT-PCR.](https://static.igem.org/mediawiki/2020/d/d6/T--Tuebingen--experiments_fig7.png)

**Figure 7: Verification of mRNA synthesis via RT-PCR.**

### Image sources

Figures 1-7: Created with BioRender.com

[![](https://static.igem.org/mediawiki/2020/0/0a/T--Tuebingen--unibound.png)](https://uni-tuebingen.de/en/universitaet/profil/freunde-und-foerderer/universitaetsbund/)

[![](https://static.igem.org/mediawiki/2020/4/47/T--Tuebingen--vwr.png)](https://vwr.com/)

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[![](https://static.igem.org/mediawiki/2019/e/eb/T--Tuebingen--Logo-Experimenta.png)](https://www.experimenta.science/en/)

[![](https://static.igem.org/mediawiki/2020/0/05/T--Tuebingen--moldevT.png)](https://www.moleculardevices.com/)

---

[![](https://static.igem.org/mediawiki/2019/0/0b/T--Tuebingen--4base_logo.png)](http://s466755640.online.de/4bl/)

[![](https://static.igem.org/mediawiki/2019/3/31/T--Tuebingen--JBS_logo.png)](https://www.jenabioscience.com/)

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[![](https://static.igem.org/mediawiki/2019/f/f8/T--Tuebingen--promega_logo.png)](https://www.promega.de/en/)

[![](https://static.igem.org/mediawiki/2020/9/97/T--Tuebingen--tenbo.png)](https://tonbobio.com/)

---

[![](https://static.igem.org/mediawiki/2020/c/c4/T--Tuebingen--Quiagen.png)](https://www.qiagen.com/us/)

[![](https://static.igem.org/mediawiki/2020/2/2c/T--Tuebingen--Dieter.png)](https://www.dieter-schwarz-stiftung.de/homepage.html)

[![](https://static.igem.org/mediawiki/2020/2/2b/T--Tuebingen--absource.png)](https://absource.de/)

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[![](https://static.igem.org/mediawiki/2020/b/b9/T--Tuebingen--Thermo_Fischer.png)](https://www.thermofisher.com/de/de/home.html)

[![](https://static.igem.org/mediawiki/2020/a/a2/T--Tuebingen--Roth.png)](https://www.carlroth.com/com/en/)

---

[![](https://static.igem.org/mediawiki/2020/9/98/T--Tuebingen--eppendorf.png)](https://www.eppendorf.com/OC-en/)

[![](https://static.igem.org/mediawiki/2020/0/04/T--Tuebingen--Zymo.png)](https://www.zymoresearch.de/)

[![](https://static.igem.org/mediawiki/2020/9/9e/T--Tuebingen--benchling.png)](https://www.benchling.com/)

[![](https://static.igem.org/mediawiki/2020/0/0d/T--Tuebingen--IDT.png)](https://eu.idtdna.com/)

[![](https://static.igem.org/mediawiki/2020/7/7e/T--Tuebingen--NEB.png)](https://www.neb-online.de/en/)

[![](https://static.igem.org/mediawiki/2019/3/3a/T--Tuebingen--snapgene_logo.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Tuebingen/Results

![](https://static.igem.org/mediawiki/2020/0/00/T--Tuebingen--loadingpacman.gif)

PacMn

# Results

[![logo](https://static.igem.org/mediawiki/2020/a/ab/T--Tuebingen--logo.png)](https://igem-tuebingen.com/index)
[##### PacMn](https://2020.igem.org/Team%3ATuebingen)

* Team & Supporters

  [Team](/Team%3ATuebingen/Team)
  [Team Statistics](/Team%3ATuebingen/Team_Statistics)
  [Attributions](/Team%3ATuebingen/Attributions)
  [Excellence](/Team%3ATuebingen/Excellence)
  [Sponsors](/Team%3ATuebingen/Sponsors)
* Project

  [Description](/Team%3ATuebingen/Description)
  [Design](/Team%3ATuebingen/Design)
  [Parts](/Team%3ATuebingen/Parts)
  [Engineering](/Team%3ATuebingen/Engineering)
  [Model](/Team%3ATuebingen/Model)
  [Experiments](/Team%3ATuebingen/Experiments)
  [Results](/Team%3ATuebingen/Results)
  [Contribution](/Team%3ATuebingen/Contribution)
  [Implementation](/Team%3ATuebingen/Implementation)
  [Notebook](/Team%3ATuebingen/Notebook)
  [Protocols](/Team%3ATuebingen/Protocols)
  [Safety](/Team%3ATuebingen/Safety)
* Outreach

  [Human Practices](/Team%3ATuebingen/Human_Practices)
  [Education](/Team%3ATuebingen/Education)
  [Collaborations](/Team%3ATuebingen/Collaborations)
* Deliverables

  [Poster](/Team%3ATuebingen/Poster)
  [Judging
  Form](https://igem.org/2020_Judging_Form?team=Tuebingen)
  [Presentation Video](/Team%3ATuebingen/Presentation)
  [Project Promotion
  Video](/Team%3ATuebingen/Promo_Video)

### Cloning

Successful cloning depended on three main steps: PCR, Gibson Assembly, and chemical transformation of E.
coli. These steps were gradually optimized until we finally perfected the procedure to clone all four of
our main composites: GA1 (BBa\_K3510002), GA2 (BBa\_K3510003), GA3 (BBa\_K3510004), and GA4 (BBa\_K3510005)
(**Fig. 1**). These composites can now be easily assembled by
anyone (see [Protocols](https://2020.igem.org/Team%3ATuebingen/Protocols)
and [Notebook](https://2020.igem.org/Team%3ATuebingen/Protocols)). For cloning of GA5 and GA6, we have proposed an experimental
procedure in our [Outlook](https://2020.igem.org/Team%3ATuebingen/Results).

![Figure 1 Plate 1](https://static.igem.org/mediawiki/2020/b/b3/T--Tuebingen--results_fig1.png)
![Figure 1 Plate 2](https://static.igem.org/mediawiki/2020/1/16/T--Tuebingen--results_fig2.png)
![Figure 1 Plate 3: Agarose gel electrophoresis of colony PCR of GA1, GA2, GA3,                     GA4                     (corrected sequences for iGEM compatibility). The products were separated by electrophoresis in                     1 %                     agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. Desired band sizes: ca. 1800 bp                     (GA1),                     ca. 1200 bp (GA2), ca. 1600 bp (GA3), and ca. 1000 bp (GA4). Cell material of the old GA1-4                     sequenced                     clones (containing a PstI restriction site) was used as positive control of PCR and gel                     electrophoresis.                     The negative control was the PCR master mix without template DNA. Approximately 88 % of the GA1-4                     test                     samples are correct.](https://static.igem.org/mediawiki/2020/a/ae/T--Tuebingen--results_fig3.png)

**Figure 1: Agarose gel electrophoresis of colony PCR of GA1, GA2, GA3,
GA4
(corrected sequences for iGEM compatibility)**. The products were separated by electrophoresis in
1 %
agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. Desired band sizes: ca. 1800 bp
(GA1),
ca. 1200 bp (GA2), ca. 1600 bp (GA3), and ca. 1000 bp (GA4). Cell material of the old GA1-4
sequenced
clones (containing a PstI restriction site) was used as positive control of PCR and gel
electrophoresis.
The negative control was the PCR master mix without template DNA. Approximately 88 % of the GA1-4
test
samples are correct.

Comparing the high cloning efficiency represented above by the colony PCR results with the cloning
efficiency in our first attempt (**Fig. 2**), we can say with confidence that our cloning experience
was
a
success. We were able to increase the efficiency by a large margin, demonstrating how important
optimization
is.

![Figure 2: Gel electrophoresis of colony PCR from GA1 (old sequence, first attempt)</b>. The products                     were separated by electrophoresis in 1 % agarose gels. As a marker, the BenchTop 1kb DNA Ladder was                     used. pUC19 is a negative control, where only the empty plasmid was transformed. Samples 12 and 13                     are labelled, as these were positive.](https://static.igem.org/mediawiki/2020/0/03/T--Tuebingen--Gel_Fig6.png)

**Figure 2: Gel electrophoresis of colony PCR from GA1 (old sequence, first attempt)**. The products
were separated by electrophoresis in 1 % agarose gels. As a marker, the BenchTop 1kb DNA Ladder was
used. pUC19 is a negative control, where only the empty plasmid was transformed. Samples 12 and 13
are labelled, as these were positive.

A major part of our optimization consisted in perfecting PCR to create fragments with the proper
overhangs for Gibson Assembly®. Our most troublesome fragment was F (FAST - Chromoprotein - Terminator
fragment), which did not show any specific gel bands in the first attempts with the UCP HiFidelity PCR
Kit (QIAGEN) (**Fig. 3**). By switching to the AllTaq PCR Master Mix Kit (QIAGEN), a more robust PCR kit
that enhances primer binding, we finally obtained a specific gel band for fragment F (**Fig. 4** B) but also
many unspecific bands. Since unspecific gel bands were observed for multiple other fragments (**Fig. 4** A),
we decided to exclude these through gel extraction of the specific gel bands. The purity before (**Fig. 5**
A) and after **Fig. 5** B) gel extraction improved considerably.

![ Figure 3: Gel electrophoresis of gradient PCR (UCP HiFidelity PCR Kit) of fragment F</b>. The                     products were separated by electrophoresis in 1 % agarose gels. As a marker, the BenchTop 1kb DNA                     Ladder was used. The specific gel bands can not be clearly differentiated from the DNA smears.](https://static.igem.org/mediawiki/2020/5/52/T--Tuebingen--results_fig5.png)

**Figure 3: Gel electrophoresis of gradient PCR (UCP HiFidelity PCR Kit) of fragment F**. The
products were separated by electrophoresis in 1 % agarose gels. As a marker, the BenchTop 1kb DNA
Ladder was used. The specific gel bands can not be clearly differentiated from the DNA smears.

![Figure 4: Gel electrophoresis of gradient PCR (UCP HiFidelity PCR Kit) of fragment C (Fig. 4A) and                     fragment F (AllTaq PCR Master Mix Kit) (Fig. 4B)</b>. The products were separated by electrophoresis                     in 1 % agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. Desired band sizes: ca. 900                     bp for fragment C (GFP - Terminator fragment) and ca. 1300 bp for fragment F. In both gel images,                     specific gel bands are observable, accompanied by non-specific bands.](https://static.igem.org/mediawiki/2020/8/82/T--Tuebingen--results_fig6.png)

**Figure 4: Gel electrophoresis of gradient PCR (UCP HiFidelity PCR Kit) of fragment C (**Fig. 4**A) and
fragment F (AllTaq PCR Master Mix Kit) (**Fig. 4**B)**. The products were separated by electrophoresis
in 1 % agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. Desired band sizes: ca. 900
bp for fragment C (GFP - Terminator fragment) and ca. 1300 bp for fragment F. In both gel images,
specific gel bands are observable, accompanied by non-specific bands.

![Figure 5: Gel electrophoresis of PCR of fragment C and fragment F before (Fig. 5A) and after (Fig. 5B) gel extraction </b>. These products were separated by electrophoresis in 1% agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. Desired band sizes: ca. 900 bp for fragment C and ca. 1300 bp for fragment F. In both gel images, specific gel bands are observable. The intensity of non-specific bands decreases considerably after gel extraction.](https://static.igem.org/mediawiki/2020/9/9b/T--Tuebingen--results_fig7.png)

**Figure 5: Gel electrophoresis of PCR of fragment C and fragment F before (**Fig. 5**A) and after (Fig.
5B) gel extraction** . These products were separated by electrophoresis in 1% agarose gels. As a
marker, the BenchTop 1kb DNA Ladder was used. Desired band sizes: ca. 900 bp for fragment C and ca.
1300 bp for fragment F. In both gel images, specific gel bands are observable. The intensity of
non-specific bands decreases considerably after gel extraction.

The last step to confirm successful cloning was sequencing. We completely sequenced the DNA of our
composite parts with the Sanger method. Here, we realized that the sequences contained a PstI
restriction site, which is incompatible with this year’s standards BioBrick RFC[10] and Type IIS
RFC[1000]. Initially, we had excluded this restriction site in the design, but we accidentally ordered
the unmodified sequences. By ordering the correct DNA sequences that contain a silent mutation in the
leucine codon of the PstI restriction site, and by sticking to our optimized cloning procedures, we were
able to correct this mistake efficiently (**Fig. 6**).

![Figure 6: Alignment of Sanger sequencing results of the correct version of GA1 with the                      incorrect reference sequence (SnapGene). Primers utilized: Microsynth standard primers M13-40                       (forward) and  M13-r (reverse), and custom-made primers FAST_mid_FW and FAST_mid_RV.                        The reference sequence is incorrect, as it contains the illegal restriction site PstI.                        The sequence of GA1 aligns perfectly, except with the PstI restriction site.](https://static.igem.org/mediawiki/2020/f/f8/T--Tuebingen--results_fig9.png)

**Figure 6: Alignment of Sanger sequencing results of the correct version of GA1 with the incorrect
reference sequence (SnapGene)**. Primers utilized: Microsynth standard primers M13-40 (forward)
and
M13-r (reverse), and custom-made primers FAST\_mid\_FW and FAST\_mid\_RV. The reference sequence is
incorrect, as it contains the illegal restriction site PstI. The sequence of GA1 aligns perfectly,
except with the PstI restriction site.

### Fluorescence measurement with a plate reader

Once all of our constructs had been cloned into *E. coli*, the next step was to assess the functionality
of our systems. For this, we measured the emitted fluorescence after growth in selective LB media
containing three different manganese concentrations: 0 µM, 10 µM, and 20µM. We also compared our older
incorrect constructs to our correctly modified constructs to analyze the effect of codon usage on
protein expression.

Contrary to our expectations, there was no clear distinction between the fluorescence emitted by *E. coli*
containing our constructs and *E. coli* containing an empty pUC19 plasmid. Moreover, the fluorescence
intensity of *E. coli* containing manganese-inducible promoters (GA1 and GA2) did not increase with higher
manganese concentrations (**Fig. 7** A-C). Since the results were inconclusive, we assume that we only
measured background noise generated by the auto-fluorescence of the cells.

![Figure 7 Part 1](https://static.igem.org/mediawiki/2020/2/27/T--Tuebingen--results_fig10.png)
![Figure 7 Part 2](https://static.igem.org/mediawiki/2020/a/a0/T--Tuebingen--results_fig11.png)
![Figure 7: Fluorescence intensity of E. coli containing different constructs after incubation in                     media with different manganese concentrations</b>. The experiments were repeated three times, each                     in triplicates. The mean values +/- standard deviation are shown. Constructs labelled as “old                     sequence” still include the PstI restriction site while constructs labelled as “new sequence” do                     not. (A) 0 µM manganese (II) chloride. (B) 10 µM manganese (II) chloride. (C) 20 µM manganese (II)                     chloride.](https://static.igem.org/mediawiki/2020/4/4d/T--Tuebingen--results_fig12.png)

**Figure 7: Fluorescence intensity of *E. coli* containing different constructs after incubation in
media with different manganese concentrations**. The experiments were repeated three times, each
in triplicates. The mean values +/- standard deviation are shown. Constructs labelled as “old
sequence” still include the PstI restriction site while constructs labelled as “new sequence” do
not. (A) 0 µM manganese (II) chloride. (B) 10 µM manganese (II) chloride. (C) 20 µM manganese (II)
chloride.

### Imaging by fluorescence microscopy

An alternative option for assessing fluorescence is by fluorescence microscopy. Here, we compared E.
coli containing the empty pUC19 plasmid (negative control) and *E. coli* with the corrected GA2 construct.
The cells were grown in selective LB media containing 10 µM manganese(II) chloride to induce our
regulation system before they were labelled with the TF-Lime fluorogen. The fluorescence intensity
observed in both cultures was very similar (**Fig. 8**). Again, background from the auto-fluorescence
emitted by the cells is presumed.

![Figure 8: Live cell imaging of cells containing pUC19 and GA2 (corrected version) under the fluorescence microscope. Laser settings: wavelengths 485 nm (absorbance) and 535 nm (emission). 1.5 x 630 zoom. (A) pUC19 control, 10 µM manganese(II) chloride. (B) GA2 (corrected version), 10 µM manganese(II) chloride.](https://static.igem.org/mediawiki/2020/9/94/T--Tuebingen--results_fig13.png)

**Figure 8: Live cell imaging of cells containing pUC19 and GA2 (corrected version)** under the
fluorescence microscope. Laser settings: wavelengths 485 nm (absorbance) and 535 nm (emission). 1.5
x 630 zoom. (A) pUC19 control, 10 µM manganese(II) chloride. (B) GA2 (corrected version), 10 µM
manganese(II) chloride.

### Chromoprotein test

Since the results obtained from fluorescence experiments were inconclusive (**Fig. 7** and **Fig. 8**), we
decided to qualitatively test the expression of the blue chromoprotein of GA1 and GA3. For this, a
sequenced clone for GA1 and GA3 (old and new versions) and a pUC19 clone were streaked on selective LB
agar plates (100 µg/ml ampicillin sodium salt) containing three different manganese(II) chloride
concentrations (0 µM, 10 µM, and 20 µM). Blue colonies were expected for colonies grown on manganese as
opposed to colonies grown on the control plates without manganese. However, the plates did not reveal
any blue coloring of the colonies growing on plates with manganese, regardless of the concentration
(**Fig. 9**).

![Figure 9: E. coli colonies containing the constructs with the chromoprotein gene (GA1 and GA3) grown on plates with different  manganese(II) chloride concentrations. The selective agar plates (100 µg/ml ampicillin sodium salt)  were prepared with three different manganese(II) chloride concentrations: 0 µM (A), 10 µM (B), or 20 µM (C)..](https://static.igem.org/mediawiki/2020/f/fb/T--Tuebingen--results_fig14.png)

**Figure 9: *E. coli* colonies containing the constructs with the chromoprotein gene (GA1 and GA3) grown
on plates with different manganese(II) chloride concentrations**. The selective agar plates (100
µg/ml
ampicillin sodium salt) were prepared with three different manganese(II) chloride concentrations: 0
µM (A), 10 µM (B), or 20 µM (C).

The failure to produce fluorescence (**Fig. 7** and **Fig. 8**) or a visible blue color (**Fig. 9**) with our GA1-4
constructs (old and corrected versions) made us take a closer look at our sequences again. We noticed
the absence of a ribosome binding site (RBS) between our riboswitch sequence and the
phytochelatin/chromoprotein gene. The mistake in our design was caused by the misconception that the
riboswitch sequence available in the parts registry would include an RBS downstream of the riboswitch.

### Stability test

We assessed the extent of transgenerational stability or toxicity of the constructs GA1, GA2, GA3, and
GA4 in comparison to the pUC19 plasmid control in E.coli by nine transfers of clones through
non-selective media with at least one overnight incubation at 37 °C in between (**Fig. 10**). The final
transfer was performed to a selective purity plate which showed single colonies after overnight
incubation at 37 °C (**Fig. 11**).

![ Figure 10: Some of the non-selective LB agar plates for the plasmid stability test of constructs GA1, GA2, GA3, GA4, and the empty pUC19 vector in E. coli. With a few exceptions for the pUC19 clone, all clones were transferred to the same plate each time, separated by a grid.](https://static.igem.org/mediawiki/2020/8/83/T--Tuebingen--results_fig15.png)

**Figure 10: Some of the non-selective LB agar plates for the plasmid stability test of constructs
GA1, GA2, GA3, GA4, and the empty pUC19 vector in *E. coli***. With a few exceptions for the pUC19
clone, all clones were transferred to the same plate each time, separated by a grid.

![Figure 11: Final selective LB agar plate after nine transfers of E. coli containing GA1, GA2, GA3, and GA4.  Single colonies were picked from this purity plate for colony PCR.](https://static.igem.org/mediawiki/2020/0/00/T--Tuebingen--results_fig16.png)

**Figure 11: Final selective LB agar plate after nine transfers of *E. coli* containing GA1, GA2, GA3,
and GA4**. Single colonies were picked from this purity plate for colony PCR.

Colony PCR was performed to evaluate the presence of the constructs after the transgenerational
stability test (**Fig. 12**).

The targets of the PCRs were observed at the desired band size of GA1 (ca. 1800 bp), GA2 (ca. 1200 bp),
GA3 (ca. 1600 bp) and GA4 (ca. 1000 bp) on the gel (**Fig. 12**). Since the bands for samples GA1 and GA3
were very faint (indicated by red arrows), and smearing and inconsistency occurred in the positive
controls, a PCR amplification error was discussed. Therefore, a repetition of this PCR is advisable.
Nonetheless, it can be cautiously surmised that the presence of the plasmids without expression of our
constructs is tolerated for nine transfers and therefore presumably does not constitute a heavy burden
for the bacteria. As the proteins encoded in the constructs cannot be expressed, no further assumptions
can be made concerning their stability or toxicity.

![Figure 12: Gel electrophoresis of colony PCR of stability experiment samples with controls. Samples for E. coli containing the constructs GA1, 2, 3, and 4 were taken from the final selective plate, while the pUC19 colony that underwent similar transfers was chosen from the penultimate non-selective plate. Positive controls for GA1, GA2, GA3, GA4  and pUC19 were picked from their respective pre-existing purity plates. Results of the colony PCR and subsequent separation via  electrophoresis in 1% agarose gels are shown in this figure. As a marker, the BenchTop 1kb DNA Ladder was used.](https://static.igem.org/mediawiki/2020/4/40/T--Tuebingen--results_fig17.png)

**Figure 12: Gel electrophoresis of colony PCR of stability experiment samples with controls**.
Samples for *E. coli* containing the constructs GA1, 2, 3, and 4 were taken from the final selective
plate, while the pUC19 colony that underwent similar transfers was chosen from the penultimate
non-selective plate. Positive controls for GA1, GA2, GA3, GA4 and pUC19 were picked from their
respective pre-existing purity plates. Results of the colony PCR and subsequent separation via
electrophoresis in 1% agarose gels are shown in this figure. As a marker, the BenchTop 1kb DNA
Ladder was used.

### mRNA experiment

With only two days left for wet lab work after noticing the missing RBS in our constructs, we designed a
Reverse Transcriptase (RT)-PCR experiment to investigate mRNA synthesis and validate our promoters,
which function independently of an RBS.

We expected an amplicon of ca. 320 bp and a difference in signal intensity between growth with 0 µM and
10 µM manganese(II) chloride for constructs with manganese-inducible promoters. However, the negative
controls had the same band sizes and different manganese concentrations did not produce different signal
intensities (**Fig. 13**). Although the band sizes of the negative controls seem to be slightly larger, this
could be explained by a variable gel morphology. Interestingly, all negative controls have similar bands
to the AllTaq PCR Master Mix negative control, which did not contain template cDNA. In summary, the
experiment was inconclusive. In order to gain more conclusive results, optimization of the PCR is
required.

![ Figure 13: Gel electrophoresis of cDNA amplification via PCR. The desired band size is ca. 320 bp. Test subjects include the old GA1-4 clones and the corrected GA2 and GA3 clones. Negative controls include a pUC19 clone, the reverse transcription (RT) reaction mix, and the AllTaq PCR master mix without template cDNA. As a marker, the BenchTop 1kb DNA Ladder was used. ](https://static.igem.org/mediawiki/2020/5/57/T--Tuebingen--results_fig18.png)

**Figure 13: Gel electrophoresis of cDNA amplification via PCR** .
The
desired band size is ca. 320 bp. Test subjects include the old GA1-4 clones and the corrected GA2
and GA3 clones. Negative controls include a pUC19 clone, the reverse transcription (RT) reaction
mix, and the AllTaq PCR master mix without template cDNA. As a marker, the BenchTop 1kb DNA Ladder
was used.

### Outlook

Ribosome binding site (RBS)

Having to include an RBS in our constructs in order to have them expressed, we evaluated two choices.
First, ordering corrected DNA fragments and repeating PCRs and Gibson Assemblies, and second, appending
an RBS either downstream of the riboswitch fragment or upstream of the FAST2 fragment by PCR
amplification with primers containing the RBS sequence. The second choice would also require Gibson
Assembly consecutively. Either way, new primers for Gibson Assembly would have to be ordered, because
the addition of the RBS changes the end of one fragment and therefore the necessary overhangs. As
amplification with long primers to add novel features usually requires a notorious PCR optimization
process, and both methods would require ordering DNA, we would favour the first option for which we
already have well-established protocols. Upon successful adaptation of our construct, the experiments
described in the following may be conducted.

**Manganese homeostasis - Potential for system
improvement**

While researching manganese homeostasis in E.
coli, we found that the bacteria reduce the expression of the manganese importer MntH in the presence of
high manganese levels [1, 2]. Presumably, this would hamper the functionality of our biosensor, which
can only detect intracellular manganese. One way to circumvent this issue would be to add the mntH gene
downstream of a constitutive promoter in our plasmids. Consequently, we would uncouple the manganese
importer from the manganese homeostasis regulatory network of *E. coli*.

Moreover, it was shown that manganese import is
increased under oxidative stress [2]. Strains lacking catalase/peroxidase (Hpx-) cannot degrade hydrogen
peroxide and accumulate H2O2 when cultured in aerobic media [3]. Therefore, we could use a sensitive
strain to assess whether the fluorescent signal is more reliable and dependent on manganese
concentrations when cells react to oxidative stress. As a control and to quantify the effect of
maintained manganese import, it would be necessary to culture the same strains under anaerobic
conditions. Here, our FAST2-tagged system comes in handy, because it is able to provide a fluorescent
signal under anaerobic conditions.

Phytochelatin

To assess whether the phytochelatin successfully binds manganese, we thought about three experiments:

**1. Evaluation of the rescue of *E. coli* manganese tolerance by induced expression of
phytochelatin.**

In *E. coli* lacking the manganese exporter MntP, high manganese concentrations in the medium lead to
reduced growth [5]. We presume that high manganese concentrations may be toxic due to the interference
of manganese with intracellular biochemical processes and enzymes. Therefore, we hypothesize that the
complexation of manganese by the phytochelatin may restore bacterial growth by preventing this
interference.

The experiment (Fig. 1) would include ten *E. coli* cultures containing different plasmids: Constructs
BBa\_K3510003 (GA2) and BBa\_K3510005 (GA4) with the phytochelatin and their control constructs with the
chromoprotein BBa\_K3510002 (GA1) and BBa\_K3510004 (GA3). Additionally, the empty pUC19 plasmid serves as
a negative control for regular cell growth. All cultures would be maintained at the same conditions with
standard media or media including 0.5 mM Manganese(II) chloride. For 4 to 5 hours, cell growth would be
assessed by measuring the OD600 every 30 min to 1 hour. With successful chelation of manganese(II) ions
by the phytochelatin, we would expect higher OD600 values and hence growth for these cultures, while the
chromoprotein controls should show the same (lower) OD600 as the pUC19 negative control.

Furthermore, the experiment should be performed with a mntP null mutated strain transformed with the
vector containing the phytochelatin or an empty pUC19 vector as a control. Here we hope to show that
phytochelatin expression rescues bacterial growth.

![ Figure 1: Experiment to determine cell growth in different manganese(II) chloride concentrations. ](https://static.igem.org/mediawiki/2020/9/99/T--Tuebingen--results_fig19.png)

**Figure 1: Experiment to determine cell growth in different manganese(II) chloride
concentrations.**

**2. Investigation of phytochelatin expressing *E. coli* and their presumed withdrawal of manganese from
the environment.**

We hypothesise that with the complexation of manganese by the phytochelatin, the bacteria withdraw the
ion from the surrounding media, therefore decreasing the extracellular manganese concentration. With the
complexation of manganese, we further assume that the MntP exporter of the ion will not be upregulated
via a feedback mechanism if we can show in the preceding experiment that phytochelatin rescues bacterial
growth in mntP null mutated strains. As control, we will employ our chromoprotein control constructs
which should turn a visible blue color in the presence of manganese without chelating the ion. Since a
detoxification will not occur in non-phytochelatin expressing controls, we expect a feedback loop which
will increase the MntP manganese exporter and decrease the genome-encoded MntH importer function. Hence,
in these controls manganese concentrations in the bacteria's environment should remain stable.

To evaluate the influence of phytochelatin expression on manganese clearance we will employ a chamber
with two reservoirs divided by a manganese ion permeable membrane. In manganese(II) chloride-containing
culture medium, *E. coli* expressing phytochelatin is grown in one chamber, while the other side harbours
the chromoprotein encoding control bacteria (**Fig. 2**). Due to the presence of manganese in the
environment of both cultures in the beginning of the experiment, we expect the chromoprotein to be
expressed in the control strain, providing a blue signal, while phytochelatin expression should be
upregulated in the other strain. Here, the expression of phytochelatin presumably serves for
complexation of manganese.

Since the permeable membrane ensures equal manganese concentrations on both sides of the membrane, we
hypothesise that the general manganese concentration decreases by the amount which is cleared by the
phytochelatin expressing strain. Furthermore, once the concentration falls below the limit necessary for
the expression of the chromoprotein in the control strain, the blue color should vanish.

As a control, the same experimental set-up will be followed using the chromoprotein control and a pUC19
empty vector transformed *E. coli*, respectively. Provided the experiment works as expected, the manganese
concentration should not fall below the detection limit of the chromoprotein strain, thus enabling
continuous expression of the blue chromoprotein.

![ Figure 2: Investigation of phytochelatin-expressing E. coli and their presumed withdrawal of manganese from the environment. ](https://static.igem.org/mediawiki/2020/1/14/T--Tuebingen--results_fig20.png)

**Figure 2: Investigation of phytochelatin-expressing *E. coli* and their presumed withdrawal of
manganese from the environment.**

**3. Measurement of intracellular manganese accumulation in phytochelatin expressing bacteria using
mass spectrometry.**

We expect manganese to accumulate in bacterial cells which express phytochelatin. A precise method to
assess intracellular manganese is mass spectrometry. Therefore we propose the following experiment
designed in accordance with a protocol by Martin, Waters et. al (2015). Phytochelatin or chromoprotein
encoding *E. coli* cultures are cultured for 2.5 hours with or without 0.5 mM manganese(II) chloride. They
are harvested via centrifugation and washing of the cells with Tris-HCl/EDTA and subsequently
resuspended in Tris-HCl. Next, the cells are lysed, and samples are prepared for mass spectrometry to
the amount of intracellular manganese (**Fig. 3**). For the analysis, the values are normalized to total
protein concentrations in the lysate.

![  Figure 3: Determination total intracellular manganese by mass spectrometry. ](https://static.igem.org/mediawiki/2020/c/c2/T--Tuebingen--results_fig21.png)

**Figure 3: Determination total intracellular manganese by mass spectrometry.**

Side project Vitamin B12:

Because successful cloning was not achieved for the two constructs of our side project, we suggest doing
it in a two-step Gibson Assembly (**Fig. 4**). Here, the fragments are prepared in the same manner, but
first the assembly of two inserts with the vector is approached. After verification of the correct
assembly of these three fragments by colony PCR and restriction digest, the construct is linearized
again by PCR. Then the second Gibson Assembly follows by inserting the third and last fragment into the
vector. As with all other Gibson Assemblies, we then would check for correct assembly and sequence the
whole insert to make sure it does not contain any mutations.

![ Figure 4: Step-wise Gibson Assembly. ](https://static.igem.org/mediawiki/2020/d/df/T--Tuebingen--results_fig22.png)

**Figure 4: Step-wise Gibson Assembly.**

### References

1. Patzer SI, Hantke K. Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the
   mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli. J Bacteriol 2001;
   183(16):4806–13.
2. Anjem A, Varghese S, Imlay JA. Manganese import is a key element of the OxyR response to hydrogen
   peroxide in Escherichia coli. Mol Microbiol 2009; 72(4):844–58.
3. DSeaver LC, Imlay JA. Hydrogen peroxide fluxes and compartmentalization
   inside growing Escherichia coli. J Bacteriol 2001; 183(24):7182–9.
4. Dambach M, Sandoval M, Updegrove TB, Anantharaman V, Aravind L, Waters LS et al. The ubiquitous
   yybP-ykoY riboswitch is a manganese-responsive regulatory element. Mol Cell 2015; 57(6):1099–109.
5. Martin JE, Waters LS, Storz G, Imlay JA. The Escherichia coli small protein MntS and exporter MntP
   optimize the intracellular concentration of manganese. PLoS Genet 2015; 11(3):e1004977.

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