iGEM 2020 — Team UNILausanne

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# Team:UNILausanne/Notebook

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# Lab book

# Plate Experiments show

Inoculate three strains for the next manipulation of the week (test the oscillator system, competent cell preparation and cloning) using the protocol of paper Potvin-Trottier 2016.

### **Week 1**

29.06.2020

- Put the three strain with the double plasmids (all DHL708 strains) to culture o/n for for testing oscillations next day

30.06.2020

- Measure fluorescence of each time point (o/n, T0, T1...) in a 96-well plate to test the oscillations. Followed the protocol and used media in Potvin-Trottier 2016. Dilutions done every 50 minutes from 10am till 5pm and fluorescence for each time point measured in plate reader

### **Week 2**

06.07.2020

- Put 2 strains (A2=DHL708 pLPT119 + pLPT 41 and A3=DHL708 pLPT107 + pLPT41) for O/N incubation, with goal to achieve OD = 0.2 in the next morning
- Made o/n culture from it

07.07.2020

- We looked at the growth overtime of the strain A2 and A3, to assess how much time is needed to go from 0,05 to 0,2. This was needed after seeing that our former Imaging media (IM) was at 2% glucose instead of 0.2% like the paper. Which explained in part why the serial dilution made on Monday 06.07.2020 did not work

08.07.2020

- Made two types of experiments: growth and dilution on tubes and on plates

09.07.2020

- Started to analyse the data collected Wednesday 08.07

### **Week 3**

14.07.2020

- We prepared some transformed cells for the non fluorescent control --> (pj2060 + p41 in both Nissle and DHL708)

16.07.2020

- Redid fluorescence experiment with:

* DHL708 pLPT119 + pLPT 41
* DHL708 pLPT107 + pLPT41
* DHL708 pJ2060 + pLPT41 (Non-fluorescent)

- Redid all new all fresh Imaging media Stocks + made pluronic F to add to this media

### **Week 4**

21.07.2020

- Redid fluorescence experiment w/ DHL708 and both media to confirm + because some of the measurements messed up a bit last time

22.07.2020

- Prepared fluorescence experiment for the 23.07.2020

23.07.2020

- Redid fluorescence experiment w/ DHL708 pLPT119/107 + pLPT41 and Nissle ΔClb pLPT119 + pLPT41 with only Imaging media this time

24.07.2020

- Made the dilution experiment to assess the growth of our 2 strains and establish a incubation protocol

### **Week 5**

27.07.2020

- Plate experiment with DHL708 (plasmid 107, 234, 41 and 145 combination and 119+mKate tested)

29.07.2020

- Planned and prepared the the cultures for tomorrow's plate experiment

30.07.2020

- Plate experiment (test of various external factors for the experiment in DHL708)

### **Week 6**

04.08.2020

- Fluorescence experiment with Nissle
- o/n cultures for experiments tomorrow

05.08.20

- Growth curve experiment with Nissle and Nissle+plasmids

06.08.2020

- Set up o/n colonies to redo growth experiment

07.08.2020

- Redid growth experiment with more time point measurements and following strains:

* Nissle pLTP234 + pLPT41
* Nissle pLTP234 + pLPT145 (simple and double transformant)
* NEB5a + 234 +145
* DHL708 pJ2060 + pLPT41 ( NF)

### **Week 7**

12.08.2020

- Planification of the plate experiment of tomorrow

13.08.2020

- Redid growth experiment with more time point measurements and following strains:

* Nissle pLTP234 + pLPT145
* Nissle pLTP119 + pLPT41
* Nissle pLPT119 + pLPT41-pelB-Azurin

### **Week 11**

07.09.2020

- Did o/n experiment of fluorescence by doing a dilution experiment

08.09.2020

- Growth experiment for plasmid stability

# Azurin Purification show

We started our azurin experiments about 3 weeks into the lab access. We used the following plasmids for all these experiments:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Plasmid number** | **Plasmid name** | **Description** | **Resistance** | **Link to map** |
| Plasmid 9 | pLIBT7\_A\_peIB-Azurin-3C-TwinStrep-His10 | plasmid expressing azurin with pelB-5D and 3X FLAG tag | Amp | [http://parts.igem.org/Part:BBa\_K3482038](http://parts.igem.org/Part%3ABBa_K3482038) |
| Plasmid 10 | pJ2060-azu-8His | plasmid pJ2060 expressing azurin with a 8His tag | Amp | [http://parts.igem.org/Part:BBa\_K3482040](http://parts.igem.org/Part%3ABBa_K3482040) |
| Plasmid 11 | pJ2060-peIB-azu-8His | plasmid pJ2060 expressing azurin coupled to a secretion tag (peIB) with a 8His tag | Amp | [http://parts.igem.org/Part:BBa\_K3482040](http://parts.igem.org/Part%3ABBa_K3482040) |
| Plasmid 12 | pJ2060-azu(ETHZ)-8His | plasmid pJ2060 expressing azurin from ETHZ with a 8His tag | Amp | [http://parts.igem.org/Part:BBa\_K3482033](http://parts.igem.org/Part%3ABBa_K3482033) |
| Plasmid 13 | pJ2060-peIB-azu(ETHZ)-8His | plasmid pJ2060 expressing azurin from ETHZ coupled to a secretion tag (peIB) with a 8His tag | Amp | [http://parts.igem.org/Part:BBa\_K3482035](http://parts.igem.org/Part%3ABBa_K3482035) |

### **Week 3**

17.07.2020

- Transformation of plasmid 9 into BL21 expression strain, left to grow over the weekend at room temperature, plated onto LBA + Amp

### **Week 4**

21.07.2020

- Set up 2 cultures of 15-20mL in TB media (see recipe in *Protocols*). Grown at 37°C in a shaking incubator until OD600 0.1-0.2
- Directly induced one culture with 5uM IPTG
- Left to shake for a further 2.5hr at 37°C, then harvested the pellet and froze at -20°C
- Cooled the second culture down to 20°C, left to shake for 30 minutes and induced overnight with 5uM IPTG, left to incubate at the same temperature.

22.07.2020

- Harvested pellet from culture 2 and proceeded with the *Protein purification* small-scale protocol and ran on 15% SDS-Page at the end, stained and destained

26.07.2020

- Large-scale purification of azurin from BL21 attempted over the weekend without conclusive results

29.07.2020

- Induction of azurin expression from plasmid pJ2060-pelB-azurin in Nissle 1917. Grown until OD600 0.1-0.2 in TB and induced with 0.2% L-arabinose

### **Week 6**

04.08.2020

- Assembled plasmids 12 & 13 by Gibson & transformation into Turbo and Nissle and plated into LBA + Amp100

05.08.2020

- Colony PCR of the transformants found to confirm the plasmid
- Sent plasmids 12 & 13 for sequencing to confirm the insertion of azurin

06.08.2020

- Set up overnight colonies of plasmids 12 and 13 to transform into final strains

07.08.2020

- Transformation of plasmids 12 and 13 into BL21 after sequencing confirmation

### **Week 7**

10.08.2020

- Transformations into BL21 unsuccessful

12.08.2020

- Transformed plasmid 12 into Nissle and BL21

13.08.2020

- Transformation of plasmid 12 into BL21 unsuccessful so started purification in Nissle
- Transformed plasmid 11 into BL21 and Nissle for azurin expression and purification

14.08.2020

- Small-scale purification of azurin from plasmid 12 in Nissle

### **Week 8**

17.08.2020

- Transformed plasmid 13 into Nissle
- Inoculated LB + antibiotics with Nissle containing plasmid 10 or plasmid 11 for purification

18.08.2020

- Attempted small-scale purification from plasmids 10 and 11 in Nissle
- Also purified a negative control (pJ2060 + p41)
- Ran results on 15% SDS-Page gel

### **Week 9**

24.08.2020

- Set up overnight cultures in LB + antibiotics for Nissle containing plasmid 10 (10mL) for large-scale purification and Nissle containing plasmid 13 for small-scale purification

25.08.2020

- Started purification protocol for both plasmids (growth at 20°C)
- 1L used for large-scale purification of azurin from plasmid 10 in Nissle
- Induced both after 4h growth with 0.2% L-arabinose
- Gel of plasmid 10 didn’t give any clear bands where expected so most likely that azurin wasn’t being produced
- Destained gel with both plasmid results overnight

### **Week 10**

1.10.2020

- Strains with the following plasmids coding for azurin were grown overnight at 37°C in LB:

* Nissle pJ2060-N2only
* Nissle p145-peIB-azu
* Nissle p145-azu
* Nissle p145-peIB-azu(ETHZ)
* Nissle p145-azu(ETHZ)
* BL21 pET22-azu
* BL21 pET22-azu + IPTG

- BL21 strain were induced the next day with 1mM IPTG for 4h at 37°C.
- The OD of the cultures was measured to adjust the amount of cell culture to lyse
- Ran results on 15% SDS-Page gel
- No clear difference in bands present in p145-azu and induced BL21 pET22-azu plasmids and Nissle pJ2060-N2-only and uninduced BL21 pET22-azu

05.10.20

- Growing the following strain until exponential phase:

* Nissle p23
* Nissle p24
* Nissle p25
* Nissle p26
* Nissle p39
* Nissle p40
* BL21 pET22-azu1
* BL21 p9
* Nissle p41

- 200 uL of inoculation were put in 20 mL LB media and grown for 5 hours
- pET22-azu1 and p9 are induced with 500uM IPTG at the same time
- BL21 pET22-azu1 and BL21 p9 are also inoculated at 1% in 5mL LB with 50 or 500uM IPTG and grown at 20°C overnight

06.10.2020

- Ran results on 15% SDS-Page gel

# MTT Assayshow

We started our MTT assay about 4 weeks.

### **Week 4**

27.07.2020

- Reception of the Caco-2 cells and preparation to put them into Caco2 cells medium to let them grow

### **Week 6**

07.08.2020

- Check cell density and split the cells

### **Week 7**

10.08.2020

- Check cell density and make 4 stocks in freezing media (10% DMSO + 90% FBS)

11.08.2020

- Prepare the dilutions of the treatments for the MTT assay

12.08.2020

- Passage n°59 of the cells, according to subculture protocol, wash the cells with 1x Dulbecco’s PBS, and transfer the cells into new growth media

### **Week 8**

17.08.2020

- Passage n°60 of Caco-2 cells, wash the cells with 1x Dulbecco’s PBS, seed them and put them into 96-wells plate for the first MTT assay

20.08.2020

- Set up the MTT assay. Treated the cells with the following different treatments:

* Controls (DMSO or media or 0.1% Triton X or lysis buffer)
* Different concentrations of Anisomycin: 50, 150, 300 and 450 [ng/ml]
* Different concentrations of Cisplatin: 2.5, 5, 10 and 100 [μM]
* Different concentration of Doxorubicin: 0.5, 1, 5 and 10 [μM]
* Different concentration of Salirasib: 2.5, 5, 10 and 50 [μM]
* Different concentration of TNFa: 1, 5, 10, 100 [ng/ml]

- We did the same treatments for THP1 cells
- Put to incubate at 37°C for 20h

21.08.2020

- Continued the MTT assay, incubated 4h
- Measuring absorbance at 570 nm using a plate reader

### **Week 9**

26.08.2020

- Redo the MTT assay for Caco-2 and THP1 cells with the same treatments but different conditions:

* Controls (DMSO or media or 0.1% Triton X)
* Different concentrations of Cisplatin: 10, 50 and 500 [μM]
* Different concentration of Doxorubicin: 3, 10 and 100 [μM]
* Different concentration of Salirasib: 1, 10 and 100 [μM]

### **Week 16**

10.10.2020

- O/n cultures in LB media with aTC and appropriated antibiotics with the following strains:

* Nissle pLPT234 + pLPT145
* Nissle pLPT234 + pLPT145-azu
* Nissle pLPT234 + pLPT145-5D-azu.1\_60del
* Nissle pLPT234 + pLPT145-NSP4-azu.1\_60del

- The different strains above reached were diluted to an OD=0.5 before lysate the bacteria

### **Week 17**

16.10.2020

- Caco2 cells viability assay with bacterial supernatant and pellet lysate, plus with controls following the MTT assay protocol
- Inoculation of Hela and HAP1 cells

### **Week 18**

18.10.2020

- O/n cultures in LB media with aTC and appropriated antibiotics with the following strains:

* Nissle pLPT234 + pLPT145
* Nissle pLPT234 + pLPT145-azu
* Nissle pLPT234 + pLPT145-5D-azu.1\_60del
* Nissle pLPT234 + pLPT145-NSP4-azu.1\_60del

19.10.2020

- Lysate the bacteria above

20.10.2020

- MTT assay: incubate Hela and HAP-1 cells bacteria supernant, pellet lysate and controls

# Kill Switchshow

### **Week 9**

25.08.2020

- Assembly of pKA1-ccdB-ccdA
- Tried with different polymerase (Q5, Vazyme and Phanta)

26.08.2020

- Assembly of the different parts to design pAND-FRT-kan

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Fragment | template | FW | REV | T\_ann | t\_elong | Amplicon size | concentration | 260/280 | 260/230 | Fmol/uL |
| FRT\_1 | pAND | PR104 | PR105 | 65°C | 40'' | 900bp | 28 ng/uL | 1.8 | 0.9 | 50 |
| FRT\_2A | pKA1 C7 | PR103 | PR106 | 55°C | 4' | 5kb | 231 ng/uL |  |  | 75 |
| FRT\_2C | pKC1 C12 | PR103 | PR106 | 55°C | 4' | 5kb | 284 ng/L |  |  | 90 |

|  |  |
| --- | --- |
| PR103 | pAND-FRT-rev |
| PR104 | kanR-FRT-fw |
| PR105 | kanR-FRT-rev |
| PR106 | pAND-FRT-fw |

### **Week 10**

03.09.2020

- Reamplification of pAND backbone (AB13) with Q5 and Phanta
- With these amplicons, assembled by Gibson Assembly
- Once the plasmid assembled, transformed into Nissle and Turbo

04.09.20

- Colony PCR

07.09

- Sequencing results

### **Week 11**

15.09.20

- The restreak of the colonies C8 and C12 showing no mutations in the sequencing did no grow back well
- Reassemble the plasmids and plate the transformants on LB infused with aTc or IPTG (since the gene can be inserted under both promoters), so that the antitoxin is expressed and not killed by the antitoxin
- The backbones fragments AB13 and AB14 were reamplified and digested using Dpn1

### **Week 12**

23.09.2020

- Reamplification of some fragments
- Gibson Assembly of these fragments
- Transformation

25.09.2020

- Colony PCR

26.09.2020

- Sequencing results good

### **Week 13**

30.09.2020

- Transformation into Nissle

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# Team:UNILausanne/Engineering

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# Engineering success

# showRepresillator system

## The original repressilator system

The repressilator system is at the heart of our experiment – naturally this meant that we had to start our lab work by testing the original system. For this, we decided to adapt the “flask experiment” found in the paper by Potvin-Trottier et al. from 2016 to use in a plate reader, so that we could test multiple conditions and mutants at once.

## Testing and redefining the protocol

To refine the flask experiment found in the source paper and adapt it to our own protocol, we had to test different growth media (EZ vs imaging media, found in  [protocols](https://2020.igem.org/Team%3AUNILausanne/Protocols) ), different ways of synchronising the oscillations (IPTG vs aTc) and different overnight incubation temperatures (ranging from 20°C to 37°C) in order to optimise the fluorescence readout.

One of the big problems we had early on was the formation of crystals in the bottom of the wells of our 96-well plate. To try and combat this we added Pluronic-F to our imaging media, a compound used for microfluidics to avoid this problem. However, we realised that this didn’t affect the results and so we continued using the media without Pluronic-F.

![Repressilator system with a three gene](https://static.igem.org/mediawiki/2020/4/4c/T--UNILausanne--Engi_Rep_Figure1withoutpluronic_F.png)

*Figure 1:* *(On the left)* *Plot of the three reporters in the pLPT234 repressilator plasmid* *with Pluronic-F in the growth media**.*

![Repressilator system with a three gene](https://static.igem.org/mediawiki/2020/6/6f/T--UNILausanne--Engi_Rep_Figure2with_pluronic_F.png)

*Figure* *2**:* *(On the right)* *Plot of the three reporters in the pLPT234 repressilator plasmid* *without Pluronic-F in the growth media**.*

However, observing the results we realised that this didn’t affect any fluorescence measurements and so we continued using the media without Pluronic-F.

## The plasmid choice journey

We started by using the plasmids pLPT119 and pLPT107 for the repressilator in **E. coli** Nissle 1917 Δclb (Part: BBa\_K3482025) and pLPT41 as the sponge (Part: BBa\_K3482026).

The plasmid pLPT107 was finally also not adapted, because of the degradation tag present at the end of the repressor genes. For this reason, we decided to replace it by the repressilator plasmid pLPT234 (Part: BBa\_K3482023).

After a few tests, we also realised that CFP in pLPT119 was not acting as expected; this is when we discovered that in fact it’s under a constitutive promote (pRNA1), in this plasmid, and so doesn’t reflect the oscillations of the Tet repressor but ends up following the OD600.

We finally defined our various models with our chosen repressilator plasmids and prepared prediction models, to determine what we would expect to observe (See Figure 3 and 4.

![Repressilator system with a three gene](https://static.igem.org/mediawiki/2020/d/da/T--UNILausanne--Rep_Figure2_plasmid_pLPT119.jpg)

*Figure 3: (A) Schematic representation of plasmid pLPT119 [BBa\_K3482025]. The promoters are represented by the arrows. The repressor genes are represented in grey. The reporter genes are colored according to the emission wavelength of their associated fluorescent protein. In this system, tetR inhibits cI, which inhibits lacI, which itself inhibits tetR. (B) Model of the oscillation of our two fluorescent proteins. CFP is expressed in a constant manner, mVenus is oscillating as before.*

![Repressilator system with a three gene](https://static.igem.org/mediawiki/2020/c/c2/T--UNILausanne--Rep_Figure1_plasmid_pLPT234.jpg
)

*Figure 4: Schematic representation of plasmid pLPT234 [BBa\_K3482023]. The promoters are represented by the arrows. The repressor genes are represented in grey. The reporter genes are colored according to the emission wavelength of their associated fluorescent proteins. In this system, tetR inhibits cI, which inhibits lacI, which itself inhibits tetR. (B) Model of the oscillation of our three fluorescent proteins. Each reporter oscillates regularly.*

Further research into the system led us to use a triple sponge plasmid, pLPT145 (Part: BBa\_K3482024), that led to similar oscillations as the pLPT41 sponge plasmid in **E. coli* DHL708*. We therefore also tested these non-modified combinations, using this sponge plasmid with either pLPT119 or pLPT234 in **E. coli** DHL708, to figure out which one showed the most reliable oscillations. We obtained oscillatory patterns of expression in the repressilator plasmids we transformed in **E. coli** DHL708 for the pLPT119 plasmid and pLPT234.

In conclusion, we succeed to characterize and optimize 3 repressilator system combinations:

* pLPT234 + pLPT41
* pLPT234 + pLPT145
* pLPT119 + pLPT41

We were able to obtain clear and regular oscillations in these three systems, as we have shown in the experiment section ( [See results on experiment section](https://2020.igem.org/Team%3AUNILausanne/Experiments)).

## The modified repressilator system

Whilst one part of our wet lab team worked on optimizing the plate experiment protocol, another part was working on the assembly of the modified plasmids for the secretion of azurin and/or with a fluorescent reporter to test if everything worked as expected.

When we first started modifying our system, we originally wanted to use mKate2 as the fluorescent reporter on our sponge plasmid to track oscillations. However, we realised after the first few plate experiments that mKate2 detection was very unreliable in pLPT107 and pLPT234. This meant that we had to rethink our plans and we eventually decided on removing mVenus from the repressilator plasmid and adding it to the sponge plasmid.

![Repressilator system with a three gene](https://static.igem.org/mediawiki/2020/1/18/T--UNILausanne--Engi_Rep_Figure5.png
)

*Figure 5: Plot of the fluorescent reporters for the repressilator plasmid pLPT119 and sponge plasmid pLPT41*

## mVenus removal and azurin integration

The removal of mVenus from either repressilator plasmid ended up being more difficult than expected. Manipulating such large fragments of DNA were the biggest challenge we faced, and we tried to overcome in multiple ways including:

* Modifying the PCR protocol for higher fidelity
* Separating the plasmid into multiple pieces to be stitched back together using Gibson assembly
* Site-directed mutagenesis to target mVenus expression

The main difficulty encountered here was the fact that the sequences were too similar, which led to the multiple difficulties to modify the sequence.

We managed to obtain the repressilator plasmid pLPT234 without mVenus and decided to test it without our modified sponge plasmids pLPT41 and pLPT145, containing each mVenus with or without azurin next to it.

Unfortunately, none of our systems gave clear fluorescence oscillations ( [See results on experiment section](https://2020.igem.org/Team%3AUNILausanne/Experiments)).

We finally decided to test a last combination: the unmodified repressilator plasmid pLPT119 with the modified sponge plasmid pLPT41, that was containing the azurin gene. As we can see on Figure 6, this repressilator system gave us clear oscillations of mVenus, as we expected.

![Repressilator system with a three gene](https://static.igem.org/mediawiki/2020/a/a5/T--UNILausanne--Rep_Figure11_Nissle_pLPT119_pLPT41-Azu.png
)

*Figure 6: Fluorescence over time plot of E.coli Nissle 1917 Δclb containing plasmids pLPT119 [BBa\_K3482025] and pLPT41-pelB-Azurin. The two lines for each reporter gene represent the two biological replicates that were done for this experiment.*

In conclusion, after many trials, tests and modifications, we managed to produce protocols, characterize and successfully transform the repressilator circuit into *E. coli* Nissle. We also managed to create a repressilatory system which was stable, while containing the azurin gene.

# showAzurin expression

## The initial design

We then planned to evaluate the azurin expression module through three main steps: 1) purifying the azurin protein from *E. coli* BL21, a protein expression strain, 2) verify that the expressed protein is indeed azurin and 3) that azurin is secreted (found in the supernatant through the addition of a pelB-5D secretion tag).

Using Golden Gate assembly, we introduced azurin along with a TwinStrep His tag onto the pET22 backbone, under a T7 expression promoter. This construct was then transformed into *E. coli* BL21 which was left to incubate overnight and produce our protein in the presence of the correct inducer. However, our first attempt at purification showed that this would be more difficult than imagined. Even though we used a protein expression strain and plasmid, we did not observe any big overexpression bands in the gel. We did, however, see some slightly smaller bands which we were led to believe that it might be azurin, and so we decided to attempt a large-scale purification using this construct.

The large-scale purification didn’t yield any azurin and so we started exploring other ways of purifying this protein from the same strain.

We then tried placing our optimized azurin (with an 8-His tag for purification) downstream of a Pbad promoter in a different plasmid - this one could not be transformed into BL21 so we decided to try purification directly from *E. coli* Nissle 1917 Δclb. We also attempted the same thing using the azurin part established by the ETH Zürich team in 2017 (link part). These plasmids at first seemed promising during the small-scale purification, but once again we did not obtain any purified azurin when we attempted to scale up.

After these attempts, we then created several new constructs with azurin combined with a 3XFLAG Tag inserted into the pET22 expression plasmid under a T7 IPTG-inducible promoter. These were then once again transformed into *E. coli* BL21, which was left to grow in media containing IPTG. However, once again in the small-scale purification, we didn’t obtain any clear overexpression bands. This time, however, we looked closer at the parameters of our experiment and tried modulating the concentration of IPTG we were using to induce protein expression.

![](https://static.igem.org/mediawiki/2020/6/69/T--UNILausanne--ES2_SDSPAGE.png)

*Figure 1A: SDS PAGE of bacterial lysate. Samples were induced for 4h with the mentioned IPTG concentrations. The OD600 of all cultures was normalized to 0.5. Samples were lysed for 10min at 95°C. and run on a 15% polyacrylamide gel for 30 minutes at 200V 1B: SDS-PAGE with the same culture conditions as the uninduced sample from Figure 1A. Purification performed using* *HisPur Ni-NTA Magnetic Beads, incubated for 5 seconds with our sample**.* *Signaled in red is the expected azurin protein. Samples were run on a 15% polyacrylamide gel for 30 minutes at 200V.*

We hypothesized that the overexpression of azurin may be toxic to this particular *E. coli* strain and thus tried the same with our chassis bacteria, *E. coli*  Nissle 1917 Δclb. From this, we saw that the same effect occurred. Thus, we suggested that only the leaky expression of the T7 promoter gave a tolerable level of expression.

## The second step

The SDS PAGE was not really representative of azurin expression because other proteins were often present at the same size of the anti-cancer peptide. So we designed azurin combined with a 3xFLAG tag at the N-terminus. We thus made the design choice to recombine a 3XFLAG flag at the N-terminus. This choice opened the ability to confirm both the secretion and identity of the azurin through proteomic analysis (Western blot and mass spectrometry) in *E. coli*  Nissle 1917 Δclb. These were proven more adapted as they are sensitive detection methods.

## Summary

We made several adjustments throughout the module in response to our experiment results. One of which was the addition of a 3XFLAG flag tag at the N-terminus of azurin genes. In the end, these results lead us to conclude on the secretion and identity of azurin.

# showKill switch system

## Kill switch system

Introducing a genetically modified living organism into the body could cause some unexpected consequences, for example the bacteria invading the blood circulation, which can cause a **septic shock**. Moreover, GMO bacteria might pose a risk of **unwanted escape and spread** in the environment when they are secreted from the body. That is why we decided to add a **biosafety and biocontainment** mechanism known as a kill switch.

Our kill switch system allows the bacteria to stay alive only in the colon where we want them to fight the colorectal cancer. When the bacteria get in the outside environment, or into the blood circulation, the kill switch will kill the bacteria.

The kill switch system does this by sensing two environmental cues: 1) the temperature, and 2) the phosphate concentration.

In the colon the temperature is high and the phosphate concentration is low, and these represent the **permissive condition** of our kill switch. On the other hand, outside the body the temperature is lower than in the colon, and in the bloodstream the phosphate concentration is higher than in the colon, and therefore low temperature and high phosphate represent the **non-permissive** conditions of our kill switch (**Figure 1**). Moreover, the ingestion of an encapsulated pill containing phosphate allows it to also kill the bacteria on demand even in the colon.

![](https://static.igem.org/mediawiki/2020/5/5c/T--UNILausanne--ESKS1.png)

**Figure 1:** Our kill switch is responsive to temperature and phosphate concentrations. The only place where the bacteria are allowed to stay alive is the colon when the temperature is high and the phosphate concentration is low (permissive condition). When one or both of these conditions are not met (non-permissive condition) the kill switch kills the cells.

To achieve this controlled cell death, we use toxin-antitoxin systems, where the cell is killed when the toxin is expressed in absence of the antitoxin. To incorporate the temperature logic, our design uses heat-inducible RNA thermosensor fused to the antitoxin genes, and heat-repressible RNA thermosensor fused toxin genes. The phosphate concentration logic is then incorporated by putting the antitoxin gene under the control of a phosphate-repressible promoter. The final design is depicted in **Figure 2**.

![](https://static.igem.org/mediawiki/2020/6/68/T--UNILausanne--ESKS2.png)

**Figure 2**: Our final kill switch plasmid design incorporating temperature and phosphate sensitivity. This system allows the bacteria to stay alive when the temperature is high and phosphate concentration is low (condition of the colon), and to kill bacteria when the temperature is low and the phosphate concentration is high (outside the body, or in the bloodstream).

Since this design is a rather sophisticated and multi-responsive subject, we use a bottom-up approach where we first characterize the effect of every single part, and then gradually add more components onto our device. To increase our chances of success, we based our system on an already well-known toxin-antitoxin system in the literature. These are the toxin-antitoxin pair ccdB/ccdA ([BBa\_K3482016](http://parts.igem.org/Part%3ABBa_K3482016)) and miniColicin E2/IM2 ([BBa\_K3482017](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3482017)). We then chose a very promising heat-inducible and heat-repressible RNA thermosensor also from the literature [1] [2]. The core promoter of a well-characterized phosphate-repressible promoter was taken from the iGEM Registry. Here, we further describe our development of the plasmid system based on miniColicin E2/IM2.

As we did not know exactly which of the two promoters, pTet and pTac, was the one with the highest leaky/basal expression, we designed our Gibson assembly such that the toxin and antitoxin genes could insert under either of promoters.

![](https://static.igem.org/mediawiki/2020/4/4a/T--UNILausanne--ESKS3.png)

**Figure 3: Assembly design of plasmid pKC1**. Gibson assembly primers were designed to allow for the insertion of the miniColicin E2 toxin and IM2 antitoxin under either pTet or pTac promoter. When aTc and IPTG are added to the SOC media and agar plate for the transformants, the insertion of antitoxin can be forced to be under the promoter which is being induced by the inducer.

When we sequenced our cloning strain transformants we observed that the ones that inserted the toxin under pTet mostly showed mutations in the toxin gene (data not shown). We then reperformed the transformation by adding either aTc or IPTG to the transformant media, and were able to select this way under which promoter the antitoxin would be inserted (**Figure 3**). As the first sequencing of the first transformants showed no mutations when the antitoxin was inserted under pTet and the toxin under pTac, we continued working with this configuration. This first kill switch plasmid is pKC1 (**Figure 3A**).

![](https://static.igem.org/mediawiki/2020/a/a0/T--UNILausanne--ESKS4.png)

**Figure 4: Bottom-up plasmid design to test the functionality of the kill switch components. a)** pAND is the empty vector containing pTac and pTet promoter without any gene. **b)** pKC1 contains the miniColicin E2 toxin under pTac and the IM2 antitoxin under pTet. **c)** pKC3 contains the heat-inducible thermosensor fused to the antitoxin gene.

To perform a first qualitative assessment of the functionality of the kill switch responsiveness towards induction of aTc and IPTG induction, we plated a lawn of *E. coli* Nissle 1917 and added orthogonally placed cardboard strips soaked with aTc and IPTG (**Figure 5**). The plate showed that close to the IPTG strip very little cells grew, and that further away from it the diffusion of aTc was able to sufficiently induce the antitoxin and rescue the cell growth.

![](https://static.igem.org/mediawiki/2020/a/a6/T--UNILausanne--ESKS5.png)

**Figure 5:** Qualitative assessment of transformed E.coli Nissle 1917 containing plasmid pKC1. Cells were grown on agar plates in the presence of both IPTG and aTc gradient.

We then performed a quantitative assessment of the effect of the stimulation of toxin and antitoxin on the growth of our *E. coli* Nissle 1917 strain by adding different amounts of aTc and IPTG inducer concentrations, and measuring growth curves in the plate reader (**Figure 6**).

![](https://static.igem.org/mediawiki/2020/d/d7/T--UNILausanne--ESKS6.png)

**Figure 6:****Dose-response growth curve of** ***E. coli*** **Nissle 1917** **Δ****clbA harboring kill switch plasmid pKC1 at 37°C.** *E. coli* Nissle with pAND (red line), *E. coli* Nissle with pKC1 (green line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

This allowed us to clearly determine the different ranges of aTc and IPTG concentration that give different growth inhibition profiles.

Finally, we then inserted the heat-inducible RNA thermosensor upstream of the antitoxin genes in the plasmid pKC3 (BBa\_K3482017). In order to prove that the heat-inducible thermosensor reduces the translation of antitoxin at low temperature and therefore the viability of the cells, we grew the strain at different temperatures reflecting the temperature inside (37°C) and outside (≤25°C) the body, respectively (**Figure 7**).

![](https://static.igem.org/mediawiki/2020/c/cc/T--UNILausanne--ESKS7.png)

**Figure 7: Dose-response growth curve of** ***E. coli*** **Nissle 1917 ΔclbA harboring kill switch plasmids pKC1 and pKC3 at 37 and 25°C.** *E. coli* Nissle with pAND (red line), *E. coli* Nissle with pKC1 (green line), and *E. coli* Nissle with pKC3 (blue line), at 37°C (dashed line) and 25°C (solid line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

At permissive temperature (37°C) the pKC3 containing cells grew a bit slower than the two controls, but their viability was not impaired. Indeed, after 10 hours the cells containing either pAND or pKC1 seemed to reach a plateau where the pKC3 cells were still growing. At lower temperature, the viability of pAND and pKC1 cells remains unchanged while pKC3 viability was significantly impaired.

By doing this **we proved that the plasmid pKC3 containing the part BBa\_K3482017 is able to control the viability of its host.**

### **References**

[1] Hoynes-O’Connor, A., Hinman, K., Kirchner, L., & Moon, T. S. (2015). De novodesign of heat-repressible RNA thermosensors in E. Coli. *Nucleic Acids Research*, *43*(12), 6166–6179.

[2] Sen, S., Apurva, D., Satija, R., Siegal, D., & Murray, R. M. (2017). Design of a Toolbox of RNA Thermometers. *ACS Synthetic Biology*, *6*(8), 1461–1470.

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# Experiments

# showRepresillator system

## Background

Treatments for **colorectal cancer** such as **chemotherapy, surgery and radiotherapy** are associated with severe side effects and are limited in effectiveness.
We aim to implement a system that can produce an anticancer drug called **azurin** in a circadian rhythm.
For this purpose, we set to use **the repressilator**.

The repressilator is a **synthetic regulatory network** of three genes encoded on a plasmid.
These three genes repress each other in a feedback loop.
As a result, the genes are cyclically expressed one after the other at regular intervals over time, which allows us to produce our anticancer drug at regular intervals.
For our system to work efficiently, a second plasmid called **a sponge plasmid** is applied together with the repressilator.
This sponge plasmid contains additional repressor binding sites which allows for the “absorption” of noise and makes the oscillations more regular and robust.

### Repressilator plasmids

For our experiment, we want to test two types of repressilator plasmids:

The first one is pLPT234 [BBa\_K3482023] (Figure 1A).
It is composed of three repressors (*tetR*, *lacI* and *cI*), that are under the regulation of three distinct promoters (*PLlacO1*, *PRNA1*and *PLtetO1* respectively).
The oscillations can be followed thanks to three fluorescent reporters (*CFP*, *mKate2* or **mVenus*)* which are under the control of the same three promoters.
The three repressors are connected in a circuit, where one repressor inhibits the next.
This feedback loop circuit leads to the oscillatory expression of three colors (Figure 1B).

![](https://static.igem.org/mediawiki/2020/c/c2/T--UNILausanne--Rep_Figure1_plasmid_pLPT234.jpg)

*Figure 1: (A) Schematic representation of plasmid pLPT234 [BBa\_K3482023]. The promoters are represented by the arrows. The repressor genes are represented in grey. The reporter genes are colored according to the emission wavelength of their associated fluorescent proteins. In this system, *tetR* inhibits cI, which inhibits *lacI*, which itself inhibits *tetR*. (B) Model of the oscillation of our three fluorescent proteins. Each reporter oscillates regularly. ( [See modeling page.](https://2020.igem.org/Team%3AUNILausanne/Model) )*

The second repressilator plasmid is pLPT119 [BBa\_K3482025] (Figure 2A).
The composition of the plasmid is similar to the plasmid pLPT234 [BBa\_K3482023].
Here there is only the fluorescent reporter gene *mVenus* associated with a repressor protein (λ), with *CFP* on the plasmid but under a constitutive promoter.
All three repressor proteins are still present, and the system still functions as in pLPT234 BBa\_K3482023], but we can only follow the oscillations using *mVenus*. (Figure 2B).

![](https://static.igem.org/mediawiki/2020/d/da/T--UNILausanne--Rep_Figure2_plasmid_pLPT119.jpg)

*Figure 2: (A) Schematic representation of plasmid pLPT119 [BBa\_K3482025]. The promoters are represented by the arrows. The repressor genes are represented in grey. The reporter genes are colored according to the emission wavelength of their associated fluorescent protein. In this system, *tetR* inhibits cI, which inhibits *lacI*, which itself inhibits *tetR*. (B) Model of the oscillation of our two fluorescent proteins. *CFP* is expressed in a constant manner, *mVenus* is oscillating as before. ( [See modeling page.](https://2020.igem.org/Team%3AUNILausanne/Model) )*

### Sponge plasmids

In order to have robust and regular oscillations, the repressilator plasmid needs to be combined with a so-called “sponge” plasmid.
This plasmid contains one or three promoters (including the repressor binding sites) already present in the repressilator plasmids.
In our project, we worked with two different sponge plasmids (Figure 3).
The first plasmid is pLPT41 [BBa\_K3482026] (Figure 3A) and contains a *PLtetO1* promoter.
The second plasmid is pLPT145 [BBa\_K3482024] (Figure 3B) and contains the three promoters present in the repressilator plasmid pLPT234 BBa\_K3482023].
We tested these two sponge plasmids in combination with our two repressilator plasmids, to determine which combination resulted in the best oscillations.

![](https://static.igem.org/mediawiki/2020/d/d8/T--UNILausanne--Rep_Figure3_plasmid_pLPT41_pLPT145.jpg)

*Figure 3: (A) Schematic representation of the sponge plasmid pLPT41 [BBa\_K3482026]. It contains the promoter *PLtetO1*. (B) Schematic representation of the sponge plasmid pLPT145 [BBa\_K3482024]. It contains the promoters *PLtetO1*, *PLlacO1* and *PRNA1**

All of the plasmids presented here were described in Potvin-Trottier 2016.
They were obtained and ordered from Addgene. They were made accessible by Prof Johan Paulson [1].
We decided to base our experiment on these plasmids and modify the system afterwards for our project.

## Procedure

### Strains

For our repressilator experiments, we worked with two different strains.- *E. coli* DHL708 (Addgene, #98417):
  This strain lacks the Clp protease (∆clpPX) and was used in the study in which we initially transformed our first repressilator constructs.
  This strain was much easier to transform and grew faster than the probiotic *E. coli* Nissle 1917 *Δclb* we aimed to use for the final constructs.
- *E. coli* Nissle 1917 *Δclb* : *E. coli* Nissle is a probiotic bacteria in the human gut.
  We want to use this strain as a chassis to express the anticancer peptide azurin in an oscillatory manner.

### Experiment goal

We had two main goals in this experiment:

First we wanted to determine if our repressilator system worked correctly in our two strains
(*E. coli* DHL708 and *E. coli* Nissle *Δclb*) and if we could detect them without using microfluidics.
To achieve this, we tested the following combinations in various conditions to optimize our protocols:

- Repressilator plasmid pLP119 [BBa\_K3482025] (Figure 2A) with sponge plasmid pLPT41 [BBa\_K3482026] (Figure 3A) in E.coli DHL708.
- Repressilator plasmid pLP234 [BBa\_K3482023] (Figure 1A) with sponge plasmid pLPT41 [BBa\_K3482026] (Figure 3A) in E.coli Nissle*Δclb*.
- Repressilator plasmid pLP234 [BBa\_K3482023] (Figure 1A) with sponge plasmid pLPT145 [BBa\_K3482024] (Figure 3B) in E.coli Nissle*Δclb*.

Our second goal was to integrate our anticancer drug (azurin) in our repressilator plasmid.
We then needed to determine if the oscillations of our gene circuit were still stable after the integration of azurin.
We tested the various following modifications:

- To test if our repressilator system was still working correctly after the integration of azurin and to verify if azurin followed an oscillatory behaviour,
  we tested the repressilator plasmid pLP234 without *mVenus* [ [pLPT234 without mVenus](https://static.igem.org/mediawiki/2020/b/b7/T--UNILausanne--234.gb)] with the modified sponge plasmids pLPT41 + *mVenus* [BBa\_K3482026 + BBa\_K3482031] (Figure 4B) , pLPT41 + *mVenus*-azurin [BBa\_K3482026 + BBa\_K3482031 + BBa\_K3482043] (Figure 4C) and pLPT145 + *mVenus*-azurin [BBa\_K3482024 + BBa\_K3482031 + BBa\_K3482043] (Figure 4D) in *E. coli* Nissle*Δclb*.
  The gene encoding for azurin is under the regulation of the *PLtetO1* promoter already present in the two sponge plasmids.
  The fluorescent reporter gene *mVenus* is under the same promoter regulation as azurin (Figure 4B, C and D).
- To test if our repressilator system was still working correctly after the integration of azurin in the sponge plasmid,
  we tested the repressilator plasmid pLPT119 [BBa\_K3482025] with the modified sponge plasmid pLPT41 [BBa\_K3482026] + pelB-azurin [BBa\_K3482026 + BBa\_K3482006 + BBa\_K3482043]
  in *E. coli* Nissle *Δclb*. The azurin is under the regulation of the *PLtetO1* promoter already present in the sponge plasmid (Figure 4A).
![](https://static.igem.org/mediawiki/2020/5/5a/T--UNILausanne--Rep_Figure4_Modified_plasmids.jpg)

*Figure 4: Schematic representation of our various combinations tested with our modified plasmids. (A) Plasmid pLPT119 [BBa\_K3482025] with modified plasmid pLPT41 [BBa\_K3482026] containing azurin and its secretion tag, under the *PLtetO1* promoter [BBa\_K3482026 + BBa\_K3482006 + BBa\_K3482043]. (B) Plasmid pLPT234 without *mVenus* with the modified plasmid pLPT41 with *mVenus* under the *PLtetO1* promoter [BBa\_K3482026 + BBa\_K3482031]. (C) Plasmid pLPT234 without *mVenus* with the modified plasmid pLPT41 with *mVenus* and azurin under the *PLtetO1* promoter [BBa\_K3482026 + BBa\_K3482031 + BBa\_K3482043]. (D) Plasmid pLPT234 without *mVenus* with the modified plasmid pLPT145 [BBa\_K3482024] with *mVenus* under the *PLtetO1* promoter [BBa\_K3482023 + BBa\_K3482043].*

### Experimental procedure

Overnight cultures of *E. coli* DHL708 or *E. coli* Nissle *Δclb* with one of the combinations of repressilator and sponge plasmids listed above were prepared in imaging media ( [See protocol](https://2020.igem.org/Team%3AUNILausanne/Protocols)).
with the corresponding antibiotics and the synchronizing molecule anhydro*tetR*acycline (aTc, 0.5 μL/mL).
The imaging media is a growth media with a very low background fluorescence.
ATc is a molecule that inhibits the *tetR* repressor; upon addition of aTc to the culture, all bacteria containing a repressilator plasmid halt oscillations at the same time point.
After removal of aTc, all the bacteria start oscillations simultaneously and thus the incubation with aTc effectively synchronizes the oscillations in a bacterial population.

In the morning, we resuspend the cells in fresh imaging media without aTc and transferred them to a 96-well plate.
We diluted the cells to reach an optical density at 600 nm (= OD600) of 0.2 and put the plate in a plate reader at 37°C and 180 rpm.
Every hour, we checked the OD600 and diluted the cells back to an OD600 of 0.2, to keep the cells in exponential phase.
This was important, because the repressilators do not produce oscillations when cells are in a stationary phase.
Fluorescence measurements were taken every hour for approximately 10 hours.

## Results

### Overview

First, we will present the results of the original repressilator plasmid pLPT234 [BBa\_K3482023] in combination with the sponge plasmids: pLPT41 [BBa\_K3482026] or pLPT145 [BBa\_K3482024].
We then compared the results of the modified version of pLPT234 [BBa\_K3482023] (without **mVenus*)* with the modified sponge plasmids (with *mVenus* and with or without azurin).
We will then present the results of the original plasmid pLPT119 [BBa\_K3482025] in combination with the sponge plasmid pLPT41 [BBa\_K3482026] containing either the azurin gene or a fluorescence reporter gene.
Finally, we will compare this system with pLPT119 [BBa\_K3482025] in combination with our modified sponge plasmid in which the azurin gene has been integrated.

### First part: Repressilator plasmid pLPT234 [BBa\_K3482023]

We tested our various repressilator systems without any modifications, starting with the combination of repressilator plasmid pLPT234 [BBa\_K3482023] and sponge plasmid pLPT41 [BBa\_K3482026] and pLPT145 [BBa\_K3482024] in *E. coli* DHL708.
In Figure 5, we show the fluorescence over time after the synchronization of the bacterial culture.

![](https://static.igem.org/mediawiki/2020/4/41/T--UNILausanne--Rep_Figure5_DHL708_pLPT234_41.png)

*Figure 5: Fluorescence over time plot of *E. coli* DHL708 containing the repressilator pLPT234 [BBa\_K3482023] and the sponge plasmid pLPT41 [BBa\_K3482026]. Synchronized overnight with 177 nM of aTC*

![](https://static.igem.org/mediawiki/2020/6/6d/T--UNILausanne--Rep_Figure6_DHL708_pLPT234_145.png)

*Figure 6: Fluorescence over time plot of *E. coli* DHL708 containing the repressilator pLPT234 [BBa\_K3482023] and the sponge plasmid pLPT145 [BBa\_K3482024]. Synchronized overnight with 177 nM of aTC.*

The combination of the repressilator plasmid pLPT234 [BBa\_K3482023] with the sponge plasmid pLPT145 [BBa\_K3482024] (Figure 6) gave clear and stable oscillations that correspond to our expectations and our model (Figure 1B,  [See modeling page.](https://2020.igem.org/Team%3AUNILausanne/Model) ).
The second combination, the plasmid pLPT234 [BBa\_K3482023] and pLPT41 [BBa\_K3482026], gave less clear and stable oscillations (Figure 5).
These results could be explained by the fact that the sponge plasmid pLPT41 [BBa\_K3482026] only has one promoter of the repressilator plasmid, compared to the sponge plasmid pLPT145 [BBa\_K3482024] that has all three.
This sponge plasmid pLPT41 [BBa\_K3482026] seems to compensate for the noise of the repressilator plasmid less than pLPT145 [BBa\_K3482024] due to the lower number of promoters.

Since we demonstrated that the repressilator system is functional, we decided to test the repressilator in combination with a sponge plasmid containing the azurin gene, which should show oscillations of azurin (Figure 4B, C and D).
The systems were integrated in our probiotic strain *E. coli* Nissle 1917 *Δclb*.

![](https://static.igem.org/mediawiki/2020/9/99/T--UNILausanne--Rep_Figure7_Nissle_pLPT234deltamVenus_145mVenus.png)

*Figure 7: Fluorescence over time plot of *E. coli* Nissle 1917 *Δclb* containing plasmids pLPT234Δ*mVenus* and pLPT145-*mVenus**

![](https://static.igem.org/mediawiki/2020/d/d6/T--UNILausanne--Rep_Figure8_Nissle_pLPT234deltamVenus_41mVenus.png)

*Figure 8: Fluorescence over time plot of *E. coli* Nissle 1917 *Δclb* containing plasmids pLPT234Δ*mVenus* and pLPT41-*mVenus**

![](https://static.igem.org/mediawiki/2020/4/44/T--UNILausanne--Rep_Figure9_Nissle_pLPT234deltamVenus_41mVenus-Azu.png)

*Figure 9: Fluorescence over time plot of *E. coli* Nissle 1917 *Δclb* containing plasmids pLPT234Δ*mVenus* and pLPT41-*mVenus*-Azurin*

We tested our modified sponge plasmids with or without azurin linked to the fluorescence gene *mVenus* separately.
In Figures 7 and 8, we can see that no clear oscillations are visible.
It might be possible that the shift of the *mVenus* reporter from the repressilator plasmid to the sponge plasmids disturbed our oscillations in this assembly.
Indeed, the sponge plasmid has a higher copy number compared to the repressilator plasmid (~20 compared to ~5).
The shift of the fluorescent reporter *mVenus* from one plasmid to another might perpetuate the dynamical equilibrium of the system and even cause burden.
Another explanation would be that the repressilator system, and synthetic biology in general, is really dependent on the context.
Modifying even slightly some synthetic systems might completely disturb the system and the results obtained [2].

In parallel, we tested the expression behavior of azurin when integrated in the sponge plasmid.
In Figure 9, we can observe again that we also obtained noisy and unstable fluorescence signals.
We were therefore not able to obtain clear oscillations with our modified repressilator and thus couldn’t observe the oscillatory behavior of azurin.
Various explanations to these results can be given. First, the shift of the reporter gene could have perturbed the repressilator system, as it seems to be the case in Figures 7 and 8.
It is also possible that the integration of azurin disturbed the oscillations.
However, the results from Figure 12 seem as noisy as the results obtained with the construct without azurin.
In conclusion, it is not possible to determine with this construct if the integration of azurin is negatively impacting the oscillations.

### Second part: Repressilator plasmid pLPT119 [BBa\_K3482025]

After observing that we were not being able to modify the three reporter repressilator plasmid pLPT234 [BBa\_K3482023] without impacting the oscillations, we decided to test the simpler version of the repressilator (pLPT119).
We then tested again the repressilator plasmid without any modification in combination with the sponge plasmid pLPT41 [BBa\_K3482026] in *E. coli* DHL708.
In Figure 5, we show the fluorescence over time after synchronization of the bacterial culture.

![](https://static.igem.org/mediawiki/2020/7/7c/T--UNILausanne--Rep_Figure10_DHL708_pLPT119_pLPT41.png)

*Figure 10: Fluorescence over time plot of *E. coli* DHL708 containing plasmids pLPT119 [BBa\_K3482025] and pLPT41 [BBa\_K3482026].*

In Figure 10, the decrease and following increase of *mVenus* fluorescence over time is clearly visible, which is consistent with our model ( [See modeling page.](https://2020.igem.org/Team%3AUNILausanne/Model) ).
The *CFP* fluorescence signal is stable for the first 9 hours, as we expected.
However after this period, its fluorescence slowly started to decrease over time.
This indicates that the repressilator begins to be unstable after approximately 10 hours of oscillations.
This problem could be resolved by resynchronizing the cells with aTc.
Looking at the fact that our experiment lasted between 10 and 12 hours, these results are still confirming that our repressilator system was working correctly for the maximum period of time where we would take measurements.

After confirming that our repressilator system containing the repressilator plasmid pLPT119 [BBa\_K3482025] in combination with the sponge plasmid pLPT41 [BBa\_K3482026] works for at least 10 hours,
we decided to test our modified system, presented in Figure 4A.
This time, the plasmids were transformed in *E. coli* Nissle *Δclb*.
As before, we expected the fluorescent reporter *mVenus* to oscillate with time, and *CFP* levels to be stable.

![](https://static.igem.org/mediawiki/2020/a/a5/T--UNILausanne--Rep_Figure11_Nissle_pLPT119_pLPT41-Azu.png)

*Figure 11: Fluorescence over time plot of E.coli Nissle 1917 *Δclb* containing plasmids pLPT119 [BBa\_K3482025] and pLPT41-pelB-Azurin. The two lines for each reporter gene represent the two biological replicates that were done for this experiment.*

In Figure 11, we can observe the results of this new repressilator system.
As before, we see that the fluorescence of *mVenus* is oscillates over time.
During our experiment, we observed that the cells were also growing a bit faster, which explains the shift of oscillations observed compared to the results of Figure 5.
The fluorescence of *CFP* is however increasing with time, and then stabilizes for the 3 last hours of measurements.
This result is a bit surprising and does not correspond exactly to our expectations.
It is possible that the presence of azurin or the faster growth of the cells at the beginning of the experiment influenced the production of *CFP* during the first hours and that the stable state was reached with a delay.

In summary, these results suggest that the pLPT119 [BBa\_K3482025] repressilator and pLPT41 [BBa\_K3482026] sponge plasmid work in both *E. coli* DHL708 and *E. coli* Nissle 1917 *Δclb*.
The oscillation is stable until around 10 hours after synchronization.
While the growth speed of *E. coli* Nissle 1917 *Δclb* is slightly increased, the oscillation period is not affected by the possible expression of azurin in the sponge plasmid.

## Conclusion

With the repressilator experiments, we could confirm that we were able to obtain oscillations with all of our unmodified repressilator plasmids, integrated in *E. coli* DHL708.
We were however not able to characterise the oscillatory behaviour of the azurin expression in the modified system with the repressilator plasmid pLPT234 [BBa\_K3482023], because the shift of the fluorescent reporter gene *mVenus* disturbed the oscillation system.
In the modified repressilator system based on the plasmid pLPT119 [BBa\_K3482025], we were able to demonstrate that transferring our modified repressilator system gave to *E. coli* Nissle 1917 *Δclb* yielded similar results as to what was observed in the cloning strain.
We could then confirm that the repressilator system is working in our probiotic and that the integration of azurin is not disturbing the oscillations.
To go further in this experiment, we should integrate an independent fluorescence reporter link to azurin, to demonstrate that the azurin is following an oscillation behavior.

## References

1. Addgene: Johan Paulsson Lab Plasmids. https://www.addgene.org/Johan\_Paulsson/.
2. Kwok, R. Five hard truths for synthetic biology. *Nature* vol. 463 288–290 (2010).

# showAzurin expression

## **Introduction**

With the intent to propose an anti-cancer therapeutic agent that is producible in an *Escherichia coli strain* and that poses no strong side effect to the patient, we found azurin as a potential anti-cancer drug. Azurin is a blue copper-binding redox protein originally found in the periplasmic membrane of *Pseudomonas aeruginosa*[1]. It has recently been discovered to have an anti-cancer effect by targeting the well-known p53 apoptosis pathway and by preferentially entering cancerous cells [2]. Because we are using *E. coli Nissle 1917Δclb*  as our chassis organism and designed our azurin to be secreted, we codon-optimized the azurin gene based on *E. coli* (ATCC\_8739\_uid58783) using the “Genome calligrapher” from ChristenLab at the ETH Zürich [3]. Because azurin is a periplasm membrane protein, it is not normally secreted by bacteria. To reach our goal of secreting azurin, we added a secretion tag (*pelB-5D*) to the C-terminus of our sequence, which originates from a strain of *Erwinia carotovora* [4], [5].

### **Overview**

Our objective is to prove the expression and secretion of our optimized azurin in an oscillatory manner. Furthermore, we wanted to prove the anti-cancer effectiveness by testing it using a cell viability assay on cancerous cells. We first performed a purification of azurin, then we characterized its expression by SDS PAGE, Western Blot, and Mass Spectrometry (MS). Lastly, we applied our construct on a colorectal cancer model cell line to assess its cytotoxicity.[learn more about this in our proof of concept](https://2020.igem.org/Team%3AUNILausanne/Proof_Of_Concept)

## **Azurin purification**

To explore the therapeutic effect of azurin on cancer cells and to be able to compare it to our design, we needed to express and purify our protein. Multiple concentrations of inducing molecules, IPTG, were tested. The SDS PAGE was used to isolate and show the presence or absence of azurin. For this, we used the expression strain *E. coli BL21* transformed with the expression vector pET22 (BBa\_3482044), containing the optimized azurin coding gene mentioned above with a 6-His tag. We induced the expression using 50 µM or 500 µM of IPTG for 4 h (Figure 1A). In a second step, we selectively purified our azurin protein using the 6-His tag in combination with His Mag Sepharose® Ni (Sigma GE28-9673-90) (Figure 1B).

![SDS-PAGE of sample from bL21 expressing azurin for purification](https://static.igem.org/mediawiki/2020/9/93/T--UNILausanne--ESA_SDSPAGEPurification.png)

*Figure 1A**: SDS PAGE of bacterial lysate. Samples were induced for 4h with the mentioned IPTG concentrations. The OD of all cultures was normalized to 0.5. Samples were lysed for 10min at 95°C. and run on a 15% polyacrylamide gel for 30 minutes at 200V **1B**: SDS-PAGE with the same culture conditions as the uninduced sample from Figure 1A. Purification performed using HisPur Ni-NTA Magnetic Beads, incubated for 5 seconds with our sample**.* *Signaled in red is the expected azurin protein. Samples were run on a 15% polyacrylamide gel for 30 minutes at 200V.*

We can observe in Figure 1A the presence of three bands around 15 kDa, which is the size range of our azurin (14 kDa). Interestingly, the uninduced sample shows the highest intensity and we hypothesized that this could be due to intrinsic toxicity upon overexpression using IPTG. Thus, the leaky expression of the T7 promoter may allow for a higher yield of our protein expression. Upon purification using the specific 6-His tag, we can see in Figure 1B only one band (red box), which corresponds to the high-intensity band previously observed. **From these experiments, we concluded that the purification of azurin was successful.**

## **Proof of expression of azurin**

To verify the expression of the azurin that we successfully cloned into our sponge plasmid and purified, we concatenated a widely used and sensitive 3XFLAG tag at the C-terminus of our azurin gene (**BBa\_3482040**). This allowed us to perform a Western blot, which allowed us to confirm the presence or absence of our expressed azurin. In addition, we modified the existing part BBa\_K2500001 (truncated azurin uploaded by the ETH Zürich team of 2017) with either the secretion tag pelB-5D or NSP4[6] and investigated its secretion capability.

First, we tested our chassis, *E.coli Nissle 1917Δclb,* which has been transformed with a modified sponge plasmid (usable in combination with a repressilator plasmid in the future) which contains our target genes constitutively expressed under a pTet promoter (BBa\_K3482040, BBa\_K3482041, BBa\_K3482038, and BBa\_K3482037). We then performed a Western blot with cultures set to an OD600 of 2.0. The culture supernatants, separated from bacterial pellets by centrifugation and remaining bacterial pellet lysate were analyzed (Figure 2).

![Western blot analysis of flag-tagged azurin from 4 different parts under 2 conditions: supernatant or cell lysate.](https://static.igem.org/mediawiki/2020/2/2c/T--UNILausanne--ESA_WBDani.png)

*Figure 2**: Western blot analysis of flag-tagged azurin from 4 different parts under 2 conditions: supernatant or cell lysate. Size range is shown around 15 kDa.  Immunoblotting after SDS-PAGE migration, membrane transfer, and analysis performed with 3XFLAG antibody.*

We can observe the presence of the azurin linked to the FLAG tag in all cell lysate extracts. In addition, we can see that the full-length azurin as well as both versions with a secretion tag (pelB or NSP4)(Figure 2, samples 3 and 4) are present in the supernatant. Importantly,we can see that no secretion occurred without a secretion tag (Figure 2, sample 2) in comparison with the full azurin protein(Figure 2, sample 1). **This leads us to conclude that our azurin is indeed expressed and secreted. In addition, we successfully added the secretion aspect to an already characterized azurin part (BBa\_2500001).**

With their expertise in immunoblotting and mass spectrometry, we were able to further assess our preliminary results. The same samples as in the previous experiments were analyzed. SDS PAGE (Figure 3) was performed first to separate proteins by size, then upon analysis, two bands corresponding to the size of azurin were cut and further characterized using mass spectrometry (Figure 4):

![Figure 3: SDS PAGE by Candiano colloidal Coomassie staining, performed by the PAF UNIL](https://static.igem.org/mediawiki/2020/6/69/T--UNILausanne--ESA_WBMS.png)

*Figure 3**: SDS PAGE by Candiano colloidal Coomassie staining, performed by the PAF UNIL. In red, bands cut for Mass spectrometry analysis. Same samples as Figure 2, purified azurin (Figure1) was also sent for analysis.*

We can observe multiple bands at the predicted size range of azurin, but no clear overrepresented band (Figure 3). Because of this, two bands of different size range A and B were cut and analyzed using Mass spectrometry (Figure 4).

Note that due to the absence of visible bands on SDS PAGE (data not shown) of the supernatant, no MS analysis was done for the supernatant. This does not mean, however, that no protein can be found in the supernatant, thus secreted, but rather that the SDS PAGE methods, in contrast with western blot (Figure 2, supernatant sample) are probably not sensitive enough to be able to see the azurin secretion.

Using MS allowed us to assess the nature of the protein content of our cell lysate as well as being able to characterize the relative quantification of the constitutive expression of our construct. Indeed, with a sequence coverage of 97.7% (difference due to the presence of truncated azurin as well as secretion and/or flag tag) based on the BBa\_3482022 sequence, **it is possible to confirm that the purified, as well as the recombined protein with 3XFLAG, are azurin.**

![MS analysis of relative protein quantity.](https://static.igem.org/mediawiki/2020/7/7e/T--UNILausanne--ESA_MSQuanti.png)

*Figure 4**: MS analysis of relative protein quantity. Based on the initial amount of our reference purified azurin of 12.5 ng, recombinant azurin amounts were calculated using ratios of normalized MS protein signal intensity by reference purified azurin.*

We can see that the presence of azurin was detected only in band A Figure 3), which was of higher size than expected. This can be due to the differentiation of migration occurring during SDS PAGE and due to conformational change which can occur when tags and flag tags are concatenated to a protein. We can also observe that the relative amount of azurin is lower for the truncated azurin containing a secretion tag. We hypothesis that due to secretion, we found less azurin in the cell lysate.

In summary, **we were able to detect the presence of azurin and quantify the relative amount of protein constitutively expressed** under the pTet promoter in *E.coli Nissle 1917Δclb*  for the BBa\_K3482040, BBa\_K3482038, and BBa\_K3482037 part.

# showKill switch system

## Achievements

* **Contribution** (bronze medal): we **provide the first and extensive characterization of the toxin-antitoxin systems** ccdB/ccdA ([BBa\_P1010](http://parts.igem.org/Part%3ABBa_P1010), [BBa\_K1075032](http://parts.igem.org/Part%3ABBa_K1075032)) and miniColicin E2/IM2 ([BBa\_K1976048](http://parts.igem.org/Part%3ABBa_K1976048), [BBa\_K1976027](http://parts.igem.org/Part%3ABBa_K1976027)) and **identified the promoter induction range for differential cell growth inhibition**. We also **demonstrate** that the widely used *E. coli* Nissle 1917ΔclbA has a ccdB sensitive gyrase genotype
* **Engineering success** (silver medal): we **characterized our new composite part** ([BBa\_K3482017](http://parts.igem.org/Part%3ABBa_K3482017))
* **Proof of concept** (gold medal): we **demonstrate that our kill switch system controls cell growth effectively depending on temperature.**

### **Introduction**

The aim of the kill switch is to constrain the activity of our engineered probiotic *Escherichia coli* Nissle 1917 Δclb to the (permissive) colonic environment. The viability of the cells should be reduced under non-permissive conditions where either the temperature is decreased or the phosphate concentration is increased (**Table 1**). The goal is to develop a system that on one hand kills the bacteria when they get in the environment where it’s colder (at least in the waste water), and on the other to kill the bacteria if they were to enter the bloodstream, where higher phosphate concentrations are present in comparison to the colon. This would also allow to remove the bacteria from the patient’s gut by providing an encapsulated pill which would release high amounts of phosphate in the colon.

**Table 1** Kill switch viability logic with temperature and phosphate concentration as inputs.

|  |  |  |  |
| --- | --- | --- | --- |
| **Condition** | **Temperature [°C]** | **Phosphate concentration** | **Predicted viability of host cell** |
| Permissive | 37 | Low | High |
| Non-permissive | 37 | High | Low |
| Non-permissive | 25 | Low | Very low |

We employ two toxin-antitoxin plasmid systems based on toxin-antitoxin pairs ccdB/ccdA ([BBa\_P1010](http://parts.igem.org/Part%3ABBa_P1010), [BBa\_K1075032](http://parts.igem.org/Part%3ABBa_K1075032)) and miniColicin E2/IM2 ([BBa\_K1976048](http://parts.igem.org/Part%3ABBa_K1976048), [BBa\_K1976027](http://parts.igem.org/Part%3ABBa_K1976027)). The ccdB toxin is a DNA gyrase inhibitor which, in absence of the ccdA antitoxin, leads to DNA breakage and cell death [1]. This ccdB toxin is active only against bacteria because DNA gyrase is only present in bacteria and lower eukaryotes [2]. The miniColicin E2 toxin is a DNA endonuclease which, in absence of the IM2 antitoxin, also leads to cell death [3]. The miniColicin E2 gene does not code for neither the translocation nor receptor binding domain, and can therefore not be secreted by the bacteria. In case the toxin were released in the gut environment, it would be digested by the highly abundant proteases [4]. Therefore, these toxins do not pose any health risk.

The toxin and antitoxin genes were cloned into pAND-MSC ([BBa\_K3482021](http://parts.igem.org/Part%3ABBa_K3482021)), a plasmid that allows for inducible expression of the genes cloned under the pTet and pTac promoters, with IPTG and aTc respectively [5]. The toxins were inserted under the control of pTac, whereas the antitoxin under that of pTet.

To incorporate the temperature logic, the heat-inducible 2U RNA thermosensor ([BBa\_K3482000](http://parts.igem.org/Part%3ABBa_K3482000)) was inserted upstream of the antitoxin genes, while the F2 heat-repressible RNA thermosensor ([BBa\_K3482001](http://parts.igem.org/Part%3ABBa_K3482001)) can be inserted upstream of the toxin genes. The heat-inducible RNA thermosensor reduces mRNA translation efficiency at lower temperature by forming a hairpin structure that conceals the ribosome binding site (RBS) [6]. Conversely, the heat-repressible RNA thermosensor reduces the mRNA translation efficiency by exposing a RNase E cleavage site, which is concealed at lower temperature by an hairpin structure [7]. Lastly, to incorporate the phosphate concentration logic, the pTet promoter can be replaced by a phosphate-repressible pPhoB promoter ([BBa\_K116401](http://parts.igem.org/Part%3ABBa_K116401)).

Our kill switch design is based on two toxin-antitoxin systems that enable us to guarantee plasmid retention of our repressilator and sponge plasmids by establishing a so-called plasmid addiction system. This is a tool based on the post-segregational killing mechanism [8]. This is an established system used to maintain plasmids without using antibiotics. It relies on the higher stability of the toxin compared to the antitoxin. This means that daughter cells who do not inherit the plasmid are killed by the remaining toxin in the cell, while the antitoxin, less stable, is degraded . Therefore, the kill switch module coding for toxin and antitoxin can be used to replace the antibiotic resistance genes of our repressilator and sponge plasmid. Consequently, our therapeutic strain will be even safer since it cannot transfer any antibiotic resistance genes to the other bacteria of the gut microbiota.

### **Objectives**

The objectives of the experiment are the following:

1. Study the cell-growth effect of different concentrations of antitoxin and toxin inducers, aTc and IPTG, respectively.
2. Assess the effect of the 2U RNA thermosensor on the expression of antitoxin at 37°C and 25°C.

## Protocol

### **Bacterial strains**

We use *E. coli* Nissle 1917 ΔclbA [9]. The clbA gene is part of an endogenous cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s), which confer the strain the ability to induce double strand breaks in eukaryotic cells. Thus, the inactivation of the clbA gene abrogates the genotoxic ability strain. *E. coli* Nissle 1917 ΔclbA has been transformed with the plasmids listed in **Table 2** and depicted in **Figure 1**.

**Table 2** Plasmids used for the kill switch experiments.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Plasmid name** | **Backbone** | **Toxin** | **Antitoxin** | **RNA thermosensor name, function** | **Reference** |
| pAND | pAND-MSC([BBa\_K3482021](http://parts.igem.org/Part%3ABBa_K3482021)) | - | - | - | [5] |
| pKA1 | pAND-MCS ([BBa\_K3482021](http://bba_k3482021/)) | ccdB([BBa\_P1010](http://parts.igem.org/Part%3ABBa_P1010)) | ccdA([BBa\_K1075032](http://parts.igem.org/Part%3ABBa_K1075032)) |  |  |
| pKC1 | pAND-MCS ([BBa\_K3482021](http://bba_k3482021/)) | miniColicin E2([BBa\_K1976048](http://parts.igem.org/Part%3ABBa_K1976048)) | IM2([BBa\_K1976027](http://parts.igem.org/Part%3ABBa_K1976027)) |  |  |
| pKC3 | pAND-MCS ([BBa\_K3482021](http://bba_k3482021/)) | miniColicin E2([BBa\_K1976048](http://parts.igem.org/Part%3ABBa_K1976048) ) | IM2([BBa\_K1976027](http://parts.igem.org/Part%3ABBa_K1976027)) | 2U, heat-inducible antitoxin expression([BBa\_K3482017](http://bba_k3482017/)) | [6] |

![toxin antitoxin graph](https://static.igem.org/mediawiki/2020/a/a1/T--UNILausanne--experiments_graph.png)

**Figure 1: Inducible toxin-antitoxin plasmids.****a)** pAND-MSC ([BBa\_K3482021](http://parts.igem.org/Part%3ABBa_K3482021)) is the empty vector used as negative control. **b)** pKA1 codes for the ccdb toxin and ccda antitoxin ([BBa\_K3482016](http://parts.igem.org/Part%3ABBa_K3482016) and [BBa\_K3482020](http://bba_k3482020/)). **c)** pKC1 codes for miniColicin E2 toxin and IM2 antitoxin ([BBa\_K3482004](http://bba_k3482004/) and [BBa\_K3482014](http://parts.igem.org/Part%3ABBa_K3482014)). **d)** pKC3 codes for miniColin E2 toxin and IM2 antitoxin coupled to the 2U heat-inducible RNA thermosensor ([BBa\_K3482000](http://parts.igem.org/Part%3ABBa_K3482000)).

For the experiments, the following strains were used:

* *Escherichia coli* Nissle 1917 ΔclbA transformed with pKA1 or pKC1 harboring toxin and antitoxin genes under the control of pTet ([BBa\_K3482012](http://parts.igem.org/Part%3ABBa_K3482012)) and pTac ([BBa\_K3482011](http://parts.igem.org/Part%3ABBa_K3482011)), respectively.
* *Escherichia coli* Nissle 1917 ΔclbA transformed with pAND. Negative control for the effect of the toxin.
* *Escherichia coli* Nissle 1917 ΔclbA transformed with pKC3 harboring miniColicin E2 toxin and heat-inducible IM2 antitoxin.

### **Material**

The required solutions are listed in **Table 3**. Further material needed is:

* 14-mL round bottom culture tubes with breathable cap

* Biotek Synergy h2, Plate reader
* Nanodrop

**Table 3** Solutions used for the kill switch experiments.

|  |  |
| --- | --- |
| **Solutions** | **Stock concentration** |
| Luria Broth (LB) |  |
| aTc stock solution | 100 ng/mL |
| IPTG stock solution | 1 M |
| Kanamycin | 50 mg/mL |

### **Procedure**

1. Pick single colonies from plates to inoculate 1 mL LB with 50 µg/mL kanamycin in tubes.
2. Incubate the culture at 37°C, shaking at 200 rpm for 3-6 h.
3. Measure the OD600 and dilute the cultures to OD600 = 0.05 in 2 mL LB with 50 µg/mL kanamycin.
4. Inoculate the 96-wells plate with 90 µL of the OD600 = 0.05 culture according to the chosen plating layout.
5. Prepare dilutions of aTc and IPTG in LB with 50 µg/mL kanamycin.
6. Add 10 µL of the 10X aTc of the desired final concentration to the appropriate wells.
7. Add 10 µL of the 10X IPTG of the desired final concentration to the appropriate wells.
8. Incubate the plate at either 37°C or 25°C under continuous shaking in the plate reader for 10 h or overnight. Set an absorbance measurement at 600 nm every 10 minutes.

### **Data analysis**

Raw data was exported using the automatic export function of Gen5 (version 5.3). These raw data were processed in Excel and analyzed in R.

## **Results**

### **ccdB/ccdA kill switch plasmid pKA1**

We first tested our pKA1 plasmid encoding for the ccdB toxin and ccdA antitoxin in *E. coli* Nissle 1917 ΔclbA. We used different concentrations of IPTG and aTc to study the effect of differential expression of the toxin and antitoxin on the growth ofour strain. As a control, we used the strain transformed with the empty vector pAND (**Figure 2**).

We observed desired growth inhibition of the strain with pKA1 at high IPTG and low aTc concentrations. The pAND strain showed as expected no alteration in growth in any of the tested conditions. The uniformity of these growth curves of the strain with pAND demonstrates that the standardized culturing and OD normalization of our protocol can yield reproducible results.

The sensitivity towards ccdB demonstrates that *E. coli* Nissle 1917 has a sensitive gyrase genotype, contrasting other *E. coli* strains such as DH5α which has a gyrA96 genotype which renders them resistant to the toxin [10]. Interestingly, cell growth rescued by expression of ccdA was only noticeable in the transition from 0 to 0.1 ng/mL and from 10 to 50 ng/mL aTc induction.

We saw an effect of IPTG on growth inhibition starting from 5 µM IPTG. At maximum IPTG concentration, we observed complete absence of growth of the pKA1 strain for the first 7 h. However, afterwards, we observed that the bacteria were able to grow. This was probably due to mutations in the kill switch system. In contrast, at lower IPTG concentrations (5 and 10 µM), we still observed an inhibitor effect on growth, which albeit being less strong, was more constant and persisted for at least 10 h. This suggests that decreasing the expression of the toxin lowers the selective pressure on the kill switch system, which makes this configuration more evolutionarily stable.

Overall, we demonstrated that the ccdB/ccdA encoding pKA1 plasmid could efficiently inhibit the growth of *E. coli* Nissle 1917 at 100 µM IPTG for up to 7 h, and provided even longer growth inhibition at 5 and 10 µM IPTG.

![](https://static.igem.org/mediawiki/2020/c/c3/T--UNILausanne--experiments_graph2.png)

**Figure 2: Dose-response growth curve of** ***E. coli*** **Nissle 1917** **Δ****clbA harboring kill switch plasmid pKA1 at 37°C.** *E. coli* Nissle with pAND (red line), *E. coli* Nissle with pKA1 (blue line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

### **MiniColicin E2/IM2 kills switch plasmid pKC1**

Secondly, we tested our pKC1 plasmid encoding for the miniColicin E2 toxin and IM2 antitoxin in *E. coli* Nissle 1917 ΔclbA. Again, we used different concentrations of IPTG and aTc to study the effect of differential expression of the toxin and antitoxin on the growth ofour strain. As a control, we used the strain transformed with the empty vector pAND (**Figure 3**).

As with *E. coli* Nissle 1917 with pKA1, we observed desired growth inhibition of the strain with pKC1 at high IPTG and low aTc concentrations. Again, as expected the pAND strain showed no alteration in growth in any of the tested conditions. Contrastingly to *E. coli* Nissle 1917 transformed with pKA1, the induction of aTc was clearly able to rescue cell growth. For increasing concentrations of IPTG, also increasing concentrations of aTc were necessary to rescue cell growth. This linear relationship was compatible with the experiments demonstrating binding of IM2 to Colicin E2 with a 1:1 stoichiometry [11]. Similar to *E. coli* Nissle 1917 pKA1, cell growth could be completely inhibited for 6-8 h (IPTG: 100 µM, aTc: 0 ng/mL) before observing rapid growth. Also similarly to pKA1, the reduction in IPTG concentration gave a lesser but more constant growth inhibition, which also persisted to at least 10 h.

Overall, we demonstrated that the miniColicin E2/IM2 encoding pKC1 plasmid could efficiently inhibit the growth of *E. coli* Nissle 1917 at 100 µM IPTG for up to 6 h and provided even longer growth inhibition at 5 µM IPTG.

![](https://static.igem.org/mediawiki/2020/1/1a/T--UNILausanne--experiments_graph3.png)

**Figure 3:****Dose-response growth curve of** ***E. coli*** **Nissle 1917** **Δ****clbA harboring kill switch plasmid pKC1 at 37°C.** *E. coli* Nissle with pAND (red line), *E. coli* Nissle with pKC1 (green line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

### **MiniColicin E2/ heat-inducible IM2 kill switch plasmid pKC3**

Finally we tested our pKC3 plasmid encoding for the miniColicin E2 toxin and heat-inducible IM2 antitoxin in *E. coli* Nissle 1917 ΔclbA. Again, we used different concentrations of IPTG and aTc to study the effect of differential expression of the toxin and antitoxin on the growth ofour strain. As a control, we used the strain transformed with the empty vector pAND. The comparison of the pKC3 with the the pKC1 strain (no thermosensor), allowed to determine the effect of the growth inhibition caused by the thermosensor (**Figure 4**).

Our desired outcome was to observe growth of the strain with pKC3 at 37°C but not at 25°C for a given combination of aTc and IPTG concentration. This behaviour was clearly observed for the low aTc and IPTG concentrations, while the strain with pKC1 (no thermosensor) did not show any growth impairment at 25°C. While it might seem surprising that the effect of the toxin is obtained also without addition of IPTG, it has to be considered that inducible promoters have a baseline (leaky) expression even when no inducer is added.Our results suggest that the leaky expression of the pTet and pTac promoter yielded the best balance of toxin and antitoxin expression for the efficient growth inhibition at low temperatures. The growth inhibition of the strain with pKC3 at 25°C could be clearly rescued by the addition of aTc. It can also be noticed that at 37°C and in absence of aTc, the pKC3 strain showed a slight growth delay compared to the strain with pKC1.This is due to the fact that even at 37°C the thermosensor slightly reduces the translation efficiency of the gene, which it is coupled to [6].

In comparison to pKA1 and pKC1 plasmids, the growth inhibiting effect of pKC3 at 25°C seems to last longer, and for the vast majority of aTc and IPTG concentration combinations no sharp increase in growth was observed at any measured time point. This might be a result of the decrease in growth rate at lower temperature, which might also aid in reducing the selective pressure on the cells to mutate the components of the kill switch.

Overall, we demonstrated that the miniColicin E2/ heat-inducible IM2 encoding pKC3 plasmid could effectively inhibit the growth of *E. coli* Nissle 1917 at 25°C for low aTc and IPTG concentrations.

![](https://static.igem.org/mediawiki/2020/7/73/T--UNILausanne--EXPkl4.png)

**Figure 4: Dose-response growth curve of** ***E. coli*** **Nissle 1917** **Δ****clbA harboring kill switch plasmids pKC1 and pKC3 at 37 and 25°C.** *E. coli* Nissle with pAND (red line), *E. coli* Nissle with pKC1 (green line), and *E. coli* Nissle with pKC3 (blue line), at 37°C (dashed line) and 25°C (solid line). The lines and shade represent the mean ± standard error. Each plot shows the absorbance at 600 nm measured every 10 min for a period of 10 hours at different aTc and IPTG concentrations.

## **Conclusion and outlook**

### **Impact of observed growth inhibition on the feasibility of our design for clinical applications**

We demonstrated that our toxin-antitoxin plasmid systems enable us to efficiently control cell viability. Doing so, we performed extensive measurements on the ccdB/ccdA and miniColicin E2/IM2 parts, which were previously uncharacterized on the iGEM Part Registry. Moreover, we provide proof of concept for a temperature sensitive kill switch, which allows to stop cell growth under the non-permissive condition of low temperature.

The main threat to our system are mutations of the kill switch components, as this can cause the system to become unresponsive to the non-permissive conditions of the kill switch system. This could pose a risk for the escape of the genetically modified bacteria in the environment, which is a widespread societal concern.

While we observed the emergence of these escape mutants with the pKA1 and pKC1 system at high IPTG concentrations (high toxin expression), longer growth inhibitions were observed when the expression of the toxin was reduced. While resulting in some growth of the bacteria, the growth rate was largely reduced. Considering the competitive environment of the gut microbiota and its quick turnover rate [12 ,13], the longer growth inhibition observed at lower toxin expression induction are most likely sufficient to guarantee the removal of the bacteria from the patient’s gut. Once in the environment (for example the wastewater system), where the temperature is likely even lower than 25°C, we expect the bacteria to not proliferate, as we clearly demonstrated that the drastically reduced growth rate at 25°C persists for at least 10 hours. To validate these hypotheses we suggest to firstly measure the retention of *E. coli* Nissle 1917 with one of the kill switch plasmid in an *in vitro* continuous fermentations with a complex gut microbiota with and without induction of the kill switch system, and secondly to measure the proliferation of *E. coli* Nissle 1917 within a complex waste water sample. Moreover, our design already includes another component which improves the evolutionary stability of the kill switch system. This component is a cold-inducible RNA thermosensor which can be coupled to the toxin. This would limit the expression of the toxin at 37°C but not at lower temperature. This would further reduce the selective pressure to mutate the toxin (or any other component which would lead to inactivation of the kill switch) under permissive conditions rendering our kill switch system even more robust.

While we demonstrate that our kill switch system can be effectively used for biocontainment in the environment, due to time constraints we have not performed measurements of the design of our system encoding for the phosphate control. However, this component has already been extensively characterized by the NUS 2017 team ([http://parts.igem.org/Part:BBa\_K2447000](http://parts.igem.org/Part%3ABBa_K2447000)), we are therefore confident that it’s implementation would succeed as planned.

As in practice IPTG and aTc cannot be supplied to induce the toxin and antitoxin, the pTet and pTac promoters need to be replaced by either a constitutive or another type of inducible promoter. The selection of these promoters can be easily done using the abundant promoter characterization in the iGEM Registry or in the literature, and can be guided by our [kill switch model](https://2020.igem.org/Team%3AUNILausanne/Model). Concretely, one can use our measurements to see for which aTc and IPTG concentration the desired growth inhibition effect is obtained. Then, these inducer concentrations can be matched to the GFP-measurements of the pTet ([BBa\_R0040](http://parts.igem.org/Part%3ABBa_R0040)) and pTac ([BBa\_K2572025](http://parts.igem.org/Part%3ABBa_K2572025)) promoters. The corresponding GFP output can then be matched to that of one of many promoters with GFP dose-response measurements, such as those in the Anderson promoter catalog (<http://parts.igem.org/Promoters/Catalog/Anderson>).

We demonstrated that all three constructed kill switch systems are able to inhibit cell growth as desired, and also showed for which level of inducer concentrations of the toxin and antitoxin expression different levels of growth inhibition can be obtained. We also argued that our kill switch system is most likely sufficient to guarantee a high level of biocontainment and its translation to a completely autonomous kill switch system for application in clinical settings to be achievable with minor adaptations.

## **Best Measurement Achievement**

With our extensive characterization of the toxin-antitoxin systems ccdB/ccdA ([BBa\_P1010](http://parts.igem.org/Part%3ABBa_P1010),

[BBa\_K1075032](http://parts.igem.org/Part%3ABBa_K1075032)) and miniColicin E2/IM2 ([BBa\_K1976048](http://parts.igem.org/Part%3ABBa_K1976048), [BBa\_K1976027](http://parts.igem.org/Part%3ABBa_K1976027)) we lay the foundations for an easy and standardized implementation of kill switch solutions based on ccdB/ccdA and miniColicinE2/IM2 for the iGEM community.

### **References**

1. [1] P. Bernard and M. Couturier, “Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes,” *J. Mol. Biol.*, 1992.
2. [2] F. Collin, S. Karkare, and A. Maxwell, “Exploiting bacterial DNA gyrase as a drug target: Current state and perspectives,” *Applied Microbiology and Biotechnology*. 2011.
3. [3] E. Cascales *et al.*, “Colicin Biology,” *Microbiol. Mol. Biol. Rev.*, 2007.
4. [4] X. Jin, W. Kightlinger, Y. C. Kwon, and S. H. Hong, “Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis,” *Synth. Biol.*, 2018.
5. [5] B. C. Stanton, A. A. K. Nielsen, A. Tamsir, K. Clancy, T. Peterson, and C. A. Voigt, “Genomic mining of prokaryotic repressors for orthogonal logic gates.,” *Nat. Chem. Biol.*, vol. 10, no. 2, pp. 99–105, 2014.
6. [6] S. Sen, D. Apurva, R. Satija, D. Siegal, and R. M. Murray, “Design of a Toolbox of RNA Thermometers,” *ACS Synth. Biol.*, vol. 6, no. 8, pp. 1461–1470, 2017.
7. [7] A. Hoynes-O’Connor, K. Hinman, L. Kirchner, and T. S. Moon, “De novodesign of heat-repressible RNA thermosensors in E. Coli,” *Nucleic Acids Res.*, vol. 43, no. 12, pp. 6166–6179, 2015.
8. [8] J. Tsang, “Bacterial plasmid addiction systems and their implications for antibiotic drug development,” *Postdoc J.*, 2017.
9. [9] M. Olier *et al.*, “Genotoxicity of escherichia coli nissle 1917 strain cannot be dissociated from its probiotic activity,” *Gut Microbes*, vol. 3, no. 6, pp. 501–509, 2012.
10. [10] Openwetware, “E. coli genotypes.” [Online]. Available: https://openwetware.org/wiki/E.\_coli\_genotypes.
11. [11] D. Duché, A. Frenkian, V. Prima, and R. Lloubès, “Release of immunity protein requires functional endonuclease colicin import machinery,” *J. Bacteriol.*, 2006.
12. [12] M. A. Bauer, K. Kainz, D. Carmona-Gutierrez, and F. Madeo, “Microbial wars: Competition in ecological niches and within the microbiome,” *Microbial Cell*. 2018.
13. [13] W. Tottey *et al.*, “Colonic transit time is a driven force of the gut microbiota composition and metabolism: In vitro evidence,” *J. Neurogastroenterol. Motil.*, 2017.

A big thank you to our sponsors for their valuable support!

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# Team:UNILausanne/Results

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# Results Overview

## Overview

* We designed and constructed plasmids for *E. coli* Nissle 1917 Δclb to produce and secrete the anti-cancer peptide [azurin](https://2020.igem.org/Team%3AUNILausanne/Proof_Of_Concept) in an oscillatory manner by using a [repressilator](https://2020.igem.org/Team%3AUNILausanne/Experiments)
* We demonstrated that the repressilator works in our chosen chassis
* We demonstrated that the azurin is produced and also secreted
* We characterized the effect of [anti-cancer drugs on colorectal cancerous Caco-2 cells](https://2020.igem.org/Team%3AUNILausanne/Proof_Of_Concept)
* We assembled a [kill switch](https://2020.igem.org/Team%3AUNILausanne/Experiments) so that our engineered bacteria will not survive outside the patient's gut

## [Repressilator](https://2020.igem.org/Team%3AUNILausanne/Experiments)

#### Overview

Chronotherapy is a promising novel approach to treat cancer. Studies suggest that administration of anti-cancer drugs at a certain point of the circadian rhythm has a positive impact on the treatment efficiency and can lower side effects. We aim to combine *E. coli* Nissle 1917 Δclb bacteria‐assisted tumor‐targeted therapy with chronotherapy to explore a new method of fighting cancer. The oscillatory release of the anti-cancer drug is achieved by using a synthetic oscillatory circuit called the “repressilator”.

#### Key Achievements

![](https://static.igem.org/mediawiki/2020/2/21/T--UNILausanne--results_overview_repressilator.jpg)

***Figure 1A:** Modelled oscillatory expression pattern of the repressilator with three fluorescent reporter genes under promoters lacl, tetR and cl.
**1B:** Oscillatory pattern obtained by transforming *E. coli* DHL708 with BBa\_K3482023 (pLPT234) and Part:BBa\_K3482024 (pLPT145), which are respectively the repressilator plasmid and the sponge plasmid. The repressilator is based on 3 repressor proteins associated to three fluorescent proteins, which are CFP, mKate2 and mVenus, under the same 3 promoters that we can see on Figure 1A (CFP is under p\_tetR, mKate2 under p\_lacl and mVenus under p\_cl) and the sponge plasmid was used to stabilize the oscillator.
The bacteria were diluted to the same OD so the fluorescence would not depend on the quantity of bacteria. All bacteria were synchronized with aTC or IPTG to start oscillations at the same time. A plate reader was used to measure OD and fluorescence for each reporter gene.
Data shows the mean and the standard deviation of 3 replicates.*

* We tested an established repressilator system and modified it with our gene of interest
* We established and optimized a protocol that allowed us to detect oscillations by the repressilator in a plate reader assay

## [Azurin secretion](https://2020.igem.org/Team%3AUNILausanne/Proof_Of_Concept)

#### Overview

Azurin is a copper-binding redox protein originally found in *Pseudomonas aeruginosa*. In *P. aeruginosa*, azurin is exported into the periplasm. Azurin reduces the growth of human breast cancer and melanoma tissue with minimal side-effects. Moreover, it can be produced in *E. coli*. To make azurin an effective drug it needs to be secreted outside the bacterial cell in the vicinity of the colorectal tumor. Therefore, we used *E. coli* Nissle 1917 Δclb as our chassis and engineered the azurin gene so it can be secreted.

#### Key Achievements

* We added either pelB-5D or NSP4 secretion tags to the azurin and showed that the expression and secretion of azurin by our designed bacteria
* We inserted the azurin with either peIB-5D or NSP4 secretion tags into the sponge plasmid of the repressilator

## [MTT assay](https://2020.igem.org/Team%3AUNILausanne/Proof_Of_Concept)

#### Overview

The effectiveness of azurin against cancer cells has only been tested on breast tumors and melanoma up to now. Azurin showed significant effects on these kinds of cancer because it can amplify the effect of p53, an intracellular protein which acts as a pro-apoptosis factor. However, the effects of azurin on colorectal cancer cells are still unknown. The susceptibility of the colorectal cancer cell line called Caco-2 to different anti-cancer drugs was characterized for the first time in the iGEM competition.

#### Key Achievements

![](https://static.igem.org/mediawiki/2020/0/07/T--UNILausanne--MTTassay_bacterialexctract_overview.jpg)

***Figure 2A:** Caco-2 cell viability (%) when they are incubated with 0.15 % Triton X or Medium or purified azurin [2 μM]. **2B:** Plasmids for each color: blue, negative control; red, negative control for the azurin secretion; green, PeIB-5D secretion tag on azurin; purple, NSP4 secretion tag on azurin.*

* We tested different concentrations of anisomycin, cisplatin, doxorubicin, salirasib and TNFa on Caco-2 cells
* We applied purified azurin on Caco-2 cells, but azurin does not have an effect on the Caco-2 cell viability at the tested concentration (Figure 2A)

## [Kill switch](https://2020.igem.org/Team%3AUNILausanne/Experiments)

#### Overview

To prevent the survival of the GMOs in the environment and in the patient's body outside the colon, we designed a kill switch. The kill switch prevents undesired survival of our chassis by killing it with a toxin/antitoxin system at two conditions: when the temperature is lower than 37°C (body temperature) and when the phosphate concentration is too high (conditions of the bloodstream). Moreover, the kill switch is intended to act as an addiction module that allows us to maintain our plasmids without the need of antibiotic resistance genes within the patient’s gut.

#### Key Achievements

* We assembled a multi-responsive sensor: it reacts on temperature and phosphate concentration
* we characterized the toxin-antitoxin systems ccdb/ccda and miniColicin E2/IM2
  and identified the promoter induction range for differential cell growth inhibition
* We demonstrated that *E. coli* Nissle 1917 ΔclB has a sensitive gyrase genotype to the ccdB toxin
* We characterized our new composite part ([BBa\_K3482017](http://parts.igem.org/Part%3ABBa_K3482017))
* We demonstrated that our kill switch system controls cell growth effectively depending on temperature

A big thank you to our sponsors for their valuable support!

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