iGEM 2020 — Team Stockholm

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# Team:Stockholm/Notebook

Notebook | iGEM Stockholm

* Project

  [Description](https://2020.igem.org/Team%3AStockholm/Description)[Design](https://2020.igem.org/Team%3AStockholm/Design)[Proof of Concept](https://2020.igem.org/Team%3AStockholm/Proof_Of_Concept)[Implementation](https://2020.igem.org/Team%3AStockholm/Implementation)[Contribution](https://2020.igem.org/Team%3AStockholm/Contribution)[Poster](https://2020.igem.org/Team%3AStockholm/Poster)
* Lab & Parts

  [Model](https://2020.igem.org/Team%3AStockholm/Model)[Engineering](https://2020.igem.org/Team%3AStockholm/Engineering)[Parts](https://2020.igem.org/Team%3AStockholm/Parts)[Experiments](https://2020.igem.org/Team%3AStockholm/Experiments)[Results](https://2020.igem.org/Team%3AStockholm/Results)[Safety](https://2020.igem.org/Team%3AStockholm/Safety)[Notebook](https://2020.igem.org/Team%3AStockholm/Notebook)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AStockholm/Human_Practices)[Education](https://2020.igem.org/Team%3AStockholm/Education)
* About

  [Team](https://2020.igem.org/Team%3AStockholm/Team)[Collaborations](https://2020.igem.org/Team%3AStockholm/Collaborations)[Sponsors](https://2020.igem.org/Team%3AStockholm/Sponsors)[Attributions](https://2020.igem.org/Team%3AStockholm/Attributions)
* Awards

  [Entrepreneurship](https://2020.igem.org/Team%3AStockholm/Entrepreneurship)[Sustainable](https://2020.igem.org/Team%3AStockholm/Sustainable)[Medal Criteria](https://2020.igem.org/Team%3AStockholm/Medal_Criteria)

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# Notebook

Experimental procedure schedule

![](https://static.igem.org/mediawiki/2020/0/0b/T--Stockholm--img--Contents--topimg_notebook.png)

##### Contents

# June

##### 18/06 - 19/06

* Acquired *Shewanella oneidensis*, *Shewanella putrefaciens*, *Escherichia coli* MFDpir for conjugation and *Escherichia coli* DH5a containing the pSRK-Km plasmid for *Shewanella oneidensis* expression
* Prepared glycerol stocks of obtained strains

##### 25/06 - 26/06

* Purified pSRK-Km from *E.coli* DH5a

# July

##### 01/07 - 03/07

* Created chemically competent *E.coli* MFDpir for transformation
* Performed test transformations of MFDpir and TOP10 chemically competent *E.coli*
* Amplified BioBricks from Uppsala iGEM 2020 team

##### 06/07 - 11/07

* Acquired *Shewanella oneidensis* wildtype (WT) and MtrB knockout strains, and *Chromobacterium Violaceum* CV026 for biological AHL assays
* Prepared glycerol stocks of obtained strains
* Amplified BioBricks from Stockholm iGEM 2019 team
* Transformed and amplified BioBricks in vivo
* Electroporation of electrocompetent cells

##### 15/07 - 17/07

* Assembly of BioBricks was attempted, but incomplete digestion gave too high background or no successful ligations

##### 23/07

* Restriction digest troubleshooting

##### 27/07 - 31/07

* Acquired gBlocks of constructs or partial constructs
* PCR amplified gBlocks
* Purification of pSB1C3 for ligating gBlocks into

# August

##### 03/08 - 07/08

* Transformed to amplify gBlocks in vivo

##### 10/08 - 14/08

* Colony PCR to select successful constructs
* Amplified gBlocks in vivo

##### 17/08-21/08

* Started MFC with *Shewanella oneidensis* WT
* PCR amplified gBlocks
* Colony PCR to select successful constructs
* Transformed to amplify gBlocks in vivo
* Attempted to assemble construct from partial constructs

##### 24/08-31/08

* Colony PCR to select successful constructs
* *Chromobacterium* assay to check for presence of AHL
* Ran Western blot to check for protein expression in constructs

# September

##### 01/09-05/09

* Transformed to amplify gBlocks in vivo
* Ran Western blot
* Colony PCR to select successful constructs
* Added LB media to MFC
* Replaced MFC media with M9 minimal media with lactate

##### 07/09-13/09

* Stopped running MFC
* Measured fluorescence intensity of constructs to check leakiness of promoters
* *Chromobacterium* assay
* Ran Western blot
* Attempted to assemble oscillation constructs
* Acquired PFOS and 1,1-biphenyl, prepared stock solutions

##### 15/09-17/09

* Colony PCR to select successful constructs

##### 22/09-27/09

* Ran viability test with pollutants
* Prepared positive control (Ftsh1-Myc) for Western blot
* Ran Western blot

##### 28/09-30/09

* Measured fluorescence intensity of constructs induced with pollutants

# October

##### 01/10-04/10

* Measured fluorescence intensity of constructs when induced with constructs producing AHL synthases and/or pollutants
* Ran Western blot
* Ran viability tests with higher pollutant concentrations

##### 05/10-11/10

* Started MFC with *Shewanella oneidensis* MtrB KO
* Repeated viability test with higher pollutant concentrations
* *Chromobacterium* assay with constructs producing AHL synthases

##### 12/10-17/10

* Ran Western blot
* Measured fluorescence intensity of constructs when induced with QS molecules and/or pollutants
* *Chromobacterium* assay for AHL

# Detailed Lab Journals

Below are the detailed lab journal files. The first part ranges from the beginning until the 17th of August, and the second part picks up from there.

[Lab Journal Part 1](https://static.igem.org/mediawiki/2020/4/43/T--Stockholm--labjournal1.pdf)

[Lab Journal Part 2](https://static.igem.org/mediawiki/2020/1/11/T--Stockholm--labjournal2.pdf)

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# Team:Stockholm/Engineering

Engineering | iGEM Stockholm

* Project

  [Description](https://2020.igem.org/Team%3AStockholm/Description)[Design](https://2020.igem.org/Team%3AStockholm/Design)[Proof of Concept](https://2020.igem.org/Team%3AStockholm/Proof_Of_Concept)[Implementation](https://2020.igem.org/Team%3AStockholm/Implementation)[Contribution](https://2020.igem.org/Team%3AStockholm/Contribution)[Poster](https://2020.igem.org/Team%3AStockholm/Poster)
* Lab & Parts

  [Model](https://2020.igem.org/Team%3AStockholm/Model)[Engineering](https://2020.igem.org/Team%3AStockholm/Engineering)[Parts](https://2020.igem.org/Team%3AStockholm/Parts)[Experiments](https://2020.igem.org/Team%3AStockholm/Experiments)[Results](https://2020.igem.org/Team%3AStockholm/Results)[Safety](https://2020.igem.org/Team%3AStockholm/Safety)[Notebook](https://2020.igem.org/Team%3AStockholm/Notebook)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AStockholm/Human_Practices)[Education](https://2020.igem.org/Team%3AStockholm/Education)
* About

  [Team](https://2020.igem.org/Team%3AStockholm/Team)[Collaborations](https://2020.igem.org/Team%3AStockholm/Collaborations)[Sponsors](https://2020.igem.org/Team%3AStockholm/Sponsors)[Attributions](https://2020.igem.org/Team%3AStockholm/Attributions)
* Awards

  [Entrepreneurship](https://2020.igem.org/Team%3AStockholm/Entrepreneurship)[Sustainable](https://2020.igem.org/Team%3AStockholm/Sustainable)[Medal Criteria](https://2020.igem.org/Team%3AStockholm/Medal_Criteria)

[ ]

# Engineering

Dissecting S-POP

![](https://static.igem.org/mediawiki/2020/6/63/T--Stockholm--img--Contents--topimg_engineer.png)

##### Contents

# Engineering Cycle

iGEM Stockholm 2020 presents its engineering cycle (Figure 1).

![Engineering cycle for our project](https://static.igem.org/mediawiki/2020/5/59/T--Stockholm--img--Engineering--cycle.png)

Figure 1: Engineering cycle for our project

In order to build a scientifically rigorous project, iGEM Stockholm proceeded by following the engineering cycle and improving little by little each and every experiment performed during the summer. We started by finding a problem we wanted to solve and gathering information about it until a clear goal could be defined. Then we divided our project into subtasks and modules. We designed experiments that would help us build and prove the viability and success of our Microbial Fuel Cell (MFC) biosensor. We modelled our main goal, the oscillatory circuit that would help detect and identify different pollutants, in-silico. We built a MFC and assembled and tested our constructs in parallel. We characterized the MFC and the constructs, analysed the results and drew conclusions for each. For each problem encountered, we followed the cycle again to optimize our experiments and get better results.

# Problem identification

In the first few months of iGEM, we gathered to find a project that was interesting and meaningful to the full team. In April-June, it became clear that we wanted a project related to the pollution of the Baltic sea, as this was a problem which we had all heard of and that directly affects the area where we live.

# Gathering information

We dived into the problem of sea pollution by reading literature and contacting researchers that worked both in the field of microbiology and pollution. We looked for reports on pollution in the Baltic sea to see what pollutants were the most problematic and what their origin was. In particular, the Helsinki Commission published in the spring a series of reports [Undeman, 2020; McLachlan & Undeman, 2020; Undeman & Johansson, 2020; Johansson & Undeman, 2020](#citation1) highlighting the levels and effects of dioxins, polychlorinated biphenyls (PCBs), flame retardants such as polybrominated diphenyl ethers and polybrominated biphenyls, diclofenac, perfluorooctanesulfonic acid (PFOS) and polyfluoroalkyl substances (PFASs). We chose to focus on two types of pollutants PCBs and PFOS, for which we found promoter parts engineered and/or studied by previous iGEM teams ([BBa\_K2911000](http://parts.igem.org/Part%3ABBa_K2911000) and [BBa\_K1155001](http://parts.igem.org/Part%3ABBa_K1155001)).

Once we chose to work on a microbial fuel cell for these pollutants' detection, we figured that two approaches could be taken: either increasing the electrical production of the cell or taking a novel approach to distinguish and quantify pollutants. Because previous approaches to increase electricity production had not been very successful, we decided to use synthetic biology to create unique electrical signals for different pollutants.

*Shewanella oneidensis MR-1* is commonly used in microbial fuel cells for electricity production thanks to its MtrB gene. However, most iGEM teams create parts that work in *E. coli* and we wanted to contribute to the creation of new parts that could be used by as many teams as possible in the future. Besides, most microbiology techniques and protocols are easily adapted and troubleshooted in *E. coli*. Thus, we developed the idea of having a modular system: a system where most of the synthetic biology work of pollutant detection would be performed on *E. coli*, and where the electrical output would be given by *Shewanella oneidensis MR-1*. To connect these two modules, we chose to take advantage of quorum sensing molecules, molecules that can transmit signals between different cells.

# Defining a goal

The final goal of our project was to engineer *E. coli* and *S. oneidensis* strains so that their growth in MFC, in the presence of pollutants could give an oscillating electrical output that would be unique for each pollutant.

# Plan and define subtasks

Since our project is by essence modular, it was easy to divide the tasks: we had to work in parallel on:

* engineering *E. coli* so that it detects pollutants and emits quorum sensing (QS) molecules as a result;
* engineering a *Shewanella oneidensis* MtrB knockout strain (unable to produce electricity in normal conditions) so that the received QS signal from *E. coli* activates a conditional electrical pathway (production of electricity only if a pollutant is detected);
* designing a genetic circuit that gives a unique oscillating output when a certain pollutant is detected;
* building a functional microbial fuel cell in which the strains could perform their activities.

Figure 2 below shows a detailed division of the tasks.

![Division of engineering tasks](https://static.igem.org/mediawiki/2020/9/94/T--Stockholm--img--Engineering--division.png)

Figure 2: Division of engineering tasks

# Design

## Circuit

We used the SnapGene software to [design](https://2020.igem.org/Team%3AStockholm/Design) the following [parts](https://2020.igem.org/Team%3AStockholm/Parts) and circuits using the Biobrick format.

Table 1: List of constructs and their aims.

| Biobrick | Code | Construct | Aim |
| --- | --- | --- | --- |
| [BBa\_K3440000](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440000) | A | Pconst-RBS-LuxI-Myc-DT | Show that LuxI can be expressed under constitutive promoter |
| [BBa\_K3440002](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440002) | B | Pprma-RBS-mCherry-DT | Test leakiness of Pprma |
| [BBa\_K3440001](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440001) | C | PbphR1-RBS-mCherry-DT | Test bphR1 promoter leakiness and compare expression with/without PCB. |
| [BBa\_K3440003](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440003) | D | Plux-RBS-GFP | Test leakiness of Plux |
| [BBa\_K3440004](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440004) | E | Prhl-RBS-GFP | Test leakiness of Prhl |
| [BBa\_K3440005](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440005) | F | Pprma-RBS-LuxI-Myc | Show that LuxI can be expressed under prma promoter, and compare expression with/without PFOS.Final construct *E. coli* PFOS. |
| [BBa\_K3440006](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440006) | G | Pconst-RBS-RhlI-Myc | Show that RhlI can be expressed under constitutive promoter |
| [BBa\_K3440007](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440007) | H | Pconst-RBS-AiiA-Myc | Show that AiiA can be expressed under constitutive promoter |
| [BBa\_K3440008](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440008) | J | Pconst-RBS-RhlR-Myc-DT | Show that RhlR can be expressed under Pconst |
| [BBa\_K3440009](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440009) | L | Pconst-RBS-bphR2-Myc-DT | Show that bphR2 can be expressed under Pconst |
| [BBa\_K3440010](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440010) | M | Pbphr1-RBS-LuxI-Myc | Show that LuxI can be expressed under bphR1 promoter, and compare expression with/without PCB. |
| [BBa\_K3440011](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440011) | N | Pconst-RBS-GFP-DT | Show that the constitutive promoter works |
| [BBa\_K3440012](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440012) | O | Pconst-RBS-RHlR-Myc-DT-Prhl-RBS-GFP | Show that Prhl promoter can be activated in the presence of AHL and RhlR expression |
| [BBa\_K3440013](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440013) | P | Pconst-RBS-LuxR-Myc | Show that LuxR can be expressed under Pconst |
| [BBa\_K3440014](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440014) | Q | Pconst-RBS-LuxR-Myc-DT-Plux-RBS-GFP | Show that Plux promoter can be activated in the presence of AHL and LuxR expression |
| [BBa\_K3440015](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440015) | K | RhlI-RBS-Aiia | Intermediate part used for construction |
| [BBa\_K3440016](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440016) | I/Ch5 | Pconst-RBS-MtrB-Myc | Show that MtrB can be expressed under constitutive promoter |
| [BBa\_K3440017](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440017) | L3B | Pconst-RBS-bphR2-Myc-DT-Pbphr1-RBS-mCherry | Show that bphR1 promoter can be activated by expression of bphR2 (and better with PCB) |
| [BBa\_K3440018](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440018) | P32 | Pconst-RBS-bphR2-Myc-DT-Pbphr1-RBS-LuxI | Full construct for detection of PCB and expression of LuxI. Final construct PCB in *E. coli* |
| [BBa\_K3440019](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440019) | Osc1 | Pconst-RBS-LuxR-DT-Plux-RBS-MtrB-DT | Show that MtrB can be expressed under Plux in the presence of AHL. |
| [BBa\_K3440020](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440020) | Osc2 | Pconst-RBS-RhlR-DT-Prhl-RBS-RhlI-RBS-AiiA-RBS-GFP-DT | GFP reporter of oscillation in *E.coli* |
| [BBa\_K3440021](http://parts.igem.org/wiki/index.php/Part%3ABBa_K3440021) | Osc3 | Plux-RBS-MtrB-DT-Pconst-RBS-RhlR-DT-Prhl-RBS-RhlI-RBS-AiiA-RBS-LuxR-DT | Electricity production under oscillatory circuit in *Shewanella*. |

Because many of these constructs were long and would each require many days of assembling biobricks, we decided to order most of them from IDT Technologies as gene blocks with convenient restriction sites in optimal positions and tags to allow characterisation. We used those gene blocks as a starting point for our experiments.

The full circuit we designed for detection of PCB (it is similar for PFOS) is the following (Figure 3): the first construct produces bphR2 constitutively. bphR2 can form a complex with the PCB pollutant, which can activate the bphR1 promoter. LuxI is placed under this promoter and produces a QS molecule. The fourth construct produces RhlR constitutively, and RhlR can bind to C4-HSL to induce Prhl promoter in the third construct, which induces expression of LuxR, RhlI and AiiA. RhlI produces C4-HSL, which creates a positive feedback loop. On the contrary, AiiA degrades C4-HSL, creating the oscillations. LuxR binds to the 3OC6-HSL produced upon detection of PCB by the first construct. The complex can activate Plux promoter, which controls the expression of MtrB, the gene responsible for electricity production in *Shewanella*.

![PCB detection circuit](https://static.igem.org/mediawiki/2020/3/3e/T--Stockholm--img--Engineering--PCB_circuit.png)

Figure 3: PCB detection circuit

## MFC

The MFC set-up was designed thanks to literature and discussions with Amirreza Khataee from the Applied Electrochemistry department at KTH. We decided to go for a liquid-air hybrid set-up, with one reservoir filled with media and another filled with air. Circulation between the MFC and the reservoirs is created by a pump at 0.1 RPM. The chosen electrodes were each made of 2 squares of graphite papers stacked together. The cation exchange membrane used was a NafionTM. The disassembled microbial fuel cell is shown in Figure 6. The circuit was assembled as shown in Figure 4.

![Disassembled microbial fuel cell components](https://static.igem.org/mediawiki/2020/5/54/T--Stockholm--img--Engineering--mfc_disassembled.png)

Figure 4: Disassembled microbial fuel cell components

# Model

The circuits were designed with the aim of creating a unique oscillatory electrical output when a certain pollutant is detected by *E. coli*. To prove that the constructs could give an oscillatory circuit, mathematical simulations were performed. An initial design inspired from literature was tested in silico and found to be unable to give unsynchronised oscillations for different pollutants. Therefore, the circuit had to be changed and improved for our purpose. The obtained [models](https://2020.igem.org/Team%3AStockholm/Model) confirmed that the design was theoretically capable of giving oscillations when pollutants are present.

# Build and experiment

## Parts

For each G-block, we performed (with regards to each particular construct's requirements) the following series of [experiments](https://2020.igem.org/Team%3AStockholm/Experiments) and characterisations (Figure 5). We first resuspended the G-blocks, PCR amplified them, and then proceeded to insert them in the pSB1C3 plasmid via restriction digestion with EcoRI and PstI enzymes. We ran gels to check the sizes of restriction digested parts, then ligated overnight with pSB1C3. All of this was possible because the G-blocks were designed with predetermined, specific restriction sites. We then transformed *E. coli* TOP 10 cells with the plasmids. We picked colonies on plates, ran PCR amplification and gels to check if the sizes were as expected and sent the potentially correct ones for sequencing at Microsynth AG. The selected colonies were used to make glycerol stocks and plasmid preparations. OD600 measurements were made for all, and then characterization experiments were performed. For parts containing GFP or mCherry genes, fluorescence measurements were made with a plate reader. For those with a Myc-tag, Western blots were made. We used *Chromobacterium violaceum* as a marker of AHL production (colonies turning violet when AHLs are detected). Finally, expression was compared between situations with and without pollutants (after viability tests in the presence of pollutants).

![G-Block processing workflow](https://static.igem.org/mediawiki/2020/5/52/T--Stockholm--img--Engineering--gblock_processing.png)

Figure 5: G-Block processing workflow

## MFC

We determined from literature on *Shewanella oneidensis*' metabolism that *S. oneidensis* could grow, using lactate as a carbon source to optimise electricity production [Pinchuk, 2010](#citation5). As a result, we chose lactate as an additive to our minimal M9 medium. We decided to check that *Shewanella oneidensis* MR1 could be used in our MFC set-up to give an electrical output thanks to its MtrB gene. In order to do so, we had to prove that a biofilm could grow on the anode and that this biofilm did not grow when the MtrB gene was knocked out. Thus, we acquired two strains of *S. oneidensis* MR1; a wild type from Ute Römling's lab at Karolinska Institutet, Stockholm, Sweden, and a MtrB knockout strain from Johannes Gescher, Karlsruhe Institute of Technology, Germany. We decided to monitor *S. oneidensis* biofilm growth by measuring the open cell voltage of the circuit with an imposed resistance of 1000 ohm. Thanks to Ohm's law U=RI, the current could then be determined.

Our first experiment consisted in putting the wild type strain on the electrodes, then assembling the MFC with LB broth in a reservoir, and finally assembling the final circuit (Figure 6) and measuring the voltage continuously. After a few days, we changed the media for fresh LB and continued to monitor the voltage. We then changed the media for minimal M9 media with lactate as a carbon source and tried to check that the biofilm growth was enhanced after a period of adaptation to the new media. Our second experiment was a control to check the necessity of having the MtrB gene for electricity production. We assembled the circuit with the MtrB knockout on the electrodes this time and measured current with LB as media.

![MFC setup, where 1/green is the pump; 2/yellow is the air bottle; 3/purple is the media bottle; 4/red is the resistor; and 5/blue is the MFC](https://static.igem.org/mediawiki/2020/7/7b/T--Stockholm--img--Engineering--mfc.png)

Figure 6: MFC setup, where 1/green is the pump; 2/yellow is the air bottle; 3/purple is the media bottle; 4/red is the resistor; and 5/blue is the MFC

# Analyse the results

Most of our constructs were successfully designed and transformed into *E. coli* TOP 10 cells. The sequencing results confirmed what our gels predicted for fifteen of our colonies. The specific results for each experiment (viability, transformation, Western blots, fluorescence) are presented in [Results](https://2020.igem.org/Team%3AStockholm/Results). We decided to focus on the MFC engineering results here.

First, we observed with the open cell voltage over time curves that a biofilm could grow when *S.oneidensis*, wild-type was inoculated in the MFC and fed with an LB media (Figure 7). The voltage increased slowly and then underwent a high increase when the LB media was renewed. Seeing that the biofilm had grown, we changed the media to a minimal M9 media with regular lactate addition. As expected, the biofilm managed to adapt to this new media. At first, the voltage dropped and then it slowly grew back as it adapted. When we disassembled the MFC, we observed the biofilm directly on the membrane (Figure 9).

Those results were compared to the curves for the MtrB knockout strain (Figure 8). The Figure shows that the MtrB knockout could not produce a biofilm because it was unable to produce electricity, as predicted by literature. This result was confirmed when we disassembled the MFC and the membrane was free from any biofilm (Figure 9).

![Current-time graph for Shewanella oneidensis MR-1 wildtype](https://static.igem.org/mediawiki/2020/e/e4/T--Stockholm--img--Results--MFC_WT.png)

Figure 7: Current-time graph for Shewanella oneidensis MR-1 wildtype

![Current-time graph for Shewanella oneidensis MR-1 MtrB knockout (cyan) and wildtype (red)](https://static.igem.org/mediawiki/2020/5/5a/T--Stockholm--img--Results--MFC_Comparison.png)

Figure 8: Current-time graph for Shewanella oneidensis MR-1 MtrB knockout (cyan) and wildtype (red)

![Membrane with A) no biofilm and B) biofilm](https://static.igem.org/mediawiki/2020/3/35/T--Stockholm--img--Engineering--biofilm.png)

Figure 9: Membrane with A) no biofilm and B) biofilm

# Discuss and conclude

### An example of engineering success: our MFC success story

We managed to create and assemble a fully functional microbial fuel cell in which we have proved that a *Shewanella oneidensis* strain can form a biofilm on the anode when it is equipped with the MtrB gene. We have optimised electricity production by cultivating this biofilm with a minimal media to which we regularly added lactate to favour the electrical pathway. Finally, we have proved that this biofilm cannot grow without the gene. Thus, our biosensor cannot give a signal without pollutants, but will be able to give an electrical output when pollutants are detected and a construct expressing in an oscillatory manner the MtrB gene can be induced when pollutants are present thanks to the design of appropriate synthetic circuits.

# Identify a problem to solve at i+1 = troubleshooting

During the summer, we had to optimise all types of experiments, from restriction digestions to Western blots. The key to troubleshooting is to find the origin of the problem. Most often, using positive and negative controls can both help you prove that your results are scientifically correct and help you find where problems occurred in an experiment. However, you might still encounter problems even with proper controls. We faced many problems with Western Blots, so we decided to share with you our very own troubleshooting checklist for Western Blots.

**Quick troubleshooting list to help you when a protein does not appear on a Western blot:**

1. Optimize your Western Blot protocol

   1. Make sure that you have appropriate positive and negative controls.
   2. Optimize your cell lysis.
   3. Think that your intensity might be too low if you have several products on the same blot, and one is overexpressed compared to the others. In that case you can either have separate gels or stop exposing the overexpressed product.
   4. If your protein might be expressed in another growth phase, adjust the timing.
   5. In our case, some proteins were expressed in the presence of AHLs and contaminants (under inducible promoters). If such is the case for you, try to increase the levels of inducers.
2. For cells that express GFP or mCherry or any colored/fluorescent protein, you might want to quickly check to the naked eye with a microscope under the right wavelength that expression occurs. Fluorescence measurements should also be performed and calibrated based on OD600.
3. Check your sequence. Sequencing your part is necessary, and you might as well do it before starting your Western Blots. The reason for this is that even if you have a band at the right size, it might still be another protein due to a substitution. Finally, if your western blot does not work, you can use sequencing to see if the problem is at the DNA level (a mutation to a stop codon could be an explanation).
4. Check your mRNA expression. If your DNA sequence is correct and you still don't have results on your blot, then you can try to see if the problem is at the RNA level by performing an RNA extraction.
5. Run a background check on your proteins to understand how they are expressed in their original host, and if possible find a paper in which it was expressed in your host. In particular, check the location of the product and the level of expression obtained in literature.
6. Check your cells. It might be that the protein product is toxic for the cell or that *E. coli* does not prefer its presence. The product might be easily degraded by protease. Thus, you might want to test growth with and without the construct and observe the number of colonies to see how your host reacts to the product. If that is the problem, then you need to change your host.
7. Coomassie staining can also help you to check your cells by counting total protein expression.
8. To increase expression of the protein, you can clone your plasmid on a low copy number plasmid or under an inducible promoter.

# References

1. Diclofenac in the Baltic Sea - Sources, transport routes and trends. Helcom Baltic Sea Environment Proceedings n°170.

   (2020). Retrieved on October 18, 2020. from <https://helcom.fi/wp-content/uploads/2020/06/Helcom_170_Diclofenac.pdf>

   [Back to text](#intext1)
2. Dioxins and PCBs in the Baltic Sea. Helcom Baltic Sea Environment Proceedings n°171.

   (2020). Retrieved on October 18, 2020. from <https://helcom.fi/wp-content/uploads/2020/06/Helcom_171_Dioxins_PCBs.pdf>

   [Back to text](#intext2)
3. Polybrominated diphenyl ethers (PBDEs) in the Baltic Sea - Sources, transport routes and trends. Helcom Baltic Sea Environment Proceedings n°172.

   (2020). Retrieved on October 18, 2020. from <https://helcom.fi/wp-content/uploads/2020/06/Helcom_172_PBDE.pdf>

   [Back to text](#intext3)
4. Perfluorooctane sulfonate (PFOS) and other perfluorinated alkyl substances (PFASs) in the Baltic Sea - Sources, transport routes and trends. Helcom Baltic Sea Environment Proceedings n°173.

   (2020). Retrieved on October 18, 2020. from <https://helcom.fi/wp-content/uploads/2020/06/Helcom_173_PFOS_PFAS.pdf>

   [Back to text](#intext4)
5. Pinchuk, G. E., Hill, E. A., Geydebrekht, O. V., De Ingeniis, J., Zhang, X., Osterman, A., ... & Reed, J. L. (2010).

   Constraint-Based Model of Shewanella oneidensis MR-1 Metabolism: A Tool for Data Analysis and Hypothesis Generation.

   PLoS Computational Biology 6(6), e1000822.

   [CrossRef](https://doi.org/10.1371/journal.pcbi.1000822)[Google Scholar](https://scholar.google.com/scholar?q=Constraint-Based Model of Shewanella oneidensis MR-1 Metabolism: A Tool for Data Analysis and Hypothesis Generation.)[Back to text](#intext5)

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# Team:Stockholm/Experiments

Experiments | iGEM Stockholm

* Project

  [Description](https://2020.igem.org/Team%3AStockholm/Description)[Design](https://2020.igem.org/Team%3AStockholm/Design)[Proof of Concept](https://2020.igem.org/Team%3AStockholm/Proof_Of_Concept)[Implementation](https://2020.igem.org/Team%3AStockholm/Implementation)[Contribution](https://2020.igem.org/Team%3AStockholm/Contribution)[Poster](https://2020.igem.org/Team%3AStockholm/Poster)
* Lab & Parts

  [Model](https://2020.igem.org/Team%3AStockholm/Model)[Engineering](https://2020.igem.org/Team%3AStockholm/Engineering)[Parts](https://2020.igem.org/Team%3AStockholm/Parts)[Experiments](https://2020.igem.org/Team%3AStockholm/Experiments)[Results](https://2020.igem.org/Team%3AStockholm/Results)[Safety](https://2020.igem.org/Team%3AStockholm/Safety)[Notebook](https://2020.igem.org/Team%3AStockholm/Notebook)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AStockholm/Human_Practices)[Education](https://2020.igem.org/Team%3AStockholm/Education)
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[ ]

# Experiments

Our entire summer

![](https://static.igem.org/mediawiki/2020/1/15/T--Stockholm--img--Contents--topimg_experiments.png)

##### Contents

# MFC

## Introduction

A Microbial Fuel Cell (MFC) works similarly to a hydrogen fuel cell, but with microbes as the terminal electron donor. The decision to use MFCs as a biosensor in our project, and the people who influenced our design are outlined in our [Human Practices](https://2020.igem.org/Team%3AStockholm/Human_Practices) page.

## Hydrogen Fuel Cells

The hydrogen fuel cell generates electricity by turning hydrogen and oxygen into water via the following reaction, which is the reverse of the electrolysis reaction:

$$ 2H\_2 + O\_2 \rightarrow 2H\_2O + energy $$ This reaction occurs in two redox half-reactions. At the anode: $$ H\_2 \rightarrow H^+ + 2e^- $$ and the cathode half-reaction: $$ \frac{1}{2} O\_2 + 2H^+ + 2e^- \rightarrow 2H\_2O $$

By constructing two chambers where one has access to hydrogen and the other to oxygen, the two half-reactions can take place in different locations as long as the other components (protons and electrons) can travel between the chambers. A selective membrane can be used that only allows protons to permeate to create an abundance of electrons at the anode. A conductive wire can connect the anode and cathode electrodes, which allows the electrons to pass through. The electrons cannot pass through any other way than the wire, and it is from that which the electricity in the form of direct current is generated [Felseghi et al., 2019](#citation1).

## Bacteria for producing electricity

In many biological systems, the energy used is in the form ATP which is formed by breaking down nutrients. The energy extracted from glycolysis and the Krebs cycle is not primarily in the form of ATP, but NADH, which is then used in the electron transport chain in oxidative phosphorylation. In some anaerobic bacteria such as *S. oneidensis*, an extracellular electron transport pathway still exists, but without oxygen as a terminal electron acceptor to form water, oxidized metals or other compounds can be reduced instead. This can result in electricity being generated as the reduced mediator can pass its electron on [Pirbadian et al., 2014](#citation2). *Shewanella oneidensis* MR-1 can also form highly conductive nanowires that can carry charge far outside the immediate area of the cell and remove the need for specific mediators. [Reguera et al., 2005](#citation3). This is in *Shewanella* done through a set of cytochromes that carries the electrons along the nanowire [Pirbadian et al., 2014](#citation2).

## Microbial Fuel Cells

Bacteria such as *Shewanella* catalyses the reduction of surrounding metals and uses them as terminal electron acceptors to get rid of electrons in their cytochromes which allows them to complete their extracellular electron transport for energy. This is a reaction catalyzed by the redox proteins in their respiratory chain, which also pumps protons out of the cell.

In our MFC, *Shewanella oneidensis* reduces the anode directly or via nanowires and the electrons then flow through the wire, creating a current [Brutinel & Gralnick, 2014](#citation4).

A simple design is the H-type MFC, which is structurally and conceptually similar to the hydrogen fuel cell. The hydrogen-rich anode chamber is replaced with the microbial growth chamber, where protons and electrons are generated through the extracellular electron transport rather than the metal catalysed hydrogen gas splitting. Water is formed at the air cathode where the electrons are rejoined with the protons and atmospheric oxygen.

![Schematic of the H-type MFC](https://static.igem.org/mediawiki/2020/9/9a/T--Stockholm--img--Experiments--mfc.png)

Figure 1: Schematic of the H-type MFC

## Setup

The electricity generation of Shewanella oneidensis was measured in a Microbial Fuel Cell (MFC). A two chamber electrochemical fuel cell was repurposed to be used for this purpose. The electrodes in each chamber consisted of stacks of 2 heat treated square graphite paper with a side length of 1.5 cm. This allowed for a tight packing of the fuel cell, while still leaving space for the media to flow between the graphite paper and allow biofilm to grow there. Separating the chambers was a NafionTM proton exchange membrane. The chamber walls were made from the thickness of the rubber gasket sealing the electrodes in place. The rubber gaskets secured the membrane in place and allowed for an airtight seal around the chambers. The graphite electrodes were connected to a gold plate, where electronics could be attached. A 1 kΩ resistor was put between the electrodes and the open circuit voltage was measured to then calculate the current. A battery tester from Landt instruments was used to collect and log the data. A detailed description of the assembly can be found in the [Engineering](https://2020.igem.org/Team%3AStockholm/Engineering#Design-MFC) page.

![Schematic of the MFC](https://static.igem.org/mediawiki/2020/2/25/T--Stockholm--img--Experiments--MFC_Schematic.png)

Figure 2: Schematic of the MFC

## Experiments

The MFC was inoculated with 100 μL culture of *Shewanella oneidensis* MR-1 directly at the anode before assembly. The fuel cell was then fed with media from a reservoir to the anode, and air to the cathode at a constant flow at 0.1 RPM, which corresponds to approximately 21 μL/min. Two runs were performed. One long run with the wildtype strain, where the media feed was exchanged (LB and M9) to determine biofilm growth, and one run with the MtrB knockout strain without any transformed plasmid to see both biofilm growth and electricity production without the crucial MtrB gene in the MFC. Some minimal growth is however expected, as MtrE can complement the complex, although with a lower efficiency than MtrB [Coursolle & Gralnick, 2012](#citation5). Only bacteria close to both the electrode and the proton exchange membrane should be able to generate current, so the amount of biofilm is proportional to the current produced. The media change is however done to confirm that biofilm has formed. Future experiments would include testing the electricity production of *Shewanella oneidensis* MR-1 ΔMtrB with MtrB under an inducible promoter (pLux) and finally with the oscillatory circuit. The open circuit voltage was logged, which later could be converted into the current produced across the 1 kΩ resistor between electrodes.

![Assembled MFC setup. Green/1: pump, Yellow/2: air reservoir, Purple/3: media reservoir, Red/4: resistor, Cyan/5: MFC](https://static.igem.org/mediawiki/2020/7/7b/T--Stockholm--img--Engineering--mfc.png)

Figure 3: Assembled MFC setup. Green/1: pump, Yellow/2: air reservoir, Purple/3: media reservoir, Red/4: resistor, Cyan/5: MFC

# Cloning

Cloning can be used to get a specific DNA fragment (insert/construct) into the vector DNA ~ Small circular chromosome with DNA for functional regions such as origin of replication and a drug resistance gene to form a recombinant DNA. This can be introduced into a host cell where the recombinant DNA will replicate and express the proteins. Two enzymes are used in this process restriction enzymes ~ Cuts DNA at specific sites by recognising 4-8 bp sequences and DNA ligase ~ Forms phosphodiester bonds between two complementary sticky end DNA fragments [Lodish et al., 2000](#citation6). For our project to express these various types of DNA sequences for RhlR, bphR1, LuxI, prmA we used the cloning techniques.

The constructs were designed based on prior BioBricks or based on the literature available. Due to the unavailability or late delivery of the biobricks we ordered our constructs from IDT. The constructs under a constitutive promoter had an additional Myc tag in the sequence and the promoter specific sequences have either a GFP or mCherry sequence for easier characterization. The full list of constructs can be found in [Parts](https://2020.igem.org/Team%3AStockholm/Parts).

To express these constructs, they were cloned into the vector plasmid pSB1C3. [Restriction digestion](https://static.igem.org/mediawiki/2020/b/ba/T--Stockholm--Restriction_digest.pdf) was performed on EcoRI and PstI sites in both the gene blocks and the plasmids. This was followed by [ligation](https://static.igem.org/mediawiki/2020/8/8f/T--Stockholm--Ligation.pdf). These plasmids were [transformed](https://static.igem.org/mediawiki/2020/d/da/T--Stockholm--Transformation.pdf) into Top10 *E. coli* and were selected on chloramphenicol plates for [colony PCR](https://static.igem.org/mediawiki/2020/3/33/T--Stockholm--Colony_PCR.pdf).

Transformation was primarily performed using [heat shock](https://static.igem.org/mediawiki/2020/d/da/T--Stockholm--Transformation.pdf) of chemically competent *E.coli* TOP10. However, when heat shock transformation was not possible, electroporation of electrocompetent *E.coli* was done instead.

The samples from the colony PCR with the right size when run on an agarose gel were sent for sequencing to Microsynth AG which were re-checked with the original sequence which if matched were used for characterization.

Once all the constructs were inserted it was necessary to test if these transformed TOP10 *E.coli* would be able to grow normally in the presence of the pollutants at varying concentrations for which we performed the viability test.

![Cloning workflow](https://static.igem.org/mediawiki/2020/3/3e/T--Stockholm--img--Experiments--cloning.png)

Figure 4: Cloning workflow

# Viability assays

Viability assays were important for our project. In this series of experiments we aimed to investigate any alterations in the growth of our bacteria of interest when cultured with the chemicals of interest at concentrations present in nature [Wahlberg, 2016; Wahlberg, 2018; Livsmedelsverket, n.d.; Silva et al., 2020](#citation7).

When cultured in a closed system, bacteria tend to have a characteristic growth curve (Figure 2). In the lag phase the bacteria adjust to the environment, while in the log phase they grow exponentially with high division rate. However, the log phase is followed by the stationary phase where the division rate is very low and the nutrients are limited. As a result of these facts, the death phase of the culture follows [Wang, Fan, Chen & Terentjev, 2015](#citation11).

![Characteristic bacteria growth curve](https://static.igem.org/mediawiki/2020/8/87/T--Stockholm--img--Experiments--growth_curve.png)

Figure 5: Characteristic bacteria growth curve

Growth-based [viability assays](https://static.igem.org/mediawiki/2020/5/57/T--Stockholm--Viability_Assay.pdf) using microplate readers are a quite common technique nowadays. It has partially replaced the traditional methods and has reduced the cost of the experiment. The cultivation of the bacteria is performed in microplates and the growth curve is designed based on the absorbance values. It is a fast, easy and cheap technique to get accurate growth curves. Finally, it allows the user to set different chemical or biological conditions in each well [Horakova et al., 2004](#citation12).

Characterization of the transformed *E.coli* cells is important to check if our genes of interest produced the right protein. For our project we mainly tested this using fluorescence for the constructs that had a GFP or mCherry after the gene of interest and a western blot for the genes that had a Myc tag sequence. Few of the constructs produce N-acylhomoserine lactones (AHLs) which cause colour change in one of the *Chromobacterium violaceum* strain and is a way of easy characterization instead of using western blot techniques.

# Fluorescence

Fluorescence emission occurs when a molecule absorbs light at high energy (i.e. short wavelength) and emits light at lower energy and usually visible wavelength [Welker, 2012](#citation13).

To characterise the activation of specific promoters in our circuit, genes coding for fluorescent proteins (GFP / mCherry) were inserted into some of our constructs. The fluorescence intensity of the constructs were quantified in whole cells, both in uninduced (i.e. to check for "leakiness" of the promoters) and induced conditions. [These measurements](https://static.igem.org/mediawiki/2020/6/6e/T--Stockholm--Fluorescence.pdf) were done in a microplate reader after calibration with fluorescent dyes, and adjusted for cell density.

![Basic fluorescense mechanism](https://static.igem.org/mediawiki/2020/e/eb/T--Stockholm--img--Experiments--fluorescence.png)

Figure 6: Basic fluorescense mechanism

# SDS PAGE and Western Blot

[Western Blots](https://static.igem.org/mediawiki/2020/b/b3/T--Stockholm--SDS_Page_and_Western_Blot.pdf) were utilised for the characterization of our constructs. In order to ensure that our gene insert was in fact expressed and translated into the desired protein we needed a method to detect the proteins that were expressed by our cells. Sodium DodecylSulfate PolyAcrylamide GEl (SDS PAGE) is an electrophoresis based separation method for size based discrimination of proteins and western blotting utilises antibody specificity in order to detect a specific protein [Mahmood & Yang, 2012](#citation14).

In order to be able to distinguish our protein of interest from our host cell proteome we designed our gene sequences to include a myc-tag at the end of the protein sequence. The myc-tag is a 1.2 kDa protein and can be fused to the N-terminus or C-terminus of a protein [Jarmander et al., 2012; UniProt Consortium, 2020](#citation15).

With a monoclonal antibody specific for the Myc tag we would be able to determine the presence of our desired protein in different constructs in different conditions, such as with and without the presence of pollutants. A blue safe protein stain could be used in order to stain the general proteome of our samples.

# Chromobacterium violaceum

Quorum sensing is dependent on the interaction between a diffusible signal molecule and a transcriptional activator protein. A commonly used signal molecule is N-acylhomoserine lactones (AHLs) which differ in their sidechains (N-acyl). *Chromobacterium violaceum* is a Gram-negative bacterium that can produce the characteristically purple pigment, violacein. The modified strain CV026, is inducible by synthetic AHL N-acyl side chains from C4-C8 in length, with varying degrees of sensitivity [McClean et al., 1997](#citation17).

*C. violaceum* was going to be utilized for its capability to show the presence of a large variety of AHLs. This would be a quick indicator for any production of AHLs without having to test protein expression in Western blot. The performed experiments with *C. violaceum* tested the needed levels of synthetic AHL for our cultures. All the characterised Top 10 *E.coli* and *Shewanella oneidensis* need to work in a microbial fuel cell, understanding the working of this system and getting a working MFC was an equally important part of our project.

# Protocols

[Viability Assay](https://static.igem.org/mediawiki/2020/5/57/T--Stockholm--Viability_Assay.pdf)

[Ligation](https://static.igem.org/mediawiki/2020/8/8f/T--Stockholm--Ligation.pdf)

[Restriction Digest](https://static.igem.org/mediawiki/2020/b/ba/T--Stockholm--Restriction_digest.pdf)

[SDS PAGE and Western Blot](https://static.igem.org/mediawiki/2020/b/b3/T--Stockholm--SDS_Page_and_Western_Blot.pdf)

[Heat Shock Transformation](https://static.igem.org/mediawiki/2020/d/da/T--Stockholm--Transformation.pdf)

[Colony PCR](https://static.igem.org/mediawiki/2020/3/33/T--Stockholm--Colony_PCR.pdf)

[Fluorescence Intensity Quantification](https://static.igem.org/mediawiki/2020/6/6e/T--Stockholm--Fluorescence.pdf)

# References

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   Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

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# Team:Stockholm/Results

Results | iGEM Stockholm

* Project

  [Description](https://2020.igem.org/Team%3AStockholm/Description)[Design](https://2020.igem.org/Team%3AStockholm/Design)[Proof of Concept](https://2020.igem.org/Team%3AStockholm/Proof_Of_Concept)[Implementation](https://2020.igem.org/Team%3AStockholm/Implementation)[Contribution](https://2020.igem.org/Team%3AStockholm/Contribution)[Poster](https://2020.igem.org/Team%3AStockholm/Poster)
* Lab & Parts

  [Model](https://2020.igem.org/Team%3AStockholm/Model)[Engineering](https://2020.igem.org/Team%3AStockholm/Engineering)[Parts](https://2020.igem.org/Team%3AStockholm/Parts)[Experiments](https://2020.igem.org/Team%3AStockholm/Experiments)[Results](https://2020.igem.org/Team%3AStockholm/Results)[Safety](https://2020.igem.org/Team%3AStockholm/Safety)[Notebook](https://2020.igem.org/Team%3AStockholm/Notebook)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AStockholm/Human_Practices)[Education](https://2020.igem.org/Team%3AStockholm/Education)
* About

  [Team](https://2020.igem.org/Team%3AStockholm/Team)[Collaborations](https://2020.igem.org/Team%3AStockholm/Collaborations)[Sponsors](https://2020.igem.org/Team%3AStockholm/Sponsors)[Attributions](https://2020.igem.org/Team%3AStockholm/Attributions)
* Awards

  [Entrepreneurship](https://2020.igem.org/Team%3AStockholm/Entrepreneurship)[Sustainable](https://2020.igem.org/Team%3AStockholm/Sustainable)[Medal Criteria](https://2020.igem.org/Team%3AStockholm/Medal_Criteria)

[ ]

# Results

The fruits of our labor

![](https://static.igem.org/mediawiki/2020/c/cc/T--Stockholm--img--Contents--topimg_results.png)

##### Contents

# MFC

Both the wildtype strain of *S. oneidensis* MR-1 (WT) and the MtrB knockout (KO) strain were cultivated in the MFC. The media was fed to the MFC from a reservoir, and the spent media from the outflow was returned to the reservoir. This was done to allow the experiment to run without much supervision, as the flow rate was still relatively high and would otherwise require refilling every day. The cathode reservoir was effectively open to the atmosphere.

## Wildtype strain

The biofilm growth of the wildtype strain was monitored for a total of 18 days (450 hours). The cultivation can be separated into four phases: two using LB to grow and confirm the biofilm growth, and two using M9 with lactate as the media feed to investigate the growth and biofilm integrity under lactate as the only carbon source. The calculated current for the entire run is shown in Figure 1.

![Measurement of current over time in red, with the different phases in cyan](https://static.igem.org/mediawiki/2020/0/0d/T--Stockholm--img--Results--MFC_WT2.png)

Figure 1: Measurement of current over time in red, with the different phases in cyan

The first phase with LB as the carbon source lasted for 260 hours. The anolyte reservoir consists of 40 mL LB media. At the initial addition of the culture to the MFC anode chamber, the current is high but rapidly drops to 0 as the excess bacteria is flushed out of the cell. After several days, the current generated starts to rise above noise levels. The current increases steadily, and is at the end at 20 μA, indicating that a successful biofilm had formed in the MFC. There is however a chance that the electricity is from cells in suspension rather than a biofilm, as the media is recycled in the reservoir.

To confirm that the current generated is from a biofilm, the reservoir with 40 mL LB media is exchanged for a new 1000 mL bottle of LB. When the bottles are changed, there is a steep drop in current. It does not drop to 0 completely, but stays at around 8-10 μA for 5 hours. This could be due to cells in suspension that were contributing to the current were removed with the fresh reservoir, or it could be due to parts of the biofilm detaching when the flow is stopped during the media change, as predicted by literature. After the initial lag phase, the current generated rapidly increased over the course of 25 hours to a peak current of 130 μA. It is after that fairly constant, perhaps having entered a stationary phase due to nutrient limitation (which can occur with a reservoir), but more likely due to other limitations related to the biofilm, such as density or biofouling of the electrode.

The third phase of the MFC run with wildtype *Shewanella oneidensis* aimed to determine whether it could survive and produce electricity in M9 minimal media with lactic acid as the only carbon source. The LB media was exchanged for 1000 mL M9 media with lactate (instead of glucose). Immediately after the media change, the current dropped rapidly, but started to plane out after a few hours.

The current generated did not recover after two days, so more lactic acid (1 mL, 10 mM) was added to the reservoir. In the following hours, the generated current increased to a steady level, indicating that substrate limitation was the cause for the low current after media change.

## Knockout

The biofilm growth (or rather, the lack of it) of the MtrB knockout (KO) strain was monitored for 130 hours (Figure 2). The setup was identical to the first phase of the wildtype strain run, with the exception of more bacteria being added at inoculation.

![Measurement of current over time in cyan](https://static.igem.org/mediawiki/2020/1/1c/T--Stockholm--img--Results--MFC_KO.png)

Figure 2: Measurement of current over time in cyan

The current starts off high but rapidly drops, similar to the wildtype run. Compared to the wildtype run however, the current doesn't increase, but remains at a low level. A comparison between KO run and the initial part of the WT run can be seen in Figure 3.

![Comparison between the initial part of the WT current (in red) and the KO current (in blue) over time](https://static.igem.org/mediawiki/2020/5/5a/T--Stockholm--img--Results--MFC_Comparison.png)

Figure 3: Comparison between the initial part of the WT current (in red) and the KO current (in blue) over time

# Cloning

Aim of the cloning experiment was to get our choice of insert/construct into the plasmid pSB1C3. If the recombinant DNA was inserted successfully they were selected using antibiotic plates.

For controls we had:

1. A negative control plate: Just the TOP10 *E.coli* cells were plated; nothing grew hence showing our plates with the antibiotics were selective.
2. The backbone plasmid pSB1C3 with RFP in it was used to check successful ligation steps. Biobrick BBa\_J04450 in pSB1C3 was used as our backbone, partial restriction digest could be easily detected with this backbone and the colonies picked for colony PCR could be narrowed down.

Restriction digest and ligation protocol was optimised for our backbone and gene blocks and they can be found in the [Notebook](https://2020.igem.org/Team%3AStockholm/Notebook).

![Transformed E.coli that took up the recombinant DNA](https://static.igem.org/mediawiki/2020/5/55/T--Stockholm--img--Results--cloning_1.png)

Figure 4: Transformed E.coli that took up the recombinant DNA

The colonies that grew on the plate could either have the construct or could be an empty backbone. To find the correct colony a colony PCR was run. The ones with the correct size constructs run on an agarose gel are compiled here, for more information on the protocol check the [Experiments](https://2020.igem.org/Team%3AStockholm/Experiments) page.

![Constructs with the right size for B, L, M, C, D and F](https://static.igem.org/mediawiki/2020/5/5e/T--Stockholm--img--Results--cloning_2.png)

Figure 5: Constructs with the right size for B, L, M, C, D and F

![Constructs with the right size for G, J, H and F](https://static.igem.org/mediawiki/2020/2/2c/T--Stockholm--img--Results--cloning_3.png)

Figure 6: Constructs with the right size for G, J, H and F

![Constructs with the right size for N,Q,P,O and plasmid 3.2](https://static.igem.org/mediawiki/2020/9/97/T--Stockholm--img--Results--cloning_4.png)

Figure 7: Constructs with the right size for N,Q,P,O and plasmid 3.2

![Constructs with the right size for Q, E, H](https://static.igem.org/mediawiki/2020/f/fb/T--Stockholm--img--Results--cloning_5.png)

Figure 8: Constructs with the right size for Q, E, H

For each construct multiple right size band samples were found, these were sent out for sequencing to find the sequence with no mis alignments or alignments that gave the same amino acid sequence as an output.

![Sequence comparison for construct O (top) and F (bottom)](https://static.igem.org/mediawiki/2020/e/e4/T--Stockholm--img--Results--cloning_6.png)

Figure 9: Sequence comparison for construct O (top) and F (bottom)

12 constructs were found to have the right alignments. A summary of the cloning experiment result is shown below.

Table 1: Summary of the cloning experiment.

| Constructs | Code | Size | Selection of sequence |
| --- | --- | --- | --- |
| Constitutive promoter-RFP | Ctrl (Control) | 1069 | / |
| Constitutive promoter- LuxI- Myc tag | A | 1158 | A3 but resequence end of LuxI w/ new primers |
| Promoter prmA- mcherry | B | 1492 | B7, B8 |
| Promoter bphR1- mcherry | C | 1361 | C3 but mismatch in promoter and gap after it |
| Promoter Lux-GFP | D | 1113 | D1 |
| Promoter Rhl-GFP | E | 1111 | E5 |
| Promoter prmA-LuxI-Myc tag | F | 1159 | F5 (F7 has a mismatch) |
| Constitutive promoter- RhlI- Myc tag | G | 1045 | G7 |
| Constitutive promoter-AiiA - Myc tag | H | 1189 | / |
| Constitutive promoter-RhlR - Myc tag | J | 1299 | (J3 has a mismatch) |
| RhlI-RBS-Aiia | K | 1779 | K5,K11 |
| Constitutive promoter-bphR2 - Myc tag | L | 1443 | L3 |
| Promoter bphR1-LuxI-Myc tag | M | 1288 | M1 |
| Constitutive promoter- GFP | N | 1266 | N3 |
| Constitutive promoter- RhlR- Myc tag- Promoter Rhl-GFP | O | 2107 | O14,O19 |
| Constitutive promoter-LuxR - Myc tag | P | 1328 | / |
| Constitutive promoter- LuxR- Myc tag- Promoter Lux-GFP | Q | 2135 | Q3 |
| Constitutive promoter-bphR2-Myc tag -double terminator-Pbphr1--LuxI | plasmid 3.2 | 2493 | P3.2-4 (middle part should be resequenced) |

# Viability assays

The successfully transformed cells with our pollutant-sensing gene inserts were checked for their survivability in the presence of the pollutants as compared to the un-transformed cells. This was done using the [Viability assay](https://2020.igem.org/Team%3AStockholm/Experiments#Viabilityassays) exploring the population growth curve in a range of pollutant concentrations.

Table 2: The following pollutant sensing constructs were used for the viability assay

| Constructs | Code | Construct Detects |
| --- | --- | --- |
| Top 10 *E. coli* BL21 (no plasmid) | Ctrl (Control) | N/A |
| Promoter prmA-LuxI-Myc tag | F | PFOS |
| Constitutive promoter-bphR2 - Myc tag - promoter bphR1-LuxI-Myc tag | Plasmid 3.2 (P 3.2) | PCB/1,1-biphenyl |

Initially, the pollutant concentration range used for the viability assays mimicked the concentration range of the pollutants as found in nature. The initial results suggested that our strains were not affected in terms of their population growth at concentrations this low. Hence, we aimed to find the threshold levels for these pollutants at which the growth would be affected, enabling us to set a maximum range for the pollutant concentration at which our strains and in turn our biosensor system would be capable of functioning. These results can be found below.

![The figure represents the growth curves of Top 10 E. coli BL21, constructs F and plasmid 3.2. Samples were incubated in different concentrations of PFOS and 1,1-biphenyl for 18 h at 37°C. The red line represents the OD<sub>600</sub> value and the blue line is after adjustment to the blank (well H7)](https://static.igem.org/mediawiki/2020/b/bb/T--Stockholm--img--Results--fluorescence.png)

Figure 10: The figure represents the growth curves of Top 10 E. coli BL21, constructs F and plasmid 3.2. Samples were incubated in different concentrations of PFOS and 1,1-biphenyl for 18 h at 37°C. The red line represents the OD600 value and the blue line is after adjustment to the blank (well H7)

![Key to layout for Figure 10 denoting samples and pollutant concentrations](https://static.igem.org/mediawiki/2020/8/87/T--Stockholm--img--Results--pollutantconc.png)

Figure 11: Key to layout for Figure 10 denoting samples and pollutant concentrations

The viability assay shows that our host cells with or without the pollutant sensing constructs are not severely affected by the addition of pollutants in terms of their population growth. An effect on the growth curve can be seen for the highest concentrations for 1,1-biphenyl at 10-3M (wells A1-3, C7-9 and F1-3) and for PFOS at 125 μM (wells B4-6, D10-12 and G4-6). This suggests that these concentrations may be in the range of toxicity threshold values for our bacterial cells. However, these concentrations are well above current concentrations of these pollutants in nature around the Baltic sea hence suggesting that our system would be able to withstand these pollutants while not impacting their growth.

Once the survivability of the transformed cells in various concentrations of the pollutants was proven; characterization of these various constructs was the next step.

# Characterization

To make sure that our final sequence was in fact expressed as well as functional we methodically divided the construct and added extra sequences that would help us with characterization such as fluorescence markers or Myc tags. This was useful in troubleshooting the subparts of the final construct that needs more modeling and analysis to give out the desired proteins. Different techniques were applied to characterize the various constructs outlined in [Experiments](https://2020.igem.org/Team%3AStockholm/Experiments).

# Fluorescence

Some of our constructs with the pollutant detection ability had mCherry sequence at the end and some of the constructs with the quorum sensing genes had GFP sequence. Fluorescence intensity of these constructs were measured both in uninduced conditions; to obtain a threshold signalling level of the promoters, and in induced conditions; to assess the degree of activation of the promoter by the inducers (i.e. AHL and/or pollutants). Inducers were added to cultures along with the constructs to be tested, and incubated overnight (Table 3). Constructs not expressing fluorescent proteins were included as controls. The results are summarised in Figure 12.

Table 3: Summary of fluorescence intensity quantification experiment. Constructs marked with an asterisk (\*) are controls

| Construct | Code | Fluorescent protein | Induced with AHL synthase constructs | Induced with synthetic AHL | Induced with pollutants |
| --- | --- | --- | --- | --- | --- |
| constitutive promoter- GFP | N | GFP | N/A | N/A | N/A |
| constitutive promoter- RhlR- Myc tag- promoter Rhl-GFP | O | GFP | co-culture with G | 2 μM C4-HSL | N/A |
| constitutive promoter- LuxR- Myc tag- promoter Lux-GFP | Q | GFP | co-culture with A | 2 μM 3OC6-HSL | N/A |
| promoter prmA- mCherry | B | mCherry | N/A | N/A | 62.5 μM PFOS |
| promoter bphR1- mCherry | C | mCherry | N/A | N/A | 10-4M 1,1-biphenyl |
| constitutive promoter- LuxI- Myc tag\* | A\* | N/A | N/A | N/A | N/A |
| constitutive promoter- RhlI- Myc tag\* | G\* | N/A | N/A | N/A | N/A |

![Net fluorescence intensity of constructs expressing (a) mCherry and (b) GFP. The values shown represent averages of 3 technical replicates, and have been adjusted for cell density. Error bars represent standard deviation](https://static.igem.org/mediawiki/2020/6/67/T--Stockholm--img--Results--mCherry_bar_graph.png)

Figure 12: Net fluorescence intensity of constructs expressing (a) mCherry and (b) GFP. The values shown represent averages of 3 technical replicates, and have been adjusted for cell density. Error bars represent standard deviation

![GFP visualisation of constructs O14, O19 and Q under UV light](https://static.igem.org/mediawiki/2020/c/c6/T--Stockholm--img--Results--fluor_eppis.png)

Figure 13: GFP visualisation of constructs O14, O19 and Q under UV light

The green fluorescence intensity of constructs O and Q increased upon induction with synthetic C4-HSL and 3OC6-HSL, respectively (Figure 12b). This suggests that the synthetic AHL molecules could bind to RhlR (constitutively expressed by construct O) and LuxR (constitutively expressed by construct Q), which could subsequently induce their respective promoters (pRhl and pLux) and therefore induce higher GFP expression. Co-culturing constructs O and Q with constructs G (which constitutively expresses RhlI) and A (which constitutively expresses LuxI) respectively induced a similar increase in GFP expression. These results suggest that AHL molecules were produced by RhlI and LuxI, which could in turn bind to RhlR and LuxR and induce the specific promoters and expression of GFP. The results of this functionality assay are supported by the Western blot results, which showed the expression of Myc-tagged LuxI (A), RhlI (G) and LuxR (Q).

The expression of mCherry by constructs B and C did not increase when induced with pollutants (Figure 12a). Construct C expresses mCherry under a bphR1 promoter, which is induced by the protein bphR2 binding to biphenyl. As no bphR2 was added, the expression of bphR1 promoter could not be induced by addition of biphenyl alone. However, the expression of mCherry by construct B (under prmA promoter) was not increased upon addition of the given concentration of PFOS. The concentration of pollutants used for characterisation were selected based on the results of the viability assays, such that they do not affect the growth of bacteria significantly. For future experiments, higher concentrations of pollutants could be added to induce the constructs. This would also help in setting a detection limit for the system.

# Western Blot

The constructs with a Myc tag at the end were characterized using the Western blot technique. The protocol was adjusted throughout the experimental period for several factors in order to optimize the protocol and ensure optimal protein expression. A positive band corresponding to the right size suggests that the gene inserted for the desired protein has been successfully translated, although functional assays are required to confirm successful protein function. This was carried out for some of the constructs through fluorescence experiments such as construct Q (LuxR) induced by the protein expressed by construct A (LuxI) (Figure 14).

Several parameters were adjusted (see our [WB troubleshooting guide](https://2020.igem.org/Team%3AStockholm/Engineering#Identifyaproblemtosolveati+1=troubleshooting)) throughout the experimental period:

* Cell culture density at lysis (in order to optimize protein expression according to cell growth phase)
* Concentration of inducer (pollutant or QS signalling molecule)
* Increased blocking time
* Increased incubation time with antibodies

The results presented here are as obtained after several repeated trials. The same positive bands for the same samples appeared over several consecutive Western blots and were hence reproducible (see [notebook](https://2020.igem.org/Team%3AStockholm/Notebook) for all Western blot results). In general, pollutant-inducible constructs were induced with the pollutants overnight.

Table 4: The constructs used for the western blot experiment, uninduced as well as induced (constructs M, F and P 3.2) by pollutants overnight. \*Positive band corresponding to correct protein size on WB. \*\*Obtained from Professor Ute Römling

| Constructs | Code | Protein Tag | Induced with pollutants (o/n) | Protein Size + Myc-tag (kDa) | Correct Sequence |
| --- | --- | --- | --- | --- | --- |
| Constitutive promoter- LuxI- Myc tag | A | c-Myc-tag | N/A | 23.1 | Yes\* |
| Constitutive promoter- RhlI- Myc tag | G | c-Myc-tag | N/A | 23.8 | Yes\* |
| Constitutive promoter-LuxR - Myc tag | P | c-Myc-tag | N/A | 29.8 | No |
| Constitutive promoter-RhlR - Myc tag | J | c-Myc-tag | N/A | 29.4 | Mismatch |
| Constitutive promoter- LuxR- Myc tag- Promoter Lux-GFP | Q | c-Myc-tag | N/A | 29.8 | Yes\* |
| Constitutive promoter- RhlR- Myc tag- Promoter Rhl-GFP | O | c-Myc-tag | N/A | 28.8 | Yes |
| Constitutive promoter-bphR2 - Myc tag | L | c-Myc-tag | N/A | 35.1 | Yes |
| Promoter bphR1-LuxI-Myc tag | M | c-Myc-tag | 10-4 M 1,1-biphenyl | 23.1 | Yes |
| Promoter prmA-LuxI-Myc tag | F | c-Myc-tag | 62.5 μM PFOS | 23.1 | Yes |
| Constitutive promoter-bphR2 - Myc tag - promoter bphR1-LuxI | Plasmid 3.2 (P 3.2) | c-Myc-tag | 10-4 M 1,1-biphenyl | 35.1 | Not fully sequenced |
| FtsH1Myc\*\* | Pos Control | c-Myc-tag | N/A | 71.2 | N/A |
| Constitutive promoter- GFP | Neg Control | None | N/A | N/A | Yes |

The results presented here are as obtained after several repeated trials. The same positive bands for the same samples appeared over several consecutive WBs and were hence reproducible (see [notebook](https://2020.igem.org/Team%3AStockholm/Notebook) for all WB results). The constructs, namely F and plasmid 3.2, designed to be induced by pollutants were tested in uninduced as well as induced conditions. The pollutant concentration used for induction corresponded to the maximum pollutant concentration threshold without an impact on their growth, as determined by the viability assay (Figure 10). Construct M, consisting only of the the bphR1 promoter, hence lacking the gene bphR2 was also tested in uninduced as well as induced conditions. This allowed for assessment of the potential inducibility of this promoter in absence of bphR2. Negative controls lacking a Myc-tag as well as positive controls (confirmed Myc tag on WB) were included.

![WB results of samples in table 4 (the one right above this) with positive bands for positive control, LuxI (A), RhlI (G) and LuxR (Q) in that order. M + biphenyl, F + PFOS and P3.2 + biphenyl were induced by pollutants according to table 4](https://static.igem.org/mediawiki/2020/a/a4/T--Stockholm--img--Results--WB.png)

Figure 14: WB results of samples in table 4 (the one right above this) with positive bands for positive control, LuxI (A), RhlI (G) and LuxR (Q) in that order. M + biphenyl, F + PFOS and P3.2 + biphenyl were induced by pollutants according to table 4

The above results show positive results i.e. successful protein translation of the proteins LuxI, RhlI as well as LuxR. Construct P was not successfully inserted as confirmed by sanger sequencing, this construct however was designed to express LuxR which was successfully transformed and translated through construct Q. It was still tested however on this blot as a form of negative control. Construct J, i.e. RhlR had a mismatch in the genetic sequence and did not generate a positive band in repeated WB trials. Constructs O14 and O19 were the same constructs and had both correct sequences yet neither yielded positive bands. Construct L consisted of the bphR2 protein required for biphenyl sensing and had a confirmed correct sequence yet did not generate a positive band. The constructs inducible by pollutants did not generate positive bands in uninduced or induced conditions. To be noted however that some parts of P3.2 was not able to be sequenced.

We used a blue safe protein stain to further troubleshoot the lack of positive bands for the constructs with correct sequences. Information of whether the general protein expression in the cell was inhibited due to the gene inserts themselves being unfavourable to our host cells could be explored through general staining of the proteome. It seemed that there was no prominent difference in the proteomic expression among different constructs.

![Blue safe protein staining of samples in table X (the one right above this) with positive bands for positive control, LuxI (A), RhlI (G) and LuxR (Q) in that order. M + biphenyl, F + PFOS and P3.2 + Biphenyl were induced by pollutants according to table x](https://static.igem.org/mediawiki/2020/5/50/T--Stockholm--img--Results--WB-Coomassie-Stain.png)

Figure 15: Blue safe protein staining of samples in table X (the one right above this) with positive bands for positive control, LuxI (A), RhlI (G) and LuxR (Q) in that order. M + biphenyl, F + PFOS and P3.2 + Biphenyl were induced by pollutants according to table x

The WB results can be interpreted in several ways. One interpretation is that the constructs lacking positive bands are in fact not expressing the desired proteins despite most of them carrying the correct genetic inserts as confirmed by sanger sequencing. There are however several other assays one would like to perform before drawing that conclusion. Future experiments in order to troubleshoot the lack of protein expression could entail:

* RNA extraction (in order to pinpoint where in the process of protein expression there could be a problem such as in the mRNA).
* Increasing the pollutant concentration for induction of constructs F and Plasmid 3.2.
* Cell lysis at different time points in their growth phase, to analyse a potential change in protein expression corresponding to different time points.
* Probing for a host protein may also aid in understanding differences in total protein expression and could help in quantifying the protein expression for the positive bands.
* The adjacent sequence to the Myc tag in our protein sequence might affect the functionality of the protein [Zordan et al., 2015](#citation1) as well as the antibody-binding ability [Schüchner et al., 2020](#citation2). Redesigning the construct sequence, altering the placement of the Myc tag, increasing the number of tags or replacing the tag to another such as a His-tag might hypothetically prove more favourable for some of our constructs, and could be further explored in future studies.

# Chromobacterium violaceum

This assay was done for the constructs that produced AHL (N-acyl homoserine lactones) which can be detected using *[Chromobacterium violaceum](https://2020.igem.org/Team%3AStockholm/Experiments#Chromobacteriumviolaceum)*. We used a stock solution of 100 μM C4-HSL and 3OC6-HSL in Milli-Q to prepare agar with varying concentrations of AHL (C4-HSL and 3OC6-HSL). A 48 well plate containing *Chromobacterium* and agar with AHL was prepared as follows:

Table 5: Schematic of *Chromobacterium* assay on agar.

| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| control (no cells), C4-HSL | control, C4-HSL | 0.1 μM, C4-HSL | 0.2 μM, C4-HSL | 1 μM, C4-HSL | 2 μM, C4-HSL | 10 μM, C4-HSL | 20 μM, C4-HSL |
| control (no cells), C4-HSL | control, C4-HSL | 0.1 μM, C4-HSL | 0.2 μM, C4-HSL | 1 μM, C4-HSL | 2 μM, C4-HSL | 10 μM, C4-HSL | 20 μM, C4-HSL |
| control (no cells), C4-HSL | control, C4-HSL | 0.1 μM, C4-HSL | 0.2 μM, C4-HSL | 1 μM, C4-HSL | 2 μM, C4-HSL | 10 μM, C4-HSL | 20 μM, C4-HSL |
| control (no cells), 3OC6-HSL | control, 3OC6-HSL | 0.1 μM, 3OC6-HSL | 0.2 μM, 3OC6-HSL | 1 μM, 3OC6-HSL | 2 μM, 3OC6-HSL | 10 μM, 3OC6-HSL | 20 μM, 3OC6-HSL |
| control (no cells), 3OC6-HSL | control, 3OC6-HSL | 0.1 μM, 3OC6-HSL | 0.2 μM, 3OC6-HSL | 1 μM, 3OC6-HSL | 2 μM, 3OC6-HSL | 10 μM, 3OC6-HSL | 20 μM, 3OC6-HSL |
| control (no cells), 3OC6-HSL | control, 3OC6-HSL | 0.1 μM, 3OC6-HSL | 0.2 μM, 3OC6-HSL | 1 μM, 3OC6-HSL | 2 μM, 3OC6-HSL | 10 μM, 3OC6-HSL | 20 μM, 3OC6-HSL |

![Plate reader readings for chromobacterium with various AHL concentrations](https://static.igem.org/mediawiki/2020/0/03/T--Stockholm--img--Results--chromo1.png)

Figure 16: Plate reader readings for chromobacterium with various AHL concentrations

Readings from plate reader did not correspond to colour change in the agar wells. Only the highest concentrations of 3OC6-HSL appeared to cause a colour change. To troubleshoot this part we went on to do a preliminary test to check if there was any colour change seen in the presence of AHL. A plate with *Chromobacterium* was grown onto which stock 100uM AHL solution was spread. Unfortunately there was no color change observed. This could possibly be because the AHL in the stock solution had degraded as it was kept at room temperature.

![Chromobacterium on agar plate. The circled area indicates the area where 3OC6-HSL was added](https://static.igem.org/mediawiki/2020/f/fd/T--Stockholm--img--Results--chromo2.png)

Figure 17: Chromobacterium on agar plate. The circled area indicates the area where 3OC6-HSL was added

This experiment was repeated with *Chromobacterium* plates using freshly prepared AHL (2μL of a 10mM AHL in acetonitrile) on plates. Purple colour was observed around the droplet areas, more for 3OC6 than C4. Also the immediate area around the droplet was found to be empty, presumably due to acetonitrile toxicity.

![Colour change seen on chromobacterium plates at 10mM AHL](https://static.igem.org/mediawiki/2020/6/6c/T--Stockholm--img--Results--chromo3.png)

Figure 18: Colour change seen on chromobacterium plates at 10mM AHL

Once the concentration at which colour change was observed was found, the constructs were tested on the chromobacterium plates. Overday cultures of A and G were tested on separate plates and a control of only *Chromobacterium* plate was used.

![Control with only Chromobacterium, A+Chromobacterium and G+Chromobacterium](https://static.igem.org/mediawiki/2020/f/f1/T--Stockholm--img--Results--chromo4.png)

Figure 19: Control with only Chromobacterium, A+Chromobacterium and G+Chromobacterium

A different experimental setup was planned with two plates on which *Chromobacterium* and the AHL or bacteria were grown adjacent to each other. This consisted of the strains transformed constructs A and G, control: Top 10 *E.coli*, C4-HSL and 30C6-HSL.

![(a) Plate setup for Chromobacterium reaction comparison. (b) No color change observed on the plates](https://static.igem.org/mediawiki/2020/7/7d/T--Stockholm--img--Results--chromo5.png)

Figure 20: (a) Plate setup for Chromobacterium reaction comparison. (b) No color change observed on the plates

There was no colour change observed on any plates; this could possibly be due to the less growth of the *Chromobacterium* and the Top 10 *E.coli* as the cultures were grown for almost 24 hours and the cells might have died before plating.

Hence from the *Chromobacterium* assay it was seen that pure AHL at a concentration of 10mM gave a clear colour change visible to naked eyes. However in case of the constructs there wasn't a huge difference. Although from the Western blot the same constructs A and G gave clear bands multiple times it could be concluded that the proteins were being formed but the concentration of the proteins formed were too low to be detected by the *Chromobacterium* plates prepared. Further literature review to better this experiment is needed to improve.

# References

1. Zordan, R. E., Beliveau, B. J., Trow, J. A., Craig, N. L., & Cormack, B. P. (2015).

   Avoiding the Ends: Internal Epitope Tagging of Proteins Using Transposon Tn7.

   Genetics 200(1), 47-58.

   [CrossRef](https://doi.org/10.1534/genetics.114.169482)[Google Scholar](https://scholar.google.com/scholar?q=Avoiding the Ends: Internal Epitope Tagging of Proteins Using Transposon Tn7.)[Back to text](#intext1)
2. Sch chner, S., Behm, C., Mudrak, I., & Ogris, E. (2020).

   The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context.

   Science Signaling 13(616), eaax9730.

   [CrossRef](https://doi.org/10.1126/scisignal.aax9730)[Google Scholar](https://scholar.google.com/scholar?q=The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context.)[Back to text](#intext2)

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