iGEM 2021 — Team Ionis_Paris

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# Team:Ionis Paris/Notebook

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# Notebook

Phase 1: Sensing of vitamin B12 and production of lactate

Phase 2: Development of a bio-electronic sensor of lactate

#### Team Ionis Paris

Our team is composed of students from 3 different IONIS schools. We wanted each part
to be
done by specialized students in order to have the strongest project possible.

#### Follow us social media:

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### **Big thanks** to our sponsors :

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# Team:Ionis Paris/Engineering

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# Engineering Sucess

![](https://static.igem.org/mediawiki/2021/d/d9/T--Ionis_Paris--build.png)

# Phase 1 : Vitamin B12 sensing

The first phase of our project relies on one question : How to specifically sense vitamin B12 in our bacteria ? After going through scientific literature and several iGEM wiki pages, we identified the Cobalamin riboswitch as the basis of our sensing module. This riboswitch relies on the sequestration of the RBS located downstream DNA sequence to regulate expression. To see more about the mechanism of the riboswitch structure, see Description part.

## Imagine :

Biobrick 1 : Inverting the riboswitch (RS) signal

![](https://static.igem.org/mediawiki/2021/6/66/T--Ionis_Paris--rbs_aaaaaaaaaaaaaaaezvzvvz.png)

Biobrick 3 : Reporting biobrick 1 signal

![](https://static.igem.org/mediawiki/2021/4/43/T--Ionis_Paris--ldh_fvzsedgzevzeve.png)

## Build :

In order to assemble our biobricks, the initial strategy we chose was to use Golden Gate Assembly. This strategy was chosen as Golden Gate Assembly is widely used for the assembly of multiple inserts. A general Golden Gate Assembly with Esp3I workflow is shown below.

![](https://static.igem.org/mediawiki/2021/f/f4/T--Ionis_Paris--sans_titre_29.png)

### Golden Gate Assembly workflow (created with Biorender)

Before this workflow, specific sequences have to be added to each fragment. These sequences are specific to Esp3I and have the following general structure :

![](https://static.igem.org/mediawiki/2021/7/7d/T--Ionis_Paris--sans_titre_30.png)

### Golden Gate Assembly overhang structure (created with biorender)

Tet operator sequences are sequences that can be difficult to synthesize. The fragment for biobrick 2 was therefore ordered with these sequences, and a first step of addition of Tet operator sequences by PCR was done. After this, specific overhangs were added to both biobricks 1 and 2 by PCR.

## Test :

Following the Golden Gate Assembly workflow, overhangs were added on each biobrick sample using PCR.

## Improve/Imagine :

After trying several times, we considered changing strategy to CPEC (Circular Polymerase Extension Cloning). CPEC was chosen because of its simplicity [1] and because needed reagents could be obtained quickly (primers, polymerase, etc.), as the deadline was approaching.

## Build :

![](https://static.igem.org/mediawiki/2021/0/06/T--Ionis_Paris--sans_titre_31.png)

### CPEC workflow (created with Biorender)

#### Step 1

1. (A) Amplification of inserts by PCR
2. (B) Addition of 30 base-pair overhangs to the insert by PCR
3. (C) Opening of backbone in two split linear fragments by PCR
4. (D) Addition of a 30 base-pair overhang to the backbone (fragment A) by PCR

#### Step 2

1. PCR purification of PCR products
2. Ligation of insert and resistance using a DNA polymerase (overhangs are used as primers) as in a PCR cycle

#### Step 3

1. Transformation of the obtained plasmid in E. coli DH5-α competent cells.

## Test (problem encountered during addition of overhangs) :

![](https://static.igem.org/mediawiki/2021/1/19/T--Ionis_Paris--sans_titre_32.png)

### Addition of overhangs on fragments of interest (L : 1kb plus DNA ladder; A : amplified insert A with overhangs (1166 bp); B : amplified insert B with overhangs (947 bp))

As we can see, the correct band sizes are present on the gel for both samples (A and B). However, non-specific bands can be seen (100 bp for fragment A and 400 bp for fragment B). Following these results, two strategies could have been applied : extracting fragments of interest on the gel or start over the experiment by improving the PCR protocol.

## Improve/Imagine :

On figure 2, we can see that bands of interest show a relatively low intensity for a PCR product. We therefore decided to improve our strategy, by adding a PCR purification step before the second PCR. This ensures the purity of template DNA and might avoid the amplification of unwanted bands.

## Test :

After a purification step on fragments of PCR products, step 2 was repeated. The corresponding agarose gel is shown below :

![](https://static.igem.org/mediawiki/2021/1/13/T--Ionis_Paris--experiment.png)

### Addition of overhangs on fragments of interest after PCR purification (L : 1kb plus DNA ladder; A : amplified insert A with overhangs (1166 bp); B : amplified insert B with overhangs (947 bp))

On the gel, we can see bands of the corresponding size, with a high intensity. No parasite bands appear. These results show that PCR purification before running our second PCR improved the addition of overhangs on previously amplified fragments.

As explained in the Results section, assembly did not work. Because of time limitations, the experiment was not repeated. A new way of improving the protocol would have been to treat backbone templates with DpnI after PCRs in order to avoid any unwanted transformation.

# Theoretical engineering cycle : Cobalamin riboswitch and Ribosome Binding Sites

Due to the specific mechanism of regulation of our sensing system, particular attention had to be given to the Ribosome Binding Sites (or RBS) design. From the beginning of our project, we knew that changes would have to be made on RBS.

## Design :

At first, we made the choice to test a standard iGEM RBS, the BBa\_B0032 RBS (medium strength), in order to avoid any issues by using a previously used RBS. Our part design was the following :

## Build :

This Biobrick should have been built using CPEC or Golden Gate Assembly (see Results).

## Test (expected) :

In order to test our plasmid, The relative expression of RFP should have been measured by measuring red fluorescence on a fluorescent plate reader (exciting wl : 488 nm; detecting wl : 588 nm). This would have been done by growing transformants overnight with various amounts of vitamin B12, and following fluorescence through time (see Results).

We can expect from this experiment to link the increase of cobalamin concentration to the increase of measured fluorescence. As we want to precisely quantify cobalamin, we presume that signal could be optimized by modifying our biobricks.

## Improve/Imagine :

The aim of this part is to obtain a precise signal from cobalamin riboswitch activity. We assume that results could have been optimized, as we tested only one riboswitch. Therefore, we tried to answer the following question : How can we improve our construct to optimize vitamin B12 quantification ?

In order to get a precise signal from our chassis, one way of optimizing the system could be the choice of designing different RBS in order to impact two different parameters. Firstly, modifying RBS is known as an effective strategy to control gene expression in bacteria [2]. Secondly, the cobalamin riboswitch is a RBS sequestering type of riboswitch : This means that protein expression is regulated through a conformational change of the RBS, as shown on the following figure.

## Design:

A series of RBS of different strengths were designed on Salislab for each gene sequence. By making different combinations between our different biobricks, an optimized system could be developed. The best possible combination could be found with the following workflow :

![](https://static.igem.org/mediawiki/2021/9/92/T--Ionis_Paris--rfp.png)

After creating this library of biobricks, the best one should be evaluated by transformation in bacteria and RFP expression measurements. Of course, the same procedure would have to be done when replacing the RFP by the lactate dehydrogenase.

## Sources

[1] Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. https://doi.org/10.1371/journal.pone.0006441

[2] Oesterle, Sabine & Gerngross, Daniel & Schmitt, Steven & Roberts, Tania & Panke, Sven. (2017). Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli. Scientific Reports. 7. 10.1038/s41598-017-12395-3.

#### Team Ionis Paris

Our team is composed of students from 3 different IONIS schools. We wanted each part
to be
done by specialized students in order to have the strongest project possible.

#### Follow us social media:

[![](https://static.igem.org/mediawiki/2021/e/e6/T--Ionis_Paris--twitter_1.png)](https://twitter.com/iGEM_IONIS)
[![](https://static.igem.org/mediawiki/2021/d/dc/T--Ionis_Paris--insta_2.png)](https://www.instagram.com/igem.ionis/)
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### **Big thanks** to our sponsors :

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# Team:Ionis Paris/Experiments

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# Experiments

#### Paris's Ionis Team

Our team is composed of students from 3 different IONIS schools. We wanted each part
to be
done by specialized students in order to have the strongest project possible.

#### Follow us social media:

Twitter: [https://twitter.com/iGEM\_IONIS/](https://twitter.com/iGEM_IONIS)

Instagram: <https://www.instagram.com/igem.ionis/>

Facebook: <https://www.facebook.com/ionisigem/>

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# Team:Ionis Paris/Results

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# Results

The objective behind our project is to develop a bioelectronic device able to quantify blood vitamin B12 (or cobalamin), thanks to the genetic modification of *Escherichia coli*  and the use of *Shewanella oneidensis*, a metal-reducing bacteria. To develop our system, the experimental approach was divided in two main phases:

1. Phase 1 : Sensing of vitamin B12 and production of lactate

2. Phase 2 : Development of a bioelectronic sensor of lactate

## Phase 1 : Sensing of vitamin B12 and production of lactate

# Biobrick design for phase 1

In order to obtain a production of lactate corresponding to the amount of vitamin B12 sensed, our final plasmid for phase 1 was design as followed :

![](https://static.igem.org/mediawiki/2021/e/e1/T--Ionis_Paris--sans_titre.png)

### Figure 1 : Final construct for phase 1

As lactate production was not a common way to report riboswitch activity, this plasmid was split in two different ones:

![](https://static.igem.org/mediawiki/2021/4/46/T--Ionis_Paris--sans_titre_1.png)

### Figure 2 : Plasmid A for the reporting of the Cobalamin riboswitch activity (containing biobrick 1 & 2 ([BBa\_K3933005](http://parts.igem.org/Part%3ABBa_K3933005)))

![](https://static.igem.org/mediawiki/2021/5/55/T--Ionis_Paris--sans_titre_2.png)

### Figure 3 : Plasmid B for the arabinose-induced production of lactate (containing biobrick 3 ([BBa\_K3933006](http://parts.igem.org/Part%3ABBa_K3933006)))

# Golden Gate Assembly of biobrick 1 and biobrick 2 :

At first, we tried to assemble biobrick 1 from iGEM kit plates, using Golden Gate Assembly. Unfortunately, we did not manage to transform our bacteria with plasmids of interest.
A linear fragment corresponding to biobrick 1 and biobrick 2 was then ordered from Twist Bioscience to be assembled using Golden Gate assembly with Esp3I. Workflow for Golden Gate Assembly is shown below :

![](https://static.igem.org/mediawiki/2021/a/a7/T--Ionis_Paris--sans_titre_3.png)

### Figure 4 : Golden Gate Assembly workflow (created with Biorender)

### A) Primer design and PCR

The Golden Gate Assembly technique mainly relies on the design of specific DNA overhangs, containing type II-S restriction enzyme sites. A more general structure for these overhangs is shown below :

![](https://static.igem.org/mediawiki/2021/b/be/T--Ionis_Paris--snas_titre_4.png)

### Figure 5 : Golden Gate Assembly overhang structure (created with biorender)

1. NNNNN (or (N)5) : Complementary ligation overhang sequences

2. Restriction site : Recognition site for the enzyme

3. NNNN (or (N)4) : Extra space needed for the enzyme to cut DNA

Tet operator sequences are sequences that can be difficult to synthesize. The fragment for biobrick 2 was therefore ordered with these sequences, and a first step of addition of Tet operator sequences by PCR was done. After this, specific overhangs were added to both biobricks 1 and 2 by PCR.

![](https://static.igem.org/mediawiki/2021/5/5d/T--Ionis_Paris--gelfigure6_schema.png)

### Figure 6 : Agarose gel after PCR (1: Addition of Tet operator on Biobricks 2. 2: Addition of Golden Gate overhangs of Biobricks 1 and Biobricks 2. L : ladder; A: Biobrick 2; B: Biobrick 1)

### B) Backbone preparation

The idea behind this step was to modify a pSB1A3 containing a RFP (BBa\_J04450), our main backbone for restriction enzyme clonings used in our lab, in order to obtain a vector usable both for restriction enzyme cloning and for Golden Gate Assembly using BsmBI/Esp3I. To do so, we ordered a 121 synthesized fragment from IDT containing random nucleotides and 4 specific sites :

![](https://static.igem.org/mediawiki/2021/0/02/T--Ionis_Paris--sans_titre_5.png)

### Figure 7 : Ordered fragment with the different wanted cut sites (SnapGene representation)

As shown on Figure 7, the fragment contains Prefix (XbaI) and Suffix (SpeI) restriction sites for insertion inside a Biobrick RFC[10] compatible backbone.

![](https://static.igem.org/mediawiki/2021/8/8d/T--Ionis_Paris--sans_titre_6.png)

### Figure 8 : Esp3I recognition and cut sites (New England Biolabs)

Figure 8 shows the recognition and cut sites of Esp3I. In order to perform Golden Gate Assembly, Esp3I sites have to be oriented in a specific way :

1. Site on the left needs to have the following sequence for the top strand : 3’ (N)₅GAGACG 5’

2. Site on the right needs to have the following sequence for the top strand : 3’ CGTCTC(N)₅ 5’

When obtained, DNA was resuspended and both the pSB1A3\_BBa\_J04450 and the DNA fragment were digested with XbaI and SpeI and ligated using a T4 DNA ligase. *E. coli* DH5-α were transformed using the obtained DNA and the construction was verified using PCR on colonies and digestion.

*### 1) PCR on colonies*

As SpeI and XbaI use leave cohesive sticky ends, primers were designed in order to check for the presence of the insert (backbone can ligate on itself), but also for the correct orientation of the insert (incorrect orientation is possible) :

![](https://static.igem.org/mediawiki/2021/5/5d/T--Ionis_Paris--sans_titre_7.png)

### Figure 9 : Primer locations on the ligated backbone (SnapGene screenshot)

Results:

![](https://static.igem.org/mediawiki/2021/9/9b/T--Ionis_Paris--sans_titre_8.png)

### Figure 10 : Agarose gel after PCR on colonies (L : ladder; A : PCR fragments; B : linearized pSB1A3\_RFP)

Results show that the fragment was correctly inserted into the backbone for nine colonies. Colonies were picked and grown into LB-Ampicilline liquid media for miniprep of plasmids.

*### 2) Gel migration*

After liquid growth, miniprep was done and plasmids were digested using Esp3I. Unfortunately, the Esp3I sample used was no longer viable : digestion was therefore done a second time later with a new enzyme sample on one clone. Figure 10 shows band sizes of undigested plasmids :

![](https://static.igem.org/mediawiki/2021/5/58/T--Ionis_Paris--sans_titre_9.png)

### Figure 11 : Agarose gel after miniprep of plasmids (L: Ladder; A: Miniprep products)

On the expected gel, the supercoiled form of the plasmid appears between 1.2 and 1.5kb. This band size is found for all clones, except for clone 19 who shows a band at higher sizes. This unexpected band can be explained by the ligation of the BBa\_J04450 part between the insert and the backbone, as XbaI and SpeI digestions leave the same sticky ends.

### C) Golden Gate Assembly

For this step, inserts and the backbone are digested with Esp3I and ligated in the same tube, following the Golden Gate Assembly protocol.

### Conclusion

Unfortunately, we did not manage to transform any bacteria after assembly. This could be explained by difficulties encountered to quantify low concentrations of DNA before assembly. We therefore changed our cloning strategy for CPEC, see more about this choice in [engineering success](https://2021.igem.org/Team%3AIonis_Paris/Engineering).

# Circular Polymerase Extension Cloning of biobrick 1 and biobrick 2 :

![](https://static.igem.org/mediawiki/2021/8/85/T--Ionis_Paris--sans_titre_10.png)

### Figure 12 : General CPEC workflow (created with Biorender)

### Step 1

(A) Amplification of inserts by PCR
(B) Addition of 30 base-pair overhangs to the insert by PCR
(C) Opening of backbone in two split linear fragments by PCR
(D) Addition of a 30 base-pair overhang to the backbone (fragment A) by PCR

### Step 2

(A) PCR purification of PCR products
(B) Ligation of insert and resistance using a DNA polymerase (overhangs are used as primers) as in a PCR cycle

### Step 3

Transformation of the obtained plasmid *in E. coli* DH5-α competent cells.

### Step 1 : preparation of PCR

*### A) Amplification of inserts by PCR*

Using 20 base-pair primers, inserts were amplified by PCR. Results are shown on figure 12.

![](https://static.igem.org/mediawiki/2021/8/8b/T--Ionis_Paris--sans_titre_11.png)

### Figure 13 : Agarose gel showing amplification of fragments of interest (L : 1kb plus NEB ladder; A : amplified insert A (1106 bp); B : amplified insert B (887 bp)) and expected gel)

Bands are seen at the correct sizes, addition of overhang can be performed.

*### B) Addition of 30 base-pair overhangs to the insert by PCR*

This step is done by using 50 base-pair primers containing the same 20 base-pair hybridization sequences as in step A) and 30 base-pair overhangs complementary to the backbone/insert sequences.

![](https://static.igem.org/mediawiki/2021/7/7f/T--Ionis_Paris--sans_titre_12.png)

### Figure 14 : Agarose gel showing addition of overhangs on fragments of interest after PCR purification (L : 1kb plus DNA ladder; A : amplified insert A with overhangs (1166 bp); B : amplified insert B with overhangs (917 bp)) with expected gel

Bands are seen at the correct sizes, fragments were correctly amplified with overhang primers.

Results show the correct band sizes for each insert. Fragments are therefore ready for CPEC reaction.

*### C) Opening of backbone in two split linear fragments by PCR*

To make sure the reaction worked, this step was done on several miniprep preparations of the psB1A3 plasmid. Primer design follows the same rules for this step as in step A) Agarose gels in Figure 14 shows results after PCR.

![](https://static.igem.org/mediawiki/2021/d/da/T--Ionis_Paris--sans_titre_13.png)

### Figure 15 : Agarose gel showing linearization of backbone fragments (L : NEB 1kb+ Ladder; A : Fragment A (1367bp); B : Fragment B (796 bp))

Results show that linearized fragments were amplified for most of the plasmids tested. Addition of CPEC overhang can be performed on fragment A.

*### D) Addition of a 30 base-pair overhang to the backbone (fragment A) by PCR*

Addition of an overhang sequence is only done on fragment A to ligate back the ampicillin resistance inside the backbone. To do so, primer design is the same as step B.

![](https://static.igem.org/mediawiki/2021/0/02/T--Ionis_Paris--sans_titre_14.png)

### Figure 16 : Agarose gel showing addition of CPEC overhang on backbone fragment A (L : NEB 1kb+ Ladder; A : Fragment A with overhang (1397bp))

Results show that fragment B was correctly amplified with overhang primers. backbone fragments were ready for cloning.

### Step 2 : Assembly by CPEC

Assembly was done by following the CPEC protocol and transformed into *E. coli*DH5-α competent cells. Figure 16 shows results after overnight culture on ampicillin agar plates.

![](https://static.igem.org/mediawiki/2021/f/fd/T--Ionis_Paris--sans_titre_15.png)

### Figure 15 : Ampicillin agar plates with transformants after incubation (C+ : positive control (pSB1A3-RFP transformants); A : Ratio A CPEC; 3 : Ratio B CPEC)

After 24 hours of incubation, colonies grow on agar plates. PCR on colony was performed on some clones (Dish 1 on figure 15) to check the correct assembly of our biobricks. We made the mistake of plating only a part of our transformation product. More transformation product was therefore plated on agar plates in order to check assemblies (Dish 2 on figure 15). The next day, colonies were picked and grown overnight in LB media with ampicillin to perform miniprep.

![](https://static.igem.org/mediawiki/2021/4/4a/T--Ionis_Paris--sans_titre_16.png)

### Figure 16 : Agarose gel showing miniprep digestion of transformants (L : Ladder; A : undigested white colonies minipreps; B : undigested red colonies minipreps; C : digested white colonies minipreps; D : digested red colonies minipreps) and expected results

On figure 16, we can see two different sizes for digested plasmids : for white colonies, a band is seen between 2.0 kb and 3.0 kb. For red colonies, a band is seen between 3.0 kb and 4.0 kb.

These results suggest that white colonies were backbones without insert (around 2200 bp) and red colonies pSB1A3-RFP (contamination with positive controls).

### Conclusion

Inserts were prepared correctly. However, Our first CPEC reaction did not result in any assembly. Due to time limitations, the experiment was not reproduced. Ways of improving this step can be found in [engineering success](https://2021.igem.org/Team%3AIonis_Paris/Engineering).

# Functional assays for biobricks 1 and 2

If assembly of biobrick 1 and 2 would have worked, we would have followed the following workflow to check riboswitch activity :

![](https://static.igem.org/mediawiki/2021/f/f9/T--Ionis_Paris--sans_titre_17.png)

### Figure 17 : Workflow for the evaluation of the cobalamin riboswitch activity (created with Biorender)

By following this workflow, we would have been able to evaluate both the activity of our riboswitch and its kinetics in one assay.

# Riboswitch activity : Expected results

If the cobalamin riboswitch is effective, we expect the increase of fluorescence with both cobalamin concentration and time. For improvement of this part, see [engineering success](https://2021.igem.org/Team%3AIonis_Paris/Engineering).

# Assembly of biobrick 3

Initially, biobrick 3 should have been assembled with Golden Gate Assembly using kit plates. As mentioned above, we were not able to transform any bacteria from kit plates. We therefore chose to order a linearized fragment from IDT. Unfortunately, we never managed to clone or amplify this fragment.

# Functional assay for biobrick 3

Lactate production by the lactate dehydrogenase would have been measured under stimulation with arabinose, for different lactose concentrations.

## Phase 2 : Development of a bioelectronic sensor for lactate

As explained before in [Description](https://2021.igem.org/Team%3AIonis_Paris/Description) and [Contribution](https://2021.igem.org/Team%3AIonis_Paris/Contribution), we aim to use the bacteria *Shewanella oneidensis (S. oneidensis)* as a reporter system.

# Experiments and results

To do so, a lab-scale prototype was setup (figure 18), following experimental setups described in the article from Zeng et al. (2019) [1]. 48 hours culture of *Shewanella oneidensis*  were diluted to obtain a DO600nm = 0.600 and 5 mL were inoculated into 45 mL of LB media in the prototype. The experiment was performed with various amounts of lactate in the media (40 mM, 20 mM, and 0 mM). Measures of voltage generated by  *Shewanella oneidensis*  were done on 500 minute periods.

![](https://static.igem.org/mediawiki/2021/9/9f/T--Ionis_Paris--sans_titre_25.png)

### Figure 18: Lab-scale prototype for bacterial electrical measures

![](https://static.igem.org/mediawiki/2021/6/6a/T--Ionis_Paris--SO_graph.png)

### Figure 19: Electrical activity of *S. oneidensis* over time (Blue : 40mM L-lactate; Grey : 20mM L-lactate; Red : No lactate)

Overall, the three curves show an increase in voltage over time. The rise in electricity production by our culture is higher and faster as concentration increases, until reaching a plateau around 300 mV. As rising in potential seems to be linked to lactate concentration, a curve was plotted using the No lactate condition as a control (figure 20).

![](https://static.igem.org/mediawiki/2021/5/5f/T--Ionis_Paris--sans_titre_22.png)

### Figure 20: Lactate specific electrical activity by *Shewanella oneidensis* over time

By looking at this curve, we can more easily see the specific impact of lactate on electrical activity without the impact of the media. This specific impact seems to be detectable in a few hours only, with a peak of specific activity at around 3 hours. After 500 minutes, the specific electrical activity from lactate seems to decrease.

Unfortunately, issues with *S. oneidensis*  stocks blocked our experiments for one month. We were therefore not able to produce more results with these experiments.

# Perspectives and expected results

In order to confirm the link between lactate concentration and electricity production by *S. oneidensis*, more experiments should be made. Firstly, more experiments should be done by testing a great variety of lactate concentrations, as we aim to precisely evaluate lactate production by phase I. Moreover, other parameters should be evaluated : varying *S. oneidensis* DO, testing different co-culture ratios with *E. coli*, or changing LB media to minimal media without a carbon source to avoid background signal due to LB media. By testing all this parameters, we aim to fine tune the accuracy of our system.

# Conclusion

Overall, results suggest that our system could be used to report lactate concentrations thanks to the production of electricity by *Shewanella oneidensis*  in a few hours. Even though this reporting seems to reach a plateau after several hours, the fact that a signal difference can be detected early is promessing, suggesting that our system could be used as a fast and efficient way of reporting genetic reporter systems. By combining our system with Phase I designs, we expect that vitamin B12 could be quantified precisely and in a few minutes to hours in the context of a personal quantifying system.

## Project achievements

+ A first lab-scale prototype for the measurement of *Shewanella oneidensis* electrical activity was successfully built

+ First measures suggest that different amounts of lactate could be measured using our lab-scale prototype

+ Attempts of automatisation using different hardware/software solutions were made

+ A final prototype was modelled and 3D printed

- We spent a lot of time on different cloning strategies that did not work due to equipment and time limitations

- Due to difficulties encountered in *Shewanella oneidensis* growth, only a few experiments were done with the prototype

## Sources

[1] Zeng, J., Banerjee, A., Kim, J. et al. A Novel Bioelectronic Reporter System in Living Cells Tested with a Synthetic Biological Comparator. Sci Rep 9, 7275 (2019). https://doi.org/10.1038/s41598-019-43771-w

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