iGEM 2023 — Team chalmers-gothenburg

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## Lab notes

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For details about the wet lab procedure, check out the lab notes below! For each week in the lab, methods and results are presented for all our experiments. The method description commonly refers to a standard protocol, which can be found on the[Experiments page.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

June

Week 1

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July

Week 2

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Week 3

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Week 4

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Week 5

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August

Week 6

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Week 7

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Week 8

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Week 9

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Week 10

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September

Week 11

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Week 12

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Step by step

Step by step: CrtE, CrtI, FAD, sgRNA, Cas9 plasmid

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Step by step: CrtYB

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When initially designing the yeast and lactic acid bacteria (LAB) implementations the creation of a symbiotic relationship between the two organisms was explored, with the goal of preventing direct competition between the two and maintaining stable populations of both production strains. The design that was eventually decided on used a modified yeast using the B12 dependent methionine synthase MetH instead of the more commonly found Met6. This made growth in media where B12 was not present unfeasible for the yeast and thus made the already negligible risk of the yeast outcompeting the LAB even less of an issue. Engineering the LAB to be dependent on the yeast was early found to be outside of the scope of what was deemed reasonable, but the naturally strong color of vitamin A would at least provide a way of confirming that both organisms were alive. Vitamin A production would therefore act as an indicator for if either the yeast died, the LAB died, B12 production stopped or yeast stopped producing vitamin A. As a proof of concept a genome scale model of vitamin A production using the Yeast8 model [1] was also established at this point where the model indicated that β-carotene production was possible without sacrificing too much in the way of cell growth.

The assembly process for the β-carotene construct started off well with the level 0 plasmids used for the MoClo pYTK [2] Golden Gate assembly being done relatively quickly. The level 1 plasmids on the other hand kept being either assembled incorrectly or weren’t being amplified sufficiently once transformed into E.coli. Several different changes were made to the methodology to try and find some success such as using higher quality enzymes, doing the procedures in triplets or ordering new reagents in case they were contaminated. Sadly none of this had the desired result and the finished vitamin A constructs were never produced.

When the process of combining and amplifying the parts required for vitamin A production via Golden Gate Assembly started to stall, the decision was made to transition the vitamin B12 project over to using Gibson assembly of larger, ordered fragments. The rationale here was that either the methodologies or reagents used for Golden Gate assembly may be one of the causes of the repeated failures, and a transition away from type IIs enzyme assembly may be advantageous.. The full B12 construct was designed to be ordered as three separate fragments which could then be combined to form the full construct. Around the same time it was also decided that since time was starting to be an issue, the B12 construct would not be stably integrated into the LAB genome but kept as transient plasmids and work as a proof of concept instead. At this point time was running out and there was only enough time for one attempt at assembling the construct, which then proceeded to yield a non-viable final product.

![](https://static.igem.wiki/teams/4902/wiki/images/design.png)

So while the results were a bit disappointing we did at the very least follow the standard “Design, Build, Test, Learn”-cycle, even though most of the learning boiled down to concluding that something does not work and never finding the true source of the error.

## References

References

* [1]: Lu, H., *et al.* "A consensus S. cerevisiae metabolic model Yeast8 and its ecosystem for comprehensively probing cellular metabolism. *Nat Commun*10: 3586." (2019).
* [2]: Michael E. Lee, William C. DeLoache, Bernardo Cervantes, and John E. Dueber. “A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly” *ACS Synthetic Biology”* (2015) DOI: 10.1021/sb500366v

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## Experiments

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The experimental workflow used during the wet lab part of our iGEM project involved several types of experiments. To get a more detailed overview of the workflow, please refer to the lab notes on the[Notebook page.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)All of the relevant experimental protocols used during our iGEM project can be found below.

### Protocols

Plating and liquid culture preparation

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Colony PCR - PHIRE II

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Competent *E.coli* cells

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Electroporation Solution (ES)

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Gel electrophoresis

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GeneJET Plasmid Miniprep kit Purification (ThermoFischer)

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Gibson Assembly

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GYT media

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*L. plantarum* competent cells and electroporation

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*L. reuteri* electrocompetent cells

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Short Guide for Yeast MoClo-Workflow

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MRS agar

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MRS broth

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OD measurement

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PHUSION polymerase protocol

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Prep of MoClo parts

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QIAprep Miniprep (QIAGEN) *E.coli*

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ThermoCycler protocol

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## Vitamin B12 project

We started this project-part by trying to make*L. reuteri*competent cells; and therefore available to receive and accept new genetic material/vectors that express genes associated with B12 synthesis.

The strains we received from SLU (Swedish University of Agricultural Sciences) are listed in the table below.

**Table 1: The LAB strains we received from SLU.**

| The LAB-strains we received from SLU |
| --- |
| *L. plantarum*LP256 |
| *L. plantarum*36e |
| *L. plantarum*LMG 9211 |
| *L. plantarum*ATCC8014 |
| *L. casei*LMG 6904 |
| *L.casei*ATCC 4646 |
| *L.reuteri*DSM 20015 |

All strains were streaked on plates, from where colonies were picked to liquid media.
[![One of the LAB-bacteria, L.reuteri DSM 20015, we received from SLU](https://static.igem.wiki/teams/4902/wiki/images/results/l-reuteri-v2.jpg)](https://static.igem.wiki/teams/4902/wiki/images/results/l-reuteri-v2.jpg)

 **Figure 1: One of the LAB-bacteria,*L. reuteri*DSM 20015, after plating.**

We followed protocol*"L. reuteri competent cells"*to do competent cells of the*L. reuteri*DSM 20015 we obtained. We electroporated 5 different vectors into*L.reuteri*competent cells, accompanied with several positive controls. There was never any growth on the plates with FL0117, yPTK095, yPTK001, or pPBT and pTRK. However, the plates with electroporated cells without any vector did grow, the positive control, hence we showed that it was possible to make competent*L.reuteri*cells.

**Table 2: Electroporation of vectors into*L. Reuteri*competent cells.**

| Plasmid electroporated in*L. reuteri* | Did it grow after electroporation? |
| --- | --- |
| pTRKH3-emGFP/PT | No growth |
| pPBT-peRNA-GG-Puro/P | No growth |
| pYTK001 | No growth |
| pYTK095 | No growth |
| FL0117 | No growth |
| No plasmids/positive control | Growth |

The genes we aimed to assemble into a backbone/vector were successfully amplified with PCR. GIbson assembly of the genes in a backbone was performed but never showed the right length on bands on gel-electrophoresis. To follow all lab procedures and attempts, please refer to the Notebook page.
[![One of the LAB-bacteria, L.reuteri DSM 20015, we received from SLU](https://static.igem.wiki/teams/4902/wiki/images/results/electro-lab2.jpg)](https://static.igem.wiki/teams/4902/wiki/images/results/electro-lab2.jpg)

**Figure 2: Electroporated*L. reuteri* cells without any vector (positive control)**

We had, as mentioned, issues with actually transforming any genetic parts into the *L. reuteri* strain. When we had got the genes needed for the gibson assembly, we tried repetitive times to assembly the genes into a backbone, but the gel electrophoresis that should show that we had the right length and that the assembly had been successful never gave us the result we wanted, therefor we never continued with attempts to electroporate our genes/vector into the lactobacteria.

## Vitamin A project

Before we could insert the Vitamin A-producing gene into*S. cerevisiae*we needed to assemble it. The final construct would consist of a backbone and the CrtI, CrtE, CrtYB, and FAD1 genes as well as sgRNA and CAS9, which can all be found in constructs. A colleague of ours provided us with all MoClo parts, which were purified and two replicates of each plasmid were stored in the freezer.

The fragments for CrtI, CrtE and FAD1 were already level 0 plasmids when we received them and were amplified in competent*E. coli*cells, after which they were grown in liquid culture. The plasmids were then extracted and stored in the freezer. The concentration was measured for all plasmids, and they all had at least one replicate with >50 ng/µl.

The CrtYB fragment was ordered and assembled with the pYTK001 backbone into a level 0 plasmid, before we could amplify and grow it. After the assembly was successful, the concentration was measured using a nanodrop. The concentration was >50 ng/µl, and the plasmid was sent to sequencing. This assembly-process can be found in more detail in the Notebook page.

We followed the MoClo protocol to create level 1 plasmids of CrtE, CrtI, FAD1, as well as the sgRNA backbone, sgRNAlvl1, Cas9 backbone and Cas9lvl1. All plasmids except for sgRNAlvl1 were amplified using competent*E. coli*cells and grown on plates.
[![Liam guiding the DNA extraction experiment.](https://static.igem.wiki/teams/4902/wiki/images/results/uv1.png)](https://static.igem.wiki/teams/4902/wiki/images/results/uv1.png)
[![Liam guiding the DNA extraction experiment.](https://static.igem.wiki/teams/4902/wiki/images/results/uv2.png)](https://static.igem.wiki/teams/4902/wiki/images/results/uv2.png)
[![Liam guiding the DNA extraction experiment.](https://static.igem.wiki/teams/4902/wiki/images/results/uv3.png)](https://static.igem.wiki/teams/4902/wiki/images/results/uv3.png)

**Figure 2: A selection of some of our plates with modified*E. coli*cells under UV light.**

We noticed that two MoClo plasmids that were used for the sgRNAlvl1, pYTK095 and pYTK047, both contained a GFP gene, which meant that both the non-modified backbone and the modified level 1 plasmid were green on the plate. To solve this, we removed the GFP gene from the pTYK095 plasmid by using the BsaI enzyme and loaded it into an electrophoresis gel. The part of the pYTK095 plasmid that we needed was separated from the GFP gene and was then purified with a gel electrophoresis purifying kit. The level 1 sgRNAlvl1 was then remade with this part instead.

The sequencing of CrtYB was a match, and was also constructed into a level 1 plasmid. However, only CasBB, sgBB and FAD1 were successful. Since we did not succeed to create all level 1 plasmids that we needed in order to create the level 2 plasmid and we had run out of time, we could not insert the vitamin A-producing gene in*S. cerevisiae.*

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