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Vitamin B12 project

    We started this project-part by trying to makeL. reutericompetent 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. plantarumLP256
    L. plantarum36e
    L. plantarumLMG 9211
    L. plantarumATCC8014
    L. caseiLMG 6904
    L.caseiATCC 4646
    L.reuteriDSM 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
    Figure 1: One of the LAB-bacteria,L. reuteriDSM 20015, after plating.

    We followed protocol"L. reuteri competent cells"to do competent cells of theL. reuteriDSM 20015 we obtained. We electroporated 5 different vectors intoL.reutericompetent 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 competentL.reutericells.
    Table 2: Electroporation of vectors intoL. Reutericompetent cells.
    Plasmid electroporated inL. 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
    Figure 2: ElectroporatedL. 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 intoS. cerevisiaewe 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 competentE. colicells, 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 competentE. colicells and grown on plates. Liam guiding the DNA extraction experiment. Liam guiding the DNA extraction experiment. Liam guiding the DNA extraction experiment.
    Figure 2: A selection of some of our plates with modifiedE. colicells 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 inS. cerevisiae.