Outlined below are the wet lab experiments conducted by iGEM Guelph in 2023 for project BloomBiota

   

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iGEM Guelph’s project is built upon basic principles of genetic engineering, and it begins with design using an in silico approach to ensure the biological parts we want to use have a high chance of combining as we plan.

 

SnapGene was used to design primers to amplify the vitamin B12 gene cluster from Salmonella typhimurium LT2 gDNA, which goes from the cbiA gene to the cobT gene. As this gene cluster is over 15,000 base pairs in length, primers were designed to amplify the gene cluster in two sections to increase the chances of effectively amplifying the fragments via polymerase chain reaction (PCR). These primers were designed to introduce a promoter upstream of the vitamin B12 gene that has shown high expression in the mammalian gut environment (Armetta et al., 2021). The size of the cluster necessitated the use of a Bacterial Artificial Chromosome (BAC), which can function correctly with large inserts, and we chose to use the pBeloBac11loxP2272 from AddGene (Coren, 2017).

 

Since BACs are low-copy plasmids, we also designed primers to amplify the plasmid to increase its concentration prior to digestion and ligation of the insert. We decided to use the Golden Gate assembly kit from New England BioLabs (NEB) for assembly (Gibson et al., 2010). This required us to ensure sufficient overlapping regions on the primers between the three amplicons (two fragments of the gene cluster and plasmid amplicon). Designing our primers as we did allowed the inserted gene cluster to disrupt the LacZα gene. This allowed us to screen white-blue colonies on X-Gal media with NEB α-competent cells after assembly and transformation. Using SnapGenes Gibson Assembly simulator, we then showed that the assembly could work as designed (Figure 2).

 

Figure 1: Plasmid map of pBeloBac11loxP2272 with vitamin B12 gene cluster inserted

   

 

For the hGIF gene, a human protein, the sequence was first located in the NCBI database. IDTs codon optimization tool was then used to optimize the codons for expression in Escherichia coli Nissle 1917 and DH5a, our chassis organisms. Following our identification of the protein’s genetic sequence, we needed to ensure that the protein was efficiently exported to the periplasm, where it has a better chance of folding correctly. A literature review was conducted, and an efficient E. coli export signal peptide (evolved pelB) to go upstream of the hGIF gene sequence was identified (Mirzadeh et al., 2020). As we chose to use the NEB Golden Gate BsaI-HFv2 assembly kit, which comes with a pGGAselect plasmid, uploaded the optimized sequence into the NEBridge Golden Gate Assembly Tool (Potapov et al., 2018; Christian et al., 2010). This tool allowed us to design primers flanking our sequence of interest with the correct restriction sites to facilitate Golden Gate assembly using a kit. The Assembly Tool also allowed for the simulation of the assembly, which indicated that there were no issues and that the assembly would work as planned in silico (Figure 3). Twist Bioscience was used for the construction of the DNA sequence, which contained the restriction sites, signal peptide and gene sequence. After assembly and transformation into NEB α-competent E. coli, we planned to use the EcoRI restriction enzyme in order to verify the transformation on an agarose gel via electrophoresis.

 

Once confirmed, the transformed E. coli can be grown in broth under various conditions before protein extraction. As proper localization of this protein is important, the culture would be spun down and the supernatant removed before a cold osmotic shock extraction is performed to extract periplasmic proteins, followed by inner membrane lysis in order to obtain the cytoplasmic contents. These three separate samples would be run on an SDS-PAGE in order to separate the proteins before a Western Blot is performed using goat anti-GIF antibodies to bind the GIF protein and rabbit anti-goat secondary antibodies for visualization. A B-PER protein solubilization assay would be performed as most proteins are soluble if in a functional conformation; this would give us a small degree of confidence that the protein was being correctly folded. To further test for expression of a functional hGIF protein, a native-PAGE assay would be performed with one sample acting as a control and another sample being exposed to vitamin B12 as the B12 would bind the hGIF if functional and increase its mass, slowing down its transfer in the native-PAGE and appearing as a band with higher molecular weight.

 

Figure 2: Plasmid map of pGGAselect with hGIF and pelB signal peptide sequences inserted

 

 

 

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References

 

Armetta, J., Schantz-Klausen, M., Shepelin, D., Vazquez-Uribe, R., Martin Iain Bahl, Martin Frederik Laursen, Tine Rask Licht, & Alexander, O. (2021). Escherichia coli Promoters with Consistent Expression throughout the Murine Gut. ACS Synthetic Biology, 10(12), 3359–3368. https://doi.org/10.1021/acssynbio.1c00325

Christian, M., Cermak, T., Doyle, E. L., Schmidt, C., Zhang, F., Hummel, A., Bogdanove, A. J., & Voytas, D. F. (2010). Targeting DNA Double-Strand Breaks with TAL Effector Nucleases. Genetics, 186(2), 757–761. https://doi.org/10.1534/genetics.110.120717

Coren, J. S. (2017). Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site. BMC Research Notes, 10(1). https://doi.org/10.1186/s13104-017-2631-8

Gibson, D. G., Smith, H. O., Hutchison, C. A., Venter, J. C., & Merryman, C. (2010). Chemical synthesis of the mouse mitochondrial genome. Nature Methods, 7(11), 901–903. https://doi.org/10.1038/nmeth.1515

Mirzadeh, K., Shilling, P. J., Elfageih, R., Cumming, A. J., Cui, H. L., Rennig, M., Nørholm, M. H. H., & Daley, D. O. (2020). Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microbial Cell Factories, 19(1). https://doi.org/10.1186/s12934-020-01339-8

Potapov, V., Ong, J. L., Kucera, R. B., Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Evans, T. C., & Lohman, G. J. S. (2018). Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology, 7(11), 2665–2674.