Next steps for BloomBiota's wet lab research
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Transforming E. coli Nissle 1917
E. coli Nissle 1917 contains two native plasmids. In order to reduce plasmid load on the cell and increase the availability of cellular resources for the added construct, the native plasmids can be removed using CRISPR/Cas9 (Lan et al., 2021). While using CRISPR/Cas9 technologies would increase the cost of the project, this method may result in increased transformation efficiency and increased construct function in the target bacteria. Another way to increase transformation efficiency would be to use electroporation. This step may prove to be essential for successful transformation.
Figure 1: Representation of plasmid being transformed into E. coli Nissle 1917 after inactivation of native plasmids.
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Demonstrating that the protein is functional
To demonstrate the functionality of the construct-produced human gastric intrinsic factor (hGIF) protein, a native PAGE could be done, or a cell culture assay suggested to the iGEM Guelph team by Dr. Stephen Seah, the Primary Principal Investigator (Nowakowski et al., 2014). In the case of native PAGE, the control would be solely the protein, and the treatment would expose the protein to vitamin B12.
The cell culture assay would be done by exposing cells to a GIF-bound vitamin B12, waiting a specified amount of time, then washing the cells to remove any exterior GIF/B12. Cells could then be lysed to see if the complex was formed inside the cell. While GIF may not be present because the cell may break it down, the vitamin B12 should be detected as it is used in many cellular processes.
Figure 2: A representation of native page and cell culture assay.
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Designing a plasmid with a form of selection other than antibiotic resistance
A shortfall of the project design was the use of an antibiotic resistance gene to conduct selection of bacteria containing the construct. Antibiotic resistance is a major, growing issue that should not be perpetuated. Instead of using an antibiotic resistance gene, selection could be conducted using an RNA-OUT selection system (Peubez et al., 2010). Alternate methods include using a Tet repressor system, and Amino-acid auxotrophy complementation (Peubez et al., 2010). An additional benefit to using an alternate selection method to antibiotic resistance is that the added antibiotic resistance gene in the plasmid can actually reduce the amount of expressed protein due to the additional stress placed on the host (Peubez et al., 2010). Not only are these alternative methods more forward thinking in the context of safety, they can also improve protein expression and plasmid recovery (Peubez et al., 2010).
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A Product with Just hGIF
One alternate path forward for BloomBiota may be to solely focus on inserting hGIF into the plasmid (pGGAselect), then transforming E. coli Nissle 1917. The vitamin B12 insert is quite large, and thus difficult to insert into a vector. If the probiotic is able to be transformed with the hGIF-containing construct, it could be simply packaged with a vitamin B12 supplement. This would allow the BloomBiota project to achieve its goal, while also reducing costs and time required to produce the engineered organism.
Figure 3: Probiotic pills with B12 added separate from E. coli Nissle 1917, which contains only the plasmid with GIF.
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References
Lan, Y.-J., Tan, S.-I., Cheng, S.-Y., Ting, W.-W., Xue, C., Lin, T.-H., Cai, M.-Z., Chen, P.-T., & Ng, I-Son. (2021). Development of Escherichia coli Nissle 1917 derivative by CRISPR/Cas9 and application for gamma-aminobutyric acid (GABA) production in antibiotic-free system. Biochemical Engineering Journal, 168, 107952–107952. https://doi.org/10.1016/j.bej.2021.107952
Peubez, I., Chaudet, N., Mignon, C., Hild, G., Husson, S., Courtois, V., De Luca, K., Speck, D., & Sodoyer, R. (2010). Antibiotic-free selection in E. coli: new considerations for optimal design and improved production. Microbial Cell Factories, 9(1), 65. https://doi.org/10.1186/1475-2859-9-65
Nowakowski, A. B., Wobig, W. J., & Petering, D. H. (2014). Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions. Metallomics : integrated biometal science, 6(5), 1068–1078. https://doi.org/10.1039/c4mt00033a