Part:BBa_K3814026:Design
ComB/PilX + RBS, ACIAD3318/ACIAD3317
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
USYD 2021's team goal was to generate naturally transformable E. coli. E. coli actually has most of the transformation and competency genes, but they are largely unexpressed under lab conditions, and attempts to induce their expression have not been successful (Sinha & Redfield, 2012).
Thus, we decided that the entire natural transformation system of another phylogenetically similar bacteria could be incorporated into E. coli. We ended up deciding on Acinetobacter baylyi, not only as an evolutionarily similar species to E. coli, but one of lower pathogenicity than other relatives (Chen et al., 2008).
The genes involved in transformation and competency in A. baylyi consist of 23 genes. Several research studies (Busch et al., 1999; Friedrich et al., 2001; Seitz & Blokesch, 2013) have highlighted that competence genes are absolutely crucial for natural transformation. If all the genes responsible for the pilus structure was functional, i.e., the pilus structure was formed, uptake rates increased 10,000 fold.
These genes are are few of the genes described above that generate a section of the pilus structure, and will be inserted into a strain of E. coli (JM109) to build this machine. Note that this single basic part has multiple genes. This was only the case because the ribosome binding sites and reading frames of the genes overlapped with one another, and it would be too difficult to separate them only to put them right after each other again. This was only done with genes in a similar situation, otherwise all genes involved with this project were separated.
Restriction enzymes were also removed to minimise off-target effects. Substitute bases were chosen to most closely match the natural codon frequency in bacteria.
Source
ADP1 Genome
References
Busch, S., Rosenplänter, C., & Averhoff, B. (1999). Identification and Characterization of ComE and ComF, Two Novel Pilin-Like Competence Factors Involved in Natural Transformation of Acinetobacter sp. Strain BD413. Applied and Environmental Microbiology, 65(10), 4568–4574. https://doi.org/10.1128/aem.65.10.4568-4574.1999
Chen, T. L., Siu, L. K., Lee, Y. T., Chen, C. P., Huang, L. Y., Wu, R. C. C., Cho, W. L., & Fung, C. P. (2008). Acinetobacter baylyi as a Pathogen for Opportunistic Infection. Journal of Clinical Microbiology, 46(9), 2938–2944. https://doi.org/10.1128/jcm.00232-08
Friedrich, A., Hartsch, T., & Averhoff, B. (2001). Natural Transformation in Mesophilic and Thermophilic Bacteria: Identification and Characterization of Novel, Closely Related Competence Genes in Acinetobacter sp. Strain BD413 and Thermus thermophilus HB27. Applied and Environmental Microbiology, 67(7), 3140–3148. https://doi.org/10.1128/aem.67.7.3140-3148.2001
Seitz, P., & Blokesch, M. (2013). DNA-uptake machinery of naturally competent Vibrio cholerae. Proceedings of the National Academy of Sciences, 110(44), 17987–17992. https://doi.org/10.1073/pnas.1315647110
Sinha, S., & Redfield, R. J. (2012). Natural DNA Uptake by Escherichia coli. PLoS ONE, 7(4), e35620. https://doi.org/10.1371/journal.pone.0035620