Difference between revisions of "Part:BBa K3814034:Design"

 
 
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<partinfo>BBa_K3814034 short</partinfo>
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<partinfo>BBa_K3814034 SequenceAndFeatures</partinfo>
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===Design Notes===
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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).
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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).
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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. In particular, Seitz & Blokesch (2013) showed in Vibrio cholerae that silencing genes comEA, comEC and comF all yielded no transformation (below detection limit) of tDNA samples. If these genes were functioning, the transformation frequency would rise above detection levels, and if the whole pilus structure was functional, uptake rates increased 10,000 fold.
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This gene is one of the few crucial genes described above that are crucial for natural transformation, and will be inserted into a strain of E. coli (JM109) to build this machine.
 +
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Restriction enzymes were also removed to minimise off-target effects. Substitute bases were chosen to most closely match the natural codon frequency in bacteria.
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===Source===
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ADP1 Genome
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===References===
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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
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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
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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
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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
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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

Latest revision as of 15:30, 21 October 2021


ComP + RBS, ACIAD3338


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 222
  • 1000
    COMPATIBLE 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. In particular, Seitz & Blokesch (2013) showed in Vibrio cholerae that silencing genes comEA, comEC and comF all yielded no transformation (below detection limit) of tDNA samples. If these genes were functioning, the transformation frequency would rise above detection levels, and if the whole pilus structure was functional, uptake rates increased 10,000 fold.

This gene is one of the few crucial genes described above that are crucial for natural transformation, and will be inserted into a strain of E. coli (JM109) to build this machine.

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