Difference between revisions of "Part:BBa K5127025:Design"
 
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===Source===
 
===Source===
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E. coli MG1655
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<i>E. coli MG1655</i>
  
  

Latest revision as of 10:18, 2 October 2024


Device for genome integration (pReplace)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

On either side of the GOI (here we have a barcode instead of GOI) there are two homology arms, HA Rev and HA For, each approximately 500bp long. These homology arms allow for precise recombination into the genome, mediated by integrase and recombinase enzymes. The integration site and the homology arms were derived from Park, Y. et al (2020). After transforming the pReplace plasmid into the Phase II competent cells (BBa_K5127015), the barcode is automatically integrated into the genome. At the same time, the bacteria are plated on 10% sucrose plates, and only those that have successfully replaced the SacB gene with the barcode will survive, as SacB is lethal in the presence of sucrose. The colonies that grow on these plates indicate successful integration. Finally, we will confirm the integration through colony PCR.


Source

E. coli MG1655


References

Park, Y., Borujeni, A. E., Gorochowski, T. E., Shin, J. & Voigt, C. A. (2020). Precision design of stable genetic circuits carried in highly-insulated E. coli genomic landing pads. Molecular Systems Biology, 16(8), e9584. https://doi.org/10.15252/msb.20209584

Taketani, M., Zhang, J., Zhang, S., Triassi, A. J., Huang, Y. J., Griffith, L. G., & Voigt, C. A. (2020). Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron. Nature Biotechnology, 38(9), 962–969. https://doi.org/10.1038/s41587-020-0468-5