Difference between revisions of "Part:BBa K3814001:Design"

 
(Summary: Adding Bulk of Design)
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===Design Notes===
 
===Design Notes===
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Restriction enzyme sites were removed
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We have designed an inducible promoter that combines the work of two researchers (Schell & Poser, 1989; Meyer et al., 2018). It uses the nahR gene and sal promoters to activate transcription in response to the inducer salicylate, and can with a few base changes in the promoter, can provide different promoter effects. See below a diagram of the design and the base changes that induce varying transcription level effects:
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[[File:T--Sydney_Australia--salpromoter.png|options|Figure 1. Inducible promoter design. According to Cebolla et al. (1997), nahR produces a transcription factor that controls the expression of genes regulated by sal promoters. In the presence of salicylate, expression of those genes is facilitated.
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Restriction enzymes were removed to minimise off-target effects. Substitute bases were chosen to most closely match the natural codon frequency in bacteria.
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===References===
 
===References===
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Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2018). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196–204. https://doi.org/10.1038/s41589-018-0168-3
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Schell, M. A., & Poser, E. F. (1989). Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. Journal of Bacteriology, 171(2), 837–846. https://doi.org/10.1128/jb.171.2.837-846.1989

Revision as of 13:44, 21 October 2021


nahR gene in P. putida + promoter + RBS


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 951
    Illegal NheI site found at 974
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We have designed an inducible promoter that combines the work of two researchers (Schell & Poser, 1989; Meyer et al., 2018). It uses the nahR gene and sal promoters to activate transcription in response to the inducer salicylate, and can with a few base changes in the promoter, can provide different promoter effects. See below a diagram of the design and the base changes that induce varying transcription level effects:

Figure 1. Inducible promoter design. According to Cebolla et al. (1997), nahR produces a transcription factor that controls the expression of genes regulated by sal promoters. In the presence of salicylate, expression of those genes is facilitated.


Restriction enzymes were removed to minimise off-target effects. Substitute bases were chosen to most closely match the natural codon frequency in bacteria.



Source

Pseudomonas putida genome

References

Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2018). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196–204. https://doi.org/10.1038/s41589-018-0168-3


Schell, M. A., & Poser, E. F. (1989). Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. Journal of Bacteriology, 171(2), 837–846. https://doi.org/10.1128/jb.171.2.837-846.1989