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

Latest revision as of 13:54, 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.

Figure 2. Sequences that generate different expression levels of a gene (RHS). Combined with the addition of TTC (Schell & Poser, 1989), this should generate a system that is tightly repressed and highly expressed in the absence and presence of salicylate.

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

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

@@ Line 10: / Line 10: @@
 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:
-[[File:T--Sydney_Australia--salpromoter.png|500x500px|"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."]]
+[[File:T--Sydney_Australia--salpromoter.png|500x500px|Caption]]
+'''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.
+[[File:T--Sydney_Australia--salpromotersequences.png|400px|Caption]]
+'''Figure 2.''' Sequences that generate different expression levels of a gene (RHS). Combined with the addition of TTC (Schell & Poser, 1989), this should generate a system that is tightly repressed and highly expressed in the absence and presence of salicylate.
+Restriction enzymes were also removed to minimise off-target effects. Substitute bases were chosen to most closely match the natural codon frequency in bacteria.