Part:BBa_K3814001:Design
nahR gene in P. putida + promoter + RBS
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 951
Illegal NheI site found at 974 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 1. 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