Regulatory

Part:BBa_K2194002:Design

Designed by: Catherine Dunaway, Anna Guseva   Group: iGEM17_Rice   (2017-10-23)


PgntK Negative Feedback Membrane Stress Promoter


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 14
    Illegal BsaI.rc site found at 216


Design Notes

We added BsmBI recognition sites before and after our Pgntk gBlock® so that we could incorporate the gBlock® into a cloning vector with complementary BsmBI sticky ends in a scarless Golden Gate reaction.

Source

We sourced the nucleotide sequence for Pgntk from the Escherichia coli BW25113 genome NCBI database (entry CP009273.1) by extracting the 200 bp uspstream of the gntK gene. This range was based on previous research that demonstrated that this region contained the full promoter sequence because the same negative feedback response was observed whether using 500 or 200 bp upstream of gntK as the promoter. [2] Then we removed the putative ribosome binding site from this promoter region. (We determined the likely RBS by using the “Salis Lab RBS Calculator” [3]). We then sent this sequence (promoter without putative RBS) to IDT to manufacture as a gBlock®.

References

[1] Nucleotide [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – . Accession No. CP009273:3570425-3570952, gntk; [cited 2017 October 31]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/CP009273.1
[2] Boyarskiy, Sergey et al. “Transcriptional Feedback Regulation of Efflux Protein Expression for Increased Tolerance to and Production of N-Butanol.” Metabolic Engineering 33 (2016): 130–137. Web. 28 Oct. 2017.
[3] https://salislab.net/software/