Intermediate

Part:BBa_K218006:Design

Designed by: Jeremy Kubik   Group: iGEM09_Calgary   (2009-10-19)
Revision as of 03:08, 22 October 2009 by Jfkubik (Talk | contribs) (→‎Design Notes)

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LuxPQ followed by terminator and LuxOU generator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2209
    Illegal BglII site found at 2427
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3509
    Illegal BsaI.rc site found at 4886
    Illegal SapI.rc site found at 2177
    Illegal SapI.rc site found at 4848


Design Notes

The LuxPQ and LuxOU operons each have an internal RBS and terminator. Additional terminators were cloned downstream of each operon to ensure insulation. The TetR promoter (Part:BBa_R0040) was cloned upstream of the LuxOU operon to allow for constitutive expression of these cytoplasmic proteins. No promoter has been cloned upstream of LuxPQ because the proteins encoded by this operon are found in the periplasm, an area of limited space. With this in mind, expression levels must be under exquisite control so the cell is not overloaded with the presence of LuxPQ. This control comes with designing a library of 256 different ∆σ70 promoters that will alter expression levels of LuxPQ. These promoters are flanked with BamHI and XhoI restriction sites and will be cloned into the pCS26 vector (Bjarnason et al., 2003), upstream of the cloning site for this signalling circuit. This design will allow for optimal AI-2 signalling.

Source

LuxPQ - GenBank Accession: NC_009784 Vibrio harveyi ATCC BAA-1116 chromosome II, REGION: 579654..580751.

LuxOU - GenBank Accession# L26221 LuxO Protein ID# AAD12736.1 LuxU Protein ID# AAD12737.2

B0015 - Registry of Standard Biological Parts

R0040 - Registry of Standard Biological Parts

References

Bjarnason, J., C. M. Southward, and M. G. Surette. 2003. Genomic profiling of iron-responsive genes in Salmonella enterica serovar Typhimurium by high-throughput screening of a random promoter library. J. Bacteriol. 185:4973-4982.

Jeremy A. Freeman and Bonnie L. Bassler. Sequence and function of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi. 1999. Journal of Bacteriology. Feb 1999, p. 899-906.

Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM. Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell. 2006 Sep 22;126(6):1095-108.