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Part:BBa_K2333435

Designed by: Ethan M Jones   Group: iGEM17_William_and_Mary   (2017-10-27)
Revision as of 01:43, 30 October 2017 by Chli (Talk | contribs)


pBad mf-Lon

This is an arabinose-inducible mf-Lon construct containing the pBad promoter. William and Mary 2017 has modified the mf-Lon gene via codon-optimization for iGEM use and added a double terminator.

Usage and Biology

This part contains mf-Lon under the control of the inducible pBad promoter. The mf-Lon protease specifically targets different protein degradation tags with varying affinities corresponding to varying degradation rates. This arabinose-inducible mf-Lon construct was used in tandem with aTc-inducible pdt reporter constructs to obtain gene expression speed measurements.

Characterization

W&M 2017 characterized this mf-Lon containing part in combination with constitutively expressed reporter constructs as well as aTc-inducible pdt reporter constructs. The graphs below show this speed data for each of the 6 tags in this series (K2333413-K2333419 and K2333427-K2333433).

Time course measurements were performed according to standard protocol, and fluorescence was normalized to steady state based upon when fluorescence no longer increased. As the no-Lon condition had not reached steady state when time course was ended, it was normalized to the final collected data point, which is likely close to the true steady state.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1245
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1184
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1019
    Illegal AgeI site found at 3102
    Illegal AgeI site found at 3186
    Illegal AgeI site found at 3392
    Illegal AgeI site found at 3417
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1001


References

[1] Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276–1281.

[2] Torella JP, Boehm CR, Lienert F, Chen J-H, Way JC, Silver PA. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Research. 2013;42(1):681–689.

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