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Part:BBa_K2333435

Designed by: Ethan M Jones   Group: iGEM17_William_and_Mary   (2017-10-27)


pBad mf-Lon

This is an arabinose-inducible E. coli optimized mf-Lon construct. Arabinose inducibility is provided by BBa_I0500, William and Mary 2017 iGEM found early on that this part had leaky expression, and thus did not perform any high-quality characterization of this part.

Usage and Biology

This part contains mf-Lon under the control of the inducible pBad promoter. The mf-Lon protease specifically targets protein degradation tags (pdts) with various 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

A concentration of 0.1mM arabinose was sufficient to create degradation of mScarlet-I and sfGFP [http://2017.igem.org/Team:William_and_Mary/Parts parts]. The concentration of 1mM arabinose increased degradation but lead to high amounts of cell death. The concentration 0.01mM arabinose generally increased expression. 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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