Part:BBa_K2333435
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1245
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1184
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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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