Difference between revisions of "Part:BBa K2333435"
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===Characterization=== | ===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 | + | 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 the respective series (K2333407-K2333412 and K2333420-K2333426). |
| − | 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. | + | Time course measurements were performed according to standard protocol, and sfGFP 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. |
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Revision as of 01:46, 30 October 2017
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 the respective series (K2333407-K2333412 and K2333420-K2333426).
Time course measurements were performed according to standard protocol, and sfGFP 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
- 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.