Difference between revisions of "Part:BBa K2333435"

Revision as of 23:45, 1 November 2017

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 .1mM arabinose was sufficient to create degradation of mScarlet-I and sfGFP [2017.igem.org/Team:William_and_Mary/Parts parts], 1mM arabinose increased degradation but lead to high amounts of cell death. .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.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2333435 short</partinfo>
-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.
+This is an arabinose-inducible <i>E. coli</i> optimized mf-Lon construct. Arabinose inducibility is provided by <partinfo>I0500</partinfo>, 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 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.
+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 .1mM arabinose was sufficient to create degradation of mScarlet-I and sfGFP [2017.igem.org/Team:William_and_Mary/Parts parts], 1mM arabinose increased degradation but lead to high amounts of cell death. .01mM Arabinose generally increased expression.
-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.
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 <span class='h3bb'>Sequence and Features</span>