Difference between revisions of "Part:BBa K2333435"
 
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<partinfo>BBa_K2333435 short</partinfo>
 
<partinfo>BBa_K2333435 short</partinfo>
  
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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.
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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===
 
===Usage and Biology===
  
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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.
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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.
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<html><img src="https://static.igem.org/mediawiki/2017/b/b0/T--William_and_Mary--circuit5.jpeg" width="600px"/></html>
  
 
===Characterization===
 
===Characterization===
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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.  
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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).
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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>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 03:49, 2 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 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.