Difference between revisions of "Part:BBa K2333414"
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===Characterization===
 
===Characterization===
  
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W&M 2017 characterized this tag's degradation rate and speed change effects in combination with inducible mf-Lon protease constructs as a part of their iGEM project. The graphs below show this speed data along with the data from the other tags in this series (<partinfo>K2333413</partinfo>-<partinfo>K2333419</partinfo>).
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W&M 2017 characterized this tagless construct in combination with inducible mf-Lon protease constructs as a control for their strength of degradation measurements. The graph below shows degradation strength data along with the data from the other tags in this series (<partinfo>K2333413</partinfo>-<partinfo>K2333419</partinfo>).
  
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<html><img src="https://static.igem.org/mediawiki/2017/f/f1/T--William_and_Mary--mScarlet-I_Speed_Normalized_Truncated.png" width="600px"/></html>
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<html><img src="https://static.igem.org/mediawiki/parts/f/fe/T--William_and_Mary--J23100_mScarlet_final.png" width="600px"/></html>
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Time course measurements were performed according to standard protocol, and mScarlet 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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<html><img src="https://static.igem.org/mediawiki/2017/d/df/T--William_and_Mary--mScarlet-I-Deg-Rate.png" width="600px"/></html>
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Revision as of 00:39, 2 November 2017


UNS J23100 mScarlet-I pdt A

This part is contained in a suite of protein degradation tagged mScarlet reporters under the control of the strong constitutive promoter BBa_J23100. These parts, in combination with inducible mf-Lon protease constructs, allowed William and Mary 2017 to characterize the degradation properties of each protein degradation tag (pdt) on a plasmid-based system. William and Mary 2017 successfully demonstrated distinct levels of protein degradation by each of the 6 pdt’s, and mScarlet reporters have been codon-optimized for E. coli and feature a double stop codon for enhanced efficiency. This specific part contains pdt-A, one of the 6 pdt's, which was used to produce a distinct effect on the speed of a tagged protein’s expression.

Usage and Biology

This part contains mScarlet-I with pdt-A under the control of the constitutive promoter BBa_J23100. The mScarlet-I reporter is a monomeric red fluorescent protein with high quantum yield, brightness, and fold-time. See Bindels, et. al (2016). Protein degradation tag A is the strongest of the 6 protein degradation tags that William and Mary 2017 characterized, and is associated with the E. Coli orthogonal protease mf-Lon (BBa_K2333011). This part also contains a double stop codon and BBa_B0015 (double terminator) in the William and Mary iGEM Universal Nucleotide Sequences (UNS) format. This enables easy cloning with Gibson Assembly, as UNS primers are designed for easy PCRs and high yield Gibson Assembly. See Torella, et. al (2013). Using this part in combination with inducible mf-Lon protease constructs, William and Mary 2017 was able to characterize the degradation properties of protein degradation tag A on a plasmid-based system. This is a part of the first experimentally-demonstrated system that allows future iGEM teams to access modular, predictive control over the temporal dynamics of their circuits by swapping parts at the genetic sequence level.

Characterization

W&M 2017 characterized this tagless construct in combination with inducible mf-Lon protease constructs as a control for their strength of degradation measurements. The graph below shows degradation strength data along with the data from the other tags in this series (BBa_K2333413-BBa_K2333419).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 47
    Illegal NheI site found at 70
    Illegal NotI site found at 605
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Bindels, D. S., Haarbosch, L., Weeren, L. V., Postma, M., Wiese, K. E., Mastop, M., . . . Gadella, T. W. (2016). MScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods, 14(1), 53-56. doi:10.1038/nmeth.4074

[2] Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276–1281.

[3] 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.