Part:BBa_K2333413
UNS J23100 mScarlet-I
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 is a tagless control construct (J23100 mScarlet with no pdt) which can be used as a comparison against protein degradation for parts with pdt's.
Usage and Biology
This part contains mScarlet-I with no pdt under the control of the constitutive promoter J23100. The mScarlet-I reporter is a monomeric red fluorescent protein with high quantum yield, brightness, and fold-time. See Bindels, et. al (2016). The 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). When used in combination with inducible mf-Lon protease constructs, this part can be used as a control in characterizing degradation properties of the 6 pdt's. 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 degradation rate and speed change effect measurements. The graphs below show this speed data along with the data from the other tags in this series (BBa_K2333413-BBa_K2333419).
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 47
Illegal NheI site found at 70
Illegal NotI site found at 605 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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