Difference between revisions of "Part:BBa K2333414"

Revision as of 23:15, 8 October 2022

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 tag's degradation strength in combination with inducible mf-Lon protease constructs as a part of their iGEM project. The graph below shows degradation strength data along with the data from the other tags in this series (BBa_K2333413-BBa_K2333419).

Toulouse_INSA-UPS’s 2022 Use of a constitutive promoter for the expression of mScarlet

BBa_K4197014 ihfb800 promoter for an efficient expression of mScarlet.

Toulouse_INSA_UPS_2022 contributed to the characterization of this part, the team showed this year that it is functionally expressed when placed under the control of the constitutive E. coli promoter ihfb800 (BBa_K4197014). To do this, the promoter was placed upon the mScarlet gene (BBa_K4197022), allowing the production of a bright red fluorescent protein detectable at the naked eye, by epifluorescence microscopy and by FACS. Check parts BBa_K4197020 and BBa_K4197021 of the full construction for more result details.

Figure 56: Second plating of pET-21 b (+)_Ara h 2_OmpA_mScarlet-I transformed cells and pET-21 b (+)_Ana o 3_OmpA_mScarlet-I transformed Stellar cells. The colonies were selected on LB-ampicillin plates. Pink coloring indicates the expression of the mScarlet-I.

More information about the project for which the part was created: DAISY (INSA-UPS 2022)

Sequence and Features

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.