Part:BBa_K2333429
UNS pTet mScarlet-I pdt B
This part is contained in a suite of protein degradation tagged mScarlet-I reporters under the control of the aTc-inducible pTet promoter combined with the pTet repressor tetR under the medium-weak strength constitutive promoter J23105. These parts were used along with the IPTG-inducible mf-Lon protease to demonstrate distinct levels of speed to steady state in reporter expression proportional to the relative strength of each pdt. William and Mary 2017 also took advantage of the inducible nature of these constructs by manipulating levels of aTc exposure in order to adjust final steady state values independently of speed control. This specific part contains pdt B, one of the 6 pdt's characterized by William and Mary 2017, which was used to produce a distinct effect on the speed of a tagged protein’s expression.
Usage and Biology
This part contains pdt-B tagged mScarlet-I under aTc inducible promoter pTet combined on the same construct with the pTet repressor tetR under the control of the medium-weak strength constitutive promoter J23105. 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 B is the second 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). 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). This part was used in William and Mary 2017's gene expression speed measurements, allowing them to control the initiation of mScarlet reporter expression using the small molecule aTc. In combination with the IPTG-inducible mf-Lon protease, this part was used by William and Mary 2017 to characterize the degradation properties of protein degradation tag B 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 rate and speed change effects with IPTG-inducible mf-Lon protease 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 (BBa_K2333427-BBa_K2333433).
Graph 1: Time course measurements were performed according to standard protocol, and fluorescence was normalized to steady state based upon when fluorescence no longer increased. Data is shown for each construct until steady state is reached (this means at least two consecutive subsequent data points do not increase fluorescence). As the no-pdt 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. Geometric mean of 10,000 cells each of three biological replicates. Shaded region represents one geometric standard deviation above and below the mean.
Graph 2: Comparison of calculated t1/2 vs degradation rate. Degradation rate was obtained as above, and t1/2 was defined as time at which each biological replicate's regression line reached half of steady state. The blue line represents an optical guide for the eye, and is not fitted. Speed is scaling with degradation rate and following a predicted trend.
Graph 3: Degradation rates were measured in the above constructs. Each data point represents the population geometric mean of at least 10,000 cells of a distinct biological replicate. Relative degradation was calculated relative to the geometric mean fluorescence of the untagged control.
Graph 4: Comparison of calculated t1/2 to construct pdt. Degradation rate was obtained, and t1/2 was defined as time at which each biological replicate's regression line reached half of steady state. Each data point represents the population geometric mean of at least 10,000 cells of a distinct biological replicate.
IFFL MEFL
Graph 5: Measurements of absolute gene expression using aTC inducible mScarlet-I constructs. Data is shown for each construct until steady state is reached (this means at least two consecutive subsequent data points do not increase fluorescence). Geometric mean of 10,000 cells each of three biological replicates. Shaded region represents one geometric standard deviation above and below the mean.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1059
Illegal NheI site found at 1082
Illegal NotI site found at 632 - 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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