Part:BBa_K2333421
UNS pTet sfGFP pdt A
This part consists of an sfGFP reporter with protein degradation tag A under the control of the aTc-inducible pTet promoter combined with the pTet repressor tetR under the medium-weak strength constitutive promoter J23105 (BBa_J23105). This part was used to measure gene expression speed with pdt A and allowed for control over the initiation of reporter expression by using the small molecule aTc. This part was used along with IPTG-inducible mf-Lon protease to demonstrate distinct levels of speed to steady state in reporter expression. 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. See [http://2017.igem.org/Team:William_and_Mary/Results William and Mary's 2017 project] for more details.
This part is one of a series of inducible sfGFP reporter pdt parts. Series range is from BBa_K2333420 to BBa_K2333426.
Usage and Biology
This part contains pdt A tagged sfGFP 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. 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). 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 sfGFP 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 A on a plasmid-based system. In order to demonstrate that protein degradation tags operated similarily regardless of the tagged protein, sfGFP reporters that were analogous to the mScarlet-I parts (BBa_K2333428 to BBa_K2333436) were built and characterized. This demonstrates that the protein degradation tags are modular and that they have differential strengths even when they are tagged on different proteins. 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 Pdt A tagged pTet sfGFP construct along with IPTG-inducible mf-Lon protease to show degradation rate and speed change effect measurements. The graph below shows this degradation characterization along with the data from the other tags in this series (BBa_K2333420 to BBa_K2333426). As expected, degradation was the greatest with the Pdt A tagged pTet sfGFP construct.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1077
Illegal NheI site found at 1100 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 133
References
[1] 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.
[2] Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276–1281.
[3] Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137–1142. http://doi.org/10.1038/nbt.2401
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