Part:BBa_K2333408

UNS J23100 sfGFP pdt B

This part is designed to facilitate easy measurement of the strength of protein degradation tag (pdt) B by measuring time to steady-state fluorescence values of sfGFP under the control of the strong constitutive promoter BBa_J23100. William and Mary iGEM 2017 used pdts as a method to control gene expression speed.This part was utilized to characterize the degradation properties of pdt B and confirm the fact that different pdts have different degradation strengths. 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 sfGFP reporter pdt parts. Series range is from BBa_K2333407 to BBa_K2333412.

Usage and Biology

Protein degradation tag B (BBa_K2333002) 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). Therefore this part has the second greatest degradation rate of the 6 protein degradation tags and it is the second fastest to reach the steady-state fluorescence value. This part contains J23100 constitutive promoter, BBa_B0034 (RBS), pdt B, a double stop codon and BBa_B0015 (double terminator) in the William and Mary iGEM Universal Nucleotide Sequences (UNS) format. See Torella, et. al (2013). In order to demonstrate that protein degradation tags operated similarly regardless of the tagged protein, sfGFP reporters that were analogous to the mScarlet-I parts (BBa_K2333413 to BBa_K2333419) 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.

Characterization

W&M 2017 characterized this Pdt B tagged BBa_J23100 sfGFP construct along with IPTG-inducible mf-Lon protease to show degradation rate and speed change effect measurements. The graph below shows this preliminary degradation characterization along with the data from the other tags in this series (BBa_K2333407-BBa_K2333412). While degradation is occurring, it does not necessarily line up with all of the expected tag strengths. This could be due to constraints surrounding plate reader measurements or inconsistencies with the sfGFP reporter itself.

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
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 106
  

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