Part:BBa_K2333407

UNS J23100 sfGFP pdt A

This part is designed to facilitate easy measurement of the strength of protein degradation tag (pdt) A by measuring time to steady-state fluorescence values of sfGFP under the control of the strong constitutive promoter 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 A 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_K2333401 to BBa_K2333406.

Usage and Biology

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). Therefore this part has the greatest degradation rate of the 6 protein degradation tags and it reaches steady-state fluorescence values the quickest. This part contains J23100 constitutive promoter, BBa_B0034 (RBS),pdt A, a double stop codon and BBa_B0015 (double terminator) in the William and Mary iGEM Universal Nucleotide Sequences (UNS) format. The sfGFP reporters have been codon-optimized for E. coli and feature a double stop codon for enhanced efficiency. 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_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.

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.