Part:BBa_K3885126

tetR-ssrA This is an improved version of tetracycline resistance transcriptional repressor (tetR), which is registered by 10_Heidelberg iGEM team (BBa_K337028: tet Repressor). SsrA is a degradation tag, added at the 3' end of tetR that degrades the repressor protein tetR to become tetR-ssrA, thus we endow the original part a new function as improvement.

Usage and Biology

Figure 1. Schematic of hydrolysis by ClpXP protein.

To ensure the normal operation of the Cell-Free system, an adjustable protein degradation mechanism is crucial. It is degraded by ClpXPAAA+ protease in Escherichia coli . The protein must contain a degradation tag in order to be recognized by our cell-free system .

Characterization

Figure 2. Schematic of gene circuits and kinetics of tetR inhibition in the Cell-Free system.
(A) The gene circuits contains three parts, sigma 28 activates promoter P28 to express tet repressor with ssrA tag, which inhibits the expression of deGFP.
(B) The gene circuits contains an additional part P70-ClpXP that makes the tetR repressor degrade, and degfp expresses.
(C) Group 1 carried tetR without ssrA and ClpXP; Group 2 carried tetR with ssrA; Group 3 didn't carry tetR as positive control; Group 4 carried tetR with ssrA and ClpXP in comparison with Group 1.

TetR with ssrA can be degraded by ClpXP, so that the flourescence intensity increased as a result of the inhibition released from tet Repressor.
Therefore, we gave the tetR a new function and improved this part. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 10
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 10
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 10
  
• 1000
  COMPATIBLE WITH RFC[1000]