Part:BBa_K337028

Tet Repressor

The TetR binds to the Tet Operator and by that causes knockdown auf gene expression. (BB-2, RFC12).

Improvement by iGEM ZJUT-China 2021

As the improvement of the 10_Heidelberg registered part (BBa_K337028: tet Repressor), we, ZJUT-China 2021 newly registered partBBa_K3885126. through adding a degradation tag ssrA to the 3' end of the tetR (Figure 1), endowing the tetR a novel function—degradation.

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 .

Results

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
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 652
  
• 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]