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.

In Figure 2. C, the highest red line ( Group 3) contains only P70a-σ28 and P28-tetO-deGFP plasmids, thus expressing deGFP with high fluorescence intensity and serving as a positive control. The second highest purple line ( Group 4) with P70a-ClpXP can degrade tetR repressor with ssrA tag, eliminate its inhibition of deGFP expression in downstream of the gene circuits, and increase fluorescence intensity. The green line at the bottom of Figure 2. C ( Group 1) indicates that the tetR repressor without ssrA tag cannot be degraded by ClpXP protein, in contrast to Group 4, indicating that the ssrA degradation tag is functional. The blue line ( Group 2) is at the bottom of Figure 2. C showed low fluorescence intensity, which was used as a negative control to indicate that the addition of tag did not affect the inhibition of tetR.
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]