Part:BBa_K3885123

ClpXP

ClpXP consists of two distinct proteins, a AAA+ ATPase called ClpX（BBa_K1911003） and a peptidase called ClpP（BBa_K1911002）. We incorporate the ClpX and ClpP to be a one-part. In protein substrates, ClpX recognizes unstructured peptide sequences (called tags or degrons), like ssrA--a specific C-terminal degradation tag, proceeds to unfold stable tertiary structure in the protein, and then spools or translocates the unfolded polypeptide chain into a sequestered proteolytic compartment in ClpP for degradation into small peptide fragments (Fig. 1).

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 EcoRI site found at 387
  Illegal EcoRI site found at 1449
  Illegal EcoRI site found at 1794
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 387
  Illegal EcoRI site found at 1449
  Illegal EcoRI site found at 1794
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 387
  Illegal EcoRI site found at 1449
  Illegal EcoRI site found at 1794
  Illegal BglII site found at 1628
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 387
  Illegal EcoRI site found at 1449
  Illegal EcoRI site found at 1794
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 387
  Illegal EcoRI site found at 1449
  Illegal EcoRI site found at 1794
  Illegal AgeI site found at 1110
  Illegal AgeI site found at 1848
  
• 1000
  COMPATIBLE WITH RFC[1000]