Part:BBa_K3885203

P70-ClpXP

The E. coli complex ClpXP, a protease from the AAA+ family, is an ideal system to achieve targeted protein degradation in Cell-Free reactions by adding, either to the N-terminus or C-terminus, a specifific degron typically 10−12 amino acids long.56 In Cell-Free systems,46 as well as in E. coli, 57 protein degradation by ClpXP is a zeroth order reaction with constant rate.
Under the influence of the P70 promoter, ClpXP protein can be expressed.The expression of ClpXP in Cell-Free system can improve the degradation rate of protein with ssrA degradation label.

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 448
  Illegal EcoRI site found at 1510
  Illegal EcoRI site found at 1855
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 448
  Illegal EcoRI site found at 1510
  Illegal EcoRI site found at 1855
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 448
  Illegal EcoRI site found at 1510
  Illegal EcoRI site found at 1855
  Illegal BglII site found at 1689
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 448
  Illegal EcoRI site found at 1510
  Illegal EcoRI site found at 1855
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 448
  Illegal EcoRI site found at 1510
  Illegal EcoRI site found at 1855
  Illegal AgeI site found at 1171
  Illegal AgeI site found at 1909
  
• 1000
  COMPATIBLE WITH RFC[1000]