Part:BBa_K3885212

P28-tetR-ssrA

Tet Repressor with ssrA degradation tag can be degraded by ClpXP (BBa_K3885123) , thus the inhibition of deGFP in the downstream region of the gene circuit is eliminated.

Usage and Biology

The ssrA tag can degrade the abnormal protein during the translation process, so that the tetR gene can be cleaved and lose its expression function, and the original expression of the repressor protein can be degraded. Therefore, we introduced the ssrA tag and constructed part P28-tetR-ssrA.

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

First, we put the part into Cell-Free system to verify its effect. From the fluorescence data, it is clear that our constructed part can function properly. SsrA tag eliminates the effect of residual deterrent proteins in the system. In the follow-up experiment, in order to further improve the degradation effect of ssrA tag, we introduced ClpXP which can accelerate the degradation of the deterrent protein and verified its binding effect.

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.

Design Page

We found that even after the Cas9 protein exercises cleavage activity to disable tetR, repressor proteins remain in the system still prevent fluorescent protein expression and have an impact on the signaling output of the system . Therefore, by reviewing the literature, we identified the ssrA degradation tag. ssrA tags are encoded by amino acids that can be added to the C-terminus of proteins to achieve degradation of abnormal proteins during translation.Thus, by adding the ssrA tag to the back end of tetR, we were able to solve the above problem.

References

Marshall R, Noireaux V. Synthetic Biology with an All E. coli TXTL System: Quantitative Characterization of Regulatory Elements and Gene Circuits. Methods Mol Biol. 2018;1772:61-93.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]