Part:BBa_K3392000
Doc-SsrA
Doc is a protein usually used in bacteria toxin-antitoxin system (TA system). This TA system is derived from the bacteriophage P1, which lysogenizes in Escherichia coli cells as a stable low-copy plasmid. Doc plays a role of toxin. SsrA is a degradation tag of 11-residue carboxy-terminal peptide tail to the incomplete nascent chain. Doc-SsrA is a part connects Doc and SsrA with TEV site. When there is no TEV in the cell, toxin protein will be degraded; after TEV is produced, it can function normally. In our biosafety switch, this design will reduce the loss of plasmid due to the leaky expression, thus reducing the escape rate.
Characterization
We can see that the leakage of DOC toxin protein is the cause of high escape rate. To improve the performance of cold-inducible kill switch, we chose to reduce the leaky expression of Doc toxin by fusing a hybrid tevS/ssrA tag to the C-terminal domain of Doc toxin (Figure 1c). The ssrA tag is used for proteasome-dependent degradation of Doc toxin by endogenous proteases ClpAP and ClpXP42, while tevS for the proteolytic removal of ssrA tag. When the temperature is lower than 34°C, TEVts, as a component of cold-inducible switch, will be highly expressed, and cleave tevS to release ssrA tag from Doc protein. By contrast, as the expression of TEVts is repressed by cI434ts at 37°C, the ssrA tag cannot be removed and Doc toxin is degraded.
Figure 1. The construction and characterization of cold-inducible kill switch. (a) Genetic circuit design of cold-inducible kill switch ver1.0. The protease tevts is cloned under pR to a p15A plasmid. The transcription factor is cloned under a strong promoter J23119 with toxin doc cloned under pR downstream to a pSC101 plasmid. (b) The escape rate of bacteria on solid LB plate after 20h. The escape rate is calculated as below: per Escape Frequency = (Colonies on nonpermissive plate ∙ dilution) / (Colonies on permissive plate ∙ dilution) 19. The escape rate of three groups are (1×107)/(2×108)=5×10-2 (i) ,(11×107)/(6×1010)=1.83×10-2 (ii) and (2×107)/(16×109)=1.25×10-3 (iii) respectively. And the Escape Frequency = Average Escape Frequency ± standard deviation = 2.318 ×10-2 ± 0.122.
Figure 2.The construction and characterization of double-status switch. (a) Genetic circuit design of the double-status switch. The heat-inducible switch that has mRFP as the output is placed on the same p15A1 plasmid as the protease TEVts.
We characterized this advanced version of kill switch (Doc-SsrA) in E. coli TOP10 and EcN at different temperatures. The escape frequency of TOP10 Doc-SsrA and EcN Doc-SsrA at 25°C were (2.68±1.56)×10^(-6) and (1.47±0.78)×10^(-6) respectively, which were comparable to the lowest escape frequency achieved by previously reported thermal sensitive kill switch, much lower than Doc without SsrA. Similarly, the escape frequency of TOP10 Doc-SsrA and EcN Doc-SsrA at 30°C were (1.13±0.68)×10^(-5) and (9.78±7.34)×10^(-6), two to three orders of magnitude lower than 10^(-3) described in previous research by Stirling et al(Fig. 1). Growth curves in liquid culture also showed that TOP10 Doc-SsrA and EcN Doc-SsrA suffered significant growth arrest at both 25°C and 30°C (Fig. 2).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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