Composite

Part:BBa_K2969046

Designed by: Liangchen Zhuo   Group: iGEM19_UCAS-China   (2019-10-16)


pCI434-doc

This part contains the coding gene of toxin doc protein under the promoter of CI434 pCI434. When there exists the repressor CI434, the transcription of doc will be inhibited. But when the doc gene is expressed, the growth of the host will be repressed and a self-kill mechanism will be started.


Characterization

Cold-inducible On-switch + Toxin

One way to address the biosafety issue is using toxin-antitoxin pairs. Toxin is a kind of protein which is the output of the genetic circuit. Under certain condition such as temperature, toxin begins to be expressed by the engineered bacteria and eventually kill those bacteria.


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In our engineering bacteria, we used Doc, a toxin interferes with basic metabolism at the level of translation, to associate with our cold-inducible on-switch, so that under low temperature, the switch is turned on to express Doc protein.


To explore the possibility of escape for evaluating the performance of our kill switch, we measured the growth of the bacteria with DOC gene in LB solid medium at different temperatures and dilutions. The results showed that the strain cultured at 25℃ grew much worse compared with the strain grown at 37℃, and the 20h average solid escape frequency is 2.318Γ—10-2 , the lowest escape rate can be limited to 10-3 level.



T--UCAS-China--biosafety3.jpeg
T--UCAS-China--biosafety4.jpeg
T--UCAS-China--biosafety5.jpeg
Figure 1. The escape rate of bacteria on solid LB plate after 20h. The escape rate is calculated using the formula 𝒑𝒆𝒓 𝑬𝒔𝒄𝒂𝒑𝒆 π‘­π’“π’†π’’π’–π’†π’π’„π’š =(π‘ͺπ’π’π’π’π’Šπ’†π’” 𝒐𝒏 π’π’π’π’‘π’†π’“π’Žπ’Šπ’”π’”π’Šπ’—π’† 𝒑𝒍𝒂𝒕𝒆 Γ— π’…π’Šπ’π’–π’•π’Šπ’π’)/(π‘ͺπ’π’π’π’π’Šπ’†π’” 𝒐𝒏 π’‘π’†π’“π’Žπ’Šπ’”π’”π’Šπ’—π’† 𝒑𝒍𝒂𝒕𝒆 Γ— π’…π’Šπ’π’–π’•π’Šπ’π’) , the escape rate of 3 groups are (1Γ—10^7)/(2Γ—10^8 )=5Γ—10^(-2) , (11Γ—10^7)/(6Γ—10^10 )=1.83Γ—10^(-2) and (2Γ—10^7)/(16Γ—10^9 )=1.25Γ—10^(-3) respectively(from above to below). And the 𝑬𝒔𝒄𝒂𝒑𝒆 π‘­π’“π’†π’’π’–π’†π’π’„π’š = π‘¨π’—π’†π’“π’‚π’ˆπ’† 𝑬𝒔𝒄𝒂𝒑𝒆 π‘­π’“π’†π’’π’–π’†π’π’„π’š Β± 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅 π’…π’†π’—π’Šπ’‚π’•π’Šπ’π’ =2.318 Γ—10^(βˆ’2) Β± 0.122.

Moreover, we measured the growth curve of the strains with DOC gene at different temperatures to characterize their growth in liquid medium, and took the strains without doc gene as the control to verify the effectiveness of the toxin system. The results showed that the growth of the two strains was almost the same at 37℃, indicating relatively low leakage of the system and low while the growth of the strains with doc gene was worse at 25℃, indicating that the system with DOC as toxin was effective.

Figure 2:The growth curve of bacteria on liquid LB. Incubated in 4 mL volume in 24-deep-well plate.


Based on the design of our cold-inducible switch and integrating toxin Doc, we developed a β€˜kill switch’ which sensitively and accurately responds to the natural signal of human body and the environment – temperature. We successfully limit the escape rate to 10-2 to 10-3. Considering the improvement of the robustness and performance of the whole system, also to ensure the ability of our ark to adapt to various situation with as little risk for both human and nature as possible, we design another strategy as follow.



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]


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