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Revision as of 17:23, 27 October 2020


pBAD/araC promoter-Inverter-mazF-terminator

It can express toxin protein according to the concentration of arabinose.It ensures that our engineering bacteria die when the concentration of glyphosate is low to a certain level.


Usage and Biology

Fig 1. pBAD_inverter_mazF_B0015

This circuit was designed to test MazF expression under inverter and inducible promoter: proBAD/araC, which in comparison with proFormaldehyde_inverter_MazF as well as proBAD/araC_MazF, ratify inverter function by arabinose promoter. Besides, it is used as the kill switch for our engineering E.coli for detection of glyphosate. When the engineering E.coli escape to the environment without arabinose, it will be killed.

Characterization

When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. We used theEcoR I and Pst I to cut the plasmid, then we got the target separate fragment.

caption

Fig.2 proBAD/araC-B0034-MazF-terminator _pSB1C3 (BBa_K3332083) colony PCR (about 2687 bp)

Protocol

1. Preparation of stock solution

Dissolve arabinose in ddH2O to make 100× stock solution（the work concentration is 0.2%）

2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.

3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 2mL arabinose stock solution into the induction group when OD600 increased to 1.0

5.Induce for 18 hours and the condition is the same as before.

6.Then, sampling 5µL culture to dilute 106 times and take 50µL diluted solution to spread across a petri plate.

7.After 14 hours, count the number of groups of E. coli

Here is the result:

Fig 3. The CFU of two engineering bacteria under induced and uninduced condition


We discovered that the number of live E.coli of pBAD/araC_inverter_mazF(without induction) and pBAD/araC_mazF(induction) decreased significantly after 8 hours, while there was no significant change in pBAD/araC_Inverter_mazF(induction) and pBAD/araC_mazF(without induction), which demonstrate that pBAD/araC_inverter_mazF has the killing function when arabinose is absent. So we can use the kill switch to prevent the engineering E.coli escaping. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961