Part:BBa_K3332083

pBAD/araC promoter-RBS-MazF-terminator

It can express mazF induced by arabinose,which is used to characterize the function of mazF.

Usage and Biology

Fig 1. pBAD _mazF_B0015(BBa_K3332083)

The application of proBAD/araC_RBS_mazF was to monitor the protein toxicity of BBa_K1096002(MazF), under the induction of arabinose, to accomplish the toxicity modeling and characterize inverter and mazF.

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 colony PCR and got the target separate fragment.

Fig 2. proBAD/araC-B0034-MazF-terminator_pSB1C3 (BBa_K3332083) colony PCR (about 2096 bp)

CFU 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 12 h.

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

4.Add 2 mL 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 50 µL 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(B1-4) and uninduced(A1-4) condition

We discovered that the number of live E.coli decreased significantly after 5 hours, while there was no significant change in the non-induced group, which can verified that MazF have the function of killing E.coli.

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