Part:BBa_K3332083

pBAD/araC promoter-RBS-MazF-terminator

It can express MazF induced by arabinose.It is used to characterize the function of the MazF.

Usage and Biology

Fig.1 Circuit

The application of proBAD/araC-RBS-MazF was to monitor the protein toxicity of MazF, under the induction of arabinose, to accomplish the toxicity modeling and characteristic inverter/MazF.

Characterization

When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing. After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the EcoR I and Pst I to cut the plasmid, then we 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 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:

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