Part:BBa_K3332079

pBAD/araC-Inverter-EYFP-terminator

It can express EYFP without arabinose and express little EYFP induced by arabinose.

Usage and Biology

This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity of induced and non-induced to characterize the function of the inverter.

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 pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by EcoR I and Pst I

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 2 mL arabinose stock solution into the induction group when OD600 increased to 0.6.

5.Induce for 6 hours and the condition is the same as before.

6.Then, sampling culture in 96-well plate reader to measure the fluorescence intensity (EYFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.

Here is the result:

We discovered that the downstream gene of proBAD/araC can be expressed in the presence of arabinose and decrease expression without arabinose. When the inverter is added to the circuit, the effect is reversed.

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