Part:BBa_K3332079

pBAD/araC-Inverter-EYFP-terminator

It can express EYFP without arabinose and express little EYFP with arabinose.

Usage and Biology

Fig 1. pBAD_inverter_eyfp_B0015(BBa_K3332079)

This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity/OD₆₀₀ of induction and non-induction bacteria, we managed to characterize the function of the inverter(BBa_Q04510).

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 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 (about 3000bp)

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 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 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:

Fig 3. Fluorescence intensity/OD₆₀₀ for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

We found that the downstream gene of proBAD/araC can be expressed when induced by arabinose, while the expression of EYFP decreases without the inducer. Besides, as we can see, the purple bar is higher on the right side(when the arabinose is absent). Therefore, we can come to the conclusion that when the inverter(BBa_Q04510) is added, 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