Part:BBa_K3332079

pBAD/araC-Inverter-EYFP-terminator

It can express cyan fluorescent protein according to the concentration of arabinose.It is used to characterize the function of the inverter.

Usage and Biology

This part was used to detect and test formaldehyde promoter expression in BL21 as well as the promoter improvement design. After formaldehyde induction, ECFP would emit fluorescence under 514 nm exciting light and indicate the strength and degree of formaldehyde promoter expression.

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-2476bp

Fig2. [BBa_K3332079] I0500_Q04510_E0430_pSB1C3 (pBAD_inverter_E0430_pSB1C3) digested by EcoR I and Pst I

Protocol:

1. Preparation of HCHO stock solution

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

3.Add 4ml of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add HCHO stock solution into the induction group（the working concentration is 0.8mM） when OD increased to 0.6. The culture condition is the same as before.

6.Then, sampling 0.5ml culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500µl sterile PBS to resuspend.

7.Measure the fluorescence intensity(ECFP)and corresponding OD600 by 96-well plate reader, then calculate the fluorescence / OD 600 value of each group.

Here is the result:

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