Part:BBa_K5291021

P_opdH

A promoter from opdH-tctCBA-tctDE manipulator system and able to sense peripheral concentration of citrate.

Usage and Biology

P_opdH is a promoter from opdH-tctCBA-tctDE manipulator system in Pseudomonas aeruginosa. It can be repressed by the product of tctD, a gene in P. aeruginosa, when there is no citrate in the environment. The product of tctD will priorly bind to citrate when there is citrate and the repression of P_opdH will decrease, and it can start the genes downstream. It is a useful element in our chassis organisms P. aeruginosa.
Given the short sequence of P_opdH, we innovatively use the dimerization of forward and reverse primer to synthesize the promoter with homologous arms by PCR in a template-free way.

Fig.1 The AGE figure of P_opdH with homologous arms.

To verify the function of P_opdH that activates the genes downstream based on the concentration citrate, we link the promoter in front of the gene encoding GFP, so that it is convenient to determine if the promoter is effective by testing the fluorescence intensity.
Fig.2 The AGE figure of colony PCR and the plasmid we construct.

We cultivate the engineered bacteria by LB-Amp broth with and without citrate (5mM, which is the best verification concentration from a paper) and examine the fluorescence intensity using enzyme labeling apparatus.


Fig.3 The diagram shown the difference of fluorescene intesity. (The gain we use is 60.)

We can know from the diagram that the solution rarely fluoresces; and the group without citrate has rare fluorescence compared with the group induced by citrate. The results mean that P_opdH is actually sense the concentration of citrate and activate the expression of the genes downstream. The gain we use when testing the fluorescence intensity, therefore the data look large. But the result is not influenced.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 16
  
• 1000
  COMPATIBLE WITH RFC[1000]