Part:BBa_K2036016

pR-GFP-LVAssrAtag

It is a GFP generator. pR is a lytic promoter with two binding sites OR1 and OR2 which can be recognized by CI. The circuit is built to characterize CI and pR interaction together with test group: CI-TT-pR-GFp-LVAssrAtag (BBa_K2036017).

Fig1:CI and pR interaction characterization circuits

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 761
  

Protein&promoter

--CI and pR

CI is a repressor from bacteriophage lambda. To test its interaction with pR promoter, we constructed CI-TT-pR-RBS-GFP-LVAssrAtag-PET-Duet-1 and take pR-RBS-GFP-LVAssrAtag-PET-Duet-1 as control to test its inhibition function.

Fig2: As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR in minor degree but the leakage expression under pR can’t be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit.

Fig3: We also detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification (seen from the figure below). From figure we can find the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI to pR from a more intuitive way.

Preliminary experiments of LVAssrA-tag

In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used plac from the Rgistery (BBa_J04500) to characterize the degradation tag LVAssrA. We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.

Fig4: From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein.