Part:BBa_K2036010

Cro-TT-pRM-RBS-GFP-LVAssrAtag

It is a negtive control of GFP expression. When inserted into a expression plasmid behind an inducible promoter,it will form a NO gate. Take PETDuet-1 as an example,after constructing PETDurt-1-Cro-TT-pRM-RBS-GFP-LVAssrAtag,IPTG can trigger T7 promoter in the plasmid, and cro can bind to a certain site within pRM, so that GFP will not be produced. And we get a NO gate of GFP generator.
HUST-Chian 2016 Build this circuit to test Cro and pRM interaction intensity with contol group:pRM-GFP-LVAssrAtag (BBa_K2036009)

Fig1:Cro&pRM interaction characterization circuit

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 1036
  

Protein&promoter

--Cro and pRM

Fig2: We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.

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.

Fig3: 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.