Difference between revisions of "Part:BBa K2036020"

Revision as of 16:19, 19 October 2016

RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag

This is a part of Prokaryote version of Signal Filter （[http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]）characterization circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag (BBa_K2036027).

Fig1:Prokaryote version of Signal Filter characterization circuit

Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.

Fig2:Prokaryote version of Signal Filter characterization plasmid

We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015). These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.

Fig: FtsH degradation test of CIII

According to the Flourescence measurement curve above, we can see clearly that GFP level of CIII test circuit increased over time and it showed significant difference from two control groups. It indicates that tandomly expressed CIII can efficiently protect CII from being degraded by Ftsh.

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

Fig: CI-pR inhibition test

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.

Fig: Fluorescence detection

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.

Fig: LVAssrAtag degradation rate measurement under plac

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.

Sequence and Features