Part:BBa_K2036027
pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag
This is the whole characterization circuit of Signal Filter prokaryote version([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki ]) as Fig1 shows.
Here is how we conduct our characterization: We employ T7 and ptrp as our input sensors and BL21 as host strain. When induced by IPTG,Cro and CII are expressed under T7 promoter and CII will give a positive feedback to enhance Cro's expression by promoting pRE. Accumulated Cro will stably bind to the bingding site within pRM thus repress GFP and at the same time RFP expression will not be interrupted (see to Fig2)
When IAA is added, CI will be expressed to further block pR. And with CII's degradation by Ftsh, GFP expression will gradually comes up to a stable state. Meanwhile, RFP level will decrease by protease degradation system through LVAssrA tag. (see to Fig3)
Protein&protein reaction
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.
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.
Protein&promoter
CII (BBa_K2036000) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.
According to the Flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK. We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.
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.
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.
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.
Tri-stable function
Ptrp2 (BBa_K1592024) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.
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.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 717
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 717
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 717
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 717
Illegal AgeI site found at 2533
Illegal AgeI site found at 2645 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3561
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