Eukaryote
Protein expression
Pichia pastoris are typical Eukaryotic expression host, but expressing plant genes are still a big challenge. So we firstly tested whether our three interest genes can be efficient expressed. We measured the secretion expression of PP2CA, ABF2 and SnRK2.2. We ran a SDS-PAGE of the culture supernatants of induced cells to identify the three recombinant proteins(Fig1). Compared to vector trasnfected cells, 3 kinds of recombinant protein plasmids transfected cells all expressed proteins at ~80kD under induction. The shift of protein bands suggested potential glycosylation modification.
Fig3
Bi-stable function:
We constructed expression plasmid and submitted this part (BBa_K2036030) to the registry. But due to the limited time, its function characterization is still under testing. However, our modeling simulation showed promising switch functions.
Prokaryote
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 the whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.
Protein&promoter
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--CII and pRE
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--CI and pR
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--Cro and pRM
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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
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--Preliminary experiments of ptrp2
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--Preliminary experiments of LVAssrA-tag
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Ptrp2(BBa_K2036000) 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.
According to the GFP expression curve, we choce 50μM final concentration to induce ptrp2.
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 placI 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 placI
From the figure above, we are sorry to find the serious placI expression 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 a interesting and useful tool for fastly down regulating certain target protein.
Application
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Beta-galactosidase activity:
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Due to the limited time before wiki freezing, we didn’t completed the test of lactic balance function of our engineered strain in vitro. But we tried to characterize plac-induced beta-galactosidase activity to prove that half of our bi-stable switch works.
We tested enzyme activity of our strain cultured at pH6.5, 7.5 and 8.5.
