Eukaryote
Protein expression
Phicia pastoris is typical Eukaryotic expression host, but expressing plant genes is still a big challenge. So we firstly test whether our three interest genes can be efficiently expressed. We measured the Exogenous expression of PP2CA, ABF2 and SnRK2.2, We found there is no significant difference between the three proteins and it is worth noting that PP2CA level is less than the other two. But considering that PP2CA is a phosphorylase, the typical enzymatic reaction rate is fast enough to reduce the effect brought by the amount deficiency.
Fig1
Fig2
And then, we ran a SDS-PAGE to identify the three proteins(Fig2). The left figure shows that our target bands are shifted to the length between 72kD and 95kD. We supposed that the protein had been glycosylated
Fig3
Fig4
Bi-stable function:
We constructed expression plasmid and submitted this part (BBa_K2036030) to the registry. But on account of long-term , we didn’t have enough time to test its function. But our modeling simulated the switch functions. See to Eukaryote circuit modeling
Prokaryote
Protein&protein reaction
--CIII and Ftsh
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. But we met problems transfer the segment from pSB1C3 to pET-Duet-1 expression plasmid. So we were late to put the result on wiki, but we would show it at the Jamboree.
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.And 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. And 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 to some degree but the leakage expression under pR can not 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).
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
Seen from the figure, we are sorry to find the serious placI expression 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.
Application
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Beta-galactosidase activity:
more details
Owing to limitation of time, we didn’t test 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.
