Eukaryote
Protein expression
Pichia pastoris are typical Eukaryotic expression host, but expression of plant genes is still a big challenge. So we firstly tested whether our three interest genes can be efficiently expressed. We measured the secretion of PP2CA, ABF2 and SnRK2.2. We ran a SDS-PAGE of the culture supernatant of induced cells to identify the expression of three recombinant proteins(Fig1). Compared to transformed cells, three kinds of recombinant protein plasmids transformed cells all expressed proteins at 80kD under induction. The shift of protein bands suggest potential glycosylation modification.
Fig1：Exogenous expression of PP2CA, SnRK2.2 and ABF2. Samples were collected from GS115 fermentation supernatant.
We analyzed our protein by SDS-PAGE，this picture is our result. From left to right,DNA marker,Wild type GS115,Wild type GS115,vector,PP2CA,ABF2,SnRK2.2.
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. See to Eukaryote circuit modeling (click here).
Prokaryote
Protein&protein interaction
We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014) and RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036013) These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.
Fig2: GFP fluorescence measurement over time gradient,excitation 485nm,emission 520nm
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. Further more, we continued constructing RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015) as control group to explore a better relative quantity between CIII and CII.
Protein&promoter interaction
CII and pRE:
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.
Fig3: GFP fluorescence measurement over time gradient,excitation 485nm,emission 520nm
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.
Fig4: GFP fluorescence detection under with 20*10 magnifying power
CI and pR:
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.
Fig5: GFP fluorescence measurement over time gradient,excitation 485nm,emission 520nm
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.
Fig6: GFP fluorescence detection under with 20*10 magnifying power
Cro and pRM:
Fig7: GFP fluorescence measurement over time gradient,excitation 485nm,emission 520nm
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
Preliminary experiments of ptrp2:
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.
Fig8: GFP fluorescence measurement over time gradient,excitation 485nm,emission 520nm
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 485nm.
Fig9: LVAssrAtag degradation rate measurement under placI
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.
Application
Beta-galactosidase activity:
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.We tested enzyme activity of our strain cultured at pH6.5, 7.5 and 8.5.
We tested enzyme activity of our strain cultured at pH6.5, 7.5 and 8.5.
Fig10: beta-galactosidase activity measurement after cultivation at 6.5, 7.5, 8.5 overnight.
As the data shows, beta-galactosidase activity of our strain cultured at pH8.5 was significantly higher than the other two groups which is corresponding to our expectations: When pH comes back to 7~9, our strain will sense the change and express beta-galactosidase.
For the results of Interlab and previous improvement,please click here
