Part:BBa_K2036030
PP2CA-TTADH1-pCyc-Kozak-Snrk2.2-TTADH1-pCyc-Kozak-ABF2-TTADH1-prd29A-Kozak-GFP-LVAssrAtag
ABF2 can be phosphorylated by Snrk2.2 to activate promoter RD29A and thus starting the expression of the target gene and also forming a positive feedback system. ABF2 can be dephosphorylated by PP2CA to stop the whole process, making the bi-stable state of our circuit.([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki])
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 trasnfected cells all expressed proteins at ~80kD under induction. The shift of protein bands suggested potential glycosylation modification.
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 more details in our Eukaryote circuit modeling.
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 PstI site found at 3756
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 3756
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 90
Illegal BglII site found at 3824
Illegal BamHI site found at 661
Illegal BamHI site found at 806
Illegal BamHI site found at 918
Illegal BamHI site found at 1816
Illegal BamHI site found at 2473
Illegal XhoI site found at 65
Illegal XhoI site found at 875 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3756
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3756
Illegal AgeI site found at 48 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 5268
Illegal BsaI.rc site found at 5939
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