Part:BBa_K2036031
PP2CA-TTADH1-pCyc
It is one of the Basic Part in the eukaryotic version of signal filter (BBa_K2036030)and it is constructed by In-Fusion method.
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. ([http://2016.igem.org/Team:HUST-China/Model/model-euk See more details in our Eukaryote circuit modeling]).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 90
Illegal BamHI site found at 661
Illegal BamHI site found at 806
Illegal BamHI site found at 918
Illegal XhoI site found at 65
Illegal XhoI site found at 875 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 48
- 1000COMPATIBLE WITH RFC[1000]
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