Part:BBa_K2036002
Prd29A
A promoter region upstream Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments .Promoter RD29A contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences which are cis-acting elements. So the promoter RD29A can be active by transcription factor and then start downstream genes expression.
Application:
we apply RD29A promoter into our eukaryotic filter .when transcription factor ABF2 is phosphorylated by kinase SnRK2.2 ,the RD29A promoter can be activated and promote the expression of SnRK2.2 and target gene. These make the system form a positive feedback and lead to stable expression of target gene.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 153
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 secretionexpression of PP2CA, ABF2 and SnRK2.2. We ran a SDS-PAGE of the culture supernatants of induced cells to identify the three recombinantproteins(Fig1). Compared to vector trasnfected cells, 3 kinds of recombinantprotein 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_K2036030to the registry.But due to the limited time,its function characterization is still under testing.However,our modeling simulationshowed promising switch functions.See to Eukaryote circuit modeling
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