Difference between revisions of "Part:BBa K2036033"

Revision as of 05:32, 25 October 2016

PP2CA-TTADH1-pCyc-Kozak-SnRK2.2-TTADH1-pCyc

Part of genetic circuit in the eukaryotic version of signal filter ([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]).
We construct this circuit to verify the interaction between PP2CA(BBa_K2036005) and SnRK2.2(BBa_K2036003) especially the inhibiting effect of PP2CA to SnRK2.2

Fig1:Charactor rating circuit of PP2CA and SnRK2.2

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 (Fig2). 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.

Fig2: SDS PAGE

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE 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 BamHI site found at 1816
Illegal BamHI site found at 2473
Illegal XhoI site found at 65
Illegal XhoI site found at 875
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 48
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 <br>
 We construct this circuit to verify the interaction between PP2CA([https://parts.igem.org/Part:BBa_K2036005 BBa_K2036005]) and SnRK2.2([https://parts.igem.org/Part:BBa_K2036003 BBa_K2036003]) especially the inhibiting effect of PP2CA to SnRK2.2<br>
-[[File:T--HUST-China--Experiments-Fig4.png|thumb|900px|center|Charactor rating circuit of PP2CA and SnRK2.2]]
+[[File:T--HUST-China--Experiments-Fig4.png|thumb|900px|center|Fig1:Charactor rating circuit of PP2CA and SnRK2.2]]
 <br>
 <h3> Protein expression</h3>
 <p>
-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.
+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 (Fig2). 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.
 </p>
-[[File: T--HUST-China--SDS.png |thumb|800px|center|Fig: SDS PAGE]]
+[[File: T--HUST-China--SDS.png |thumb|800px|center|Fig2: SDS PAGE]]
 <br>
 <h3> Bi-stable function:</h3>