Part:BBa_K3221104
promoter GAL1, CNS1-linker-CNS2 coding sequence, V5 tag, 6xHis tag and CYC terminator.
CNS1 and CNS2 are two enzymes responsible for the synthesis of COR(cordycepin). The two enzymes were found by CAS Key Laboratory of Insect Developmental and Evolutionary Biology. It was described that Cns2 catalyzes 3'-AMP to 2'-C-3'-dA and then CNS1 catalyzes 2'-C-3'-dA into COR. So this part is able to express enzymes needed for catalyze 3'-AMP into COR. This part allow the two enzymes express together as a fusion protein.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 4045
Illegal SpeI site found at 1266
Illegal SpeI site found at 1795
Illegal SpeI site found at 2449 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 1266
Illegal SpeI site found at 1795
Illegal SpeI site found at 2449 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1843
Illegal BglII site found at 2430
Illegal BglII site found at 2848
Illegal BamHI site found at 2932
Illegal XhoI site found at 4039 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 4045
Illegal SpeI site found at 1266
Illegal SpeI site found at 1795
Illegal SpeI site found at 2449 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 4045
Illegal SpeI site found at 1266
Illegal SpeI site found at 1795
Illegal SpeI site found at 2449
Illegal AgeI site found at 150
Illegal AgeI site found at 1909
Illegal AgeI site found at 4108 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology:
Cns1 and Cns2 are two enzymes responsible for the synthesis of COR(cordycepin) in the medicinal fungus Cordyceps militari. The two enzymes were found by CAS Key Laboratory of Insect Developmental and Evolutionary Biology. It was described that Cns2 catalyzes 3'-AMP to 2'-C-3'-dA and then Cns1 catalyzes 2'-C-3'-dA into COR. So, this part is able to express enzymes needed for catalyze 3'-AMP into COR. We want to demonstrate that express these two enzymes together can improve the efficiency of the reaction, so we constructed this part. The two enzymes are able to be expressed together but separately in this part. There are one GAL1 promoter and one T7 promoter locate upstream of each gene. One 6×His tag and one CYC terminator are located downstream of each gene. There is a V5 tag downstream of CNS2 as well.
Results:
Plasmid construction:
We construct plasmid pYES2-cns1-Cns2 by double enzyme digestion and ligate gene cns1 and cns2 to backbone pYES2-NTA. Then we transformed the constructed plasmid into E.coli DH5α for amplification. Plasmid extraction, double enzyme digestion confirmation and sequencing were carried then to verify the correct ligation(Figure.1.).
Confirm that Cns1 and Cns2 were successfully expressed in Saccharomyces Cerevisiae BY4741:
After the construction of plasmid pYES2-cns1-cns2, we must detect that whether the two enzymes are able to express in Saccharomyces Cerevisiae. Therefore, we transform the plasmid PYES2-cns1-cns2 into Saccharomyces Cerevisiae BY4741 and induced the expression by galactose. Furthermore, we performed SDS-PAGE and Western Blot to detect the protein expression of the two enzymes in yeast lysate(Figure.2.).
Demonstrate that enzymes Cns1 and Cns2 are functional in Saccharomyces Cerevisiae:
In order to verify that the two enzymes expressed in Saccharomyces Cerevisiae is able to catalyze 3'-AMP into COR, we carried out HPLC (High Performance Liquid Chromatography), a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture, to test the reduction of the substrate and the increment of the product.
//function/biosynthesis
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