Difference between revisions of "Part:BBa K5143025"
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Revision as of 13:33, 1 October 2024
Plasmid D
Description
Our team has nicknamed this plasmid “plasmid D”. This part was designed to be used in Saccharomyces cerevisiae . Its main components are fwYellow (BBa_K5143023) and Cp19k-MaSp1
(BBa_K5143022) fused together (BBa_K5143024).
By digesting this part with XhoI restriction enzyme, the linearized fragment could be transformed into the yeast in order to recombinate with the Ura locus in S. cerevisiae BY4741 strain. Then, the yeast will express the alphafactor-fwYellow-CBD-P2A-alphafactor-MaSp1-CBD gene. P2A (BBa_K5143012) system enables the ribosomal-switch on the mRNA, and leads to the formation of two different proteins : alphafactor-fwYellow-CBD and alphafactor-Cp19k_MaSp1-CBD.
In our project, these two proteins will be secreted by the yeast and will bind to the cellulose (thanks to the fused CBD) in order to functionalize the cellulose.
Construction
The codons were optimised for synthesis and expression in Saccharomyces cerevisiae .
The pUC57 backbone was synthesized with the alphafactor-fwYellow-CBD sequence in it. Then, this plasmid was linearised by PCR in order to clone the alphafactor-Bioglue-CBD in it. The alphafactor-Bioglue-CBD had been previously synthesized. Afterwards, plasmids obtained in clones has been sequenced in order to verify the insertion of the Bioglue fragment in the plasmid.
This composite part is composed of the following parts:
- the GAPpromotor-alphafactor-fwYellow-CBD-P2A-alphafactor-Cp19k_MaSp1-CBD BBa_K5143024
- the pUC57 backbone BBa_K5143005