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Assignment 7 - Enzyme Expression

1- What is the genotype of JM109(DE3) cells and how does this allow the plasmid given to you in project 2 to express the b-galactosidase protein?

2- What genotypic element in the Cos-7 cells allows replication of the plasmid given to you in project 2 in these cells?

3- The following components are used in a microbial eukaryote to express proteins and export them out of the cell. Assemble the components in an order that would allow efficient transcription, translation, export from the cell as well as recovery of the secreted protein from the media. The isolated protein will then need to be able to be cleaved such that the purified protein can be recovered without the extra peptides that were not part of the original inserted fragment.

alpha factor - signal sequence for secretion of the protein from the cell.

V5 epitope that allows the easy detection of the recombinant protein with an anti-V5 antibody.

Polyhistidine (6xHis) for rapid purification of the recombinant protein from the media with Ni bound Agarose.

Enterokinase recognition site that allows cleavage of the recombinant protein.

Strong AUG1 promoter that transcribes and provides the AUG for translation.

Ampicillin resistant gene.

pUC ori for replication in bacteria.

Multiple cloning site where genes will be inserted to make recombinant proteins.

Ade2 gene for auxotrophic selection.