Answer to Question #191325 in Molecular Biology for Hasibul Badhan

Question

Describe how you can clone the gene encoding the protein R in the vector pQE-70. Your description should also indicate the choice of restriction enzymes that can be used, the process of screening of E.coli cells harbouring the recombinant plasmid.

Answer 1

PCR amplification of specific gene encoding the protein R, Gel Extraction of PCR amplicons of the specific gene, Restriction digestion of PCR amplified gene fragment (without affecting the coding region of the gene) using the restriction enzyme which is present in MCS site of the vector pQE-70.

Restriction digestion of vector DNA, pQE-70 using the same restriction site which is used for PCR amplified gene product, Ligation of restricted PCR amplified gene fragment and restricted pQE-70 vector DNA

Preparation of competent E. coli DH5α cells follows, Transformation of gene is then inserted into vector DNA into the intermediate host, E. coli DH5α

Confirmation of a specific DNA fragment in pQE-70 vector DNA - recombinant plasmid is in E. coli DH5α strain. A his-tag, present in pQE-70 vector DNA is a string of histidine residues at either C or N terminus of a recombinant protein. His-tag has a high affinity for these metal ions and binds strongly to the IMAC column. Whereas, other proteins in the lysate will not bind to the resin, or bind only weakly. Hence, after cell lysis, the lysate obtained could be used to screen the expressed protein through his-tag in the IMAC column.