Question

Describe how you can use PCR to obtain a gene fragment X that can be cloned into a
plasmid vector for expression of the recombinant protein. Include in your description
how the PCR reaction will be performed, how the amplicon will be analyzed, the use of
restriction enzymes, how cloning will be achieved, how you will know that you have a
recombinant containing the insert gene fragment X and how you will detect the
recombinant protein.

Answer 1

We will clone the gene in the following steps

1) amplification of gene if interst

First we will amplify the gene of interst using specific primers of the gene of interst. We will design the priners in such a way that , they must contain the restriction enzyme site present in the plasmid ( in which we have to insert the gene).

2) after amplification we will digest the gene as well as plasmid by using same enzyme, this will produce sticky ends and help in ligation of both the molecules and lead to formation of recombinant DNA ( plasmid + gene).

3) after transformation, E coli cell containg desired plasmid will be recognized by alpha complementation.

Plasmid has Lac alpha gene which code for alpha protein under Lac Z promoter.

Bacteria in which transformation done code for beta part of galactosidase gene. So alpha part produce by plasmid interact with beta part and form functional beta galactosidase gene. When these bacteria grow in media containing X gal substrate ,in presence of lactose will produce blue colonies. Here functional bets galactosidase gene act on X gal convert it in to blue colour.

If we insert a gene in lac alpha gene which contains MCS ( multiple cloning site). Lead to loss of formation of alpha subunit and no beta galactosidase gene and no formatio of blue colour leads to white colonies. So this is a single step selection, blue colonies represent non recombinant plasmid and white colonies represent recombinant plamid has gene of interst.

This whole process has shown in attached image below

This has been explained below