Question

1) Please explain how to transform bacterial cells with plasmid DNA.

2) Please explain how to purify plasmid DNA from bacterial cells.

3) How can you use PCR to clone a 2000bp gene into a plasmid vector?

4) Please explain how a fluorescent tag can be used to visualize protein localization in a cell.

Answer 1

Answer 1:

Process of bacterial transformation using plasmid:

Transformation of bacteria is the process by which its genome is modified by uptake of genes from external environment.

The steps of bacterial transformation using a plasmid involves:

1. Bacterial cells are made capable or competent to uptake recombinant plasmid , by heat shock or eletroporation. Both these methods create temporary pores in the plasma membrane of bacterial cell, through which foreign DNA molecule can enter.

2. Construction of a recombinant molecule :

This involves insertion of gene of interest into the plasmid.

It is achieved by the use of restriction endonuclease, which cuts gene of interest and plasmid DNA at specific recognition sites.

The gene of interest is then ligated to plasmid using DNA ligase enzyme.

This recombinant molecule is then screened against non-recombinants using antibiotics.

3. Transformation of bacteria:

These recombinant molecules are then allowed to transform competent bacterial cells.

Successful uptake of recombinant plasmid transforms bacteria.