Question

What is site directed mutagenesis and how is it used in research?

How does LacZ fragment complement the competent bacteria’s genome and result in different colored colonies?

Answer 1

Site-directed mutagenesis is a method where specific and intentional changes within the DNA sequence of a gene and any gene products is made. It is also known as site-specific mutagenesis or oligonucleotide-directed mutagenesis,

This method is extensively used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and also for protein engineering. Specific mutations in DNA induce the function and properties of a particular DNA sequence or a protein to be investigated in a rational approach. Moreover, single amino-acid changes by site-directed mutagenesis in proteins also help to understand the importance of post-translational modifications. On the other hand  Proteins may be engineered to produce mutants forms that are destined for a specific application.

Blue-white screening is an efficient technique for the identification of recombinant bacteria. It depends on the activity of β-galactosidase, occurring in E. coli, which cleaves lactose into glucose and galactose. The presence of lactose triggers the lacZ operon in E. coli and this results in the production of β-galactoisdase enzyme which metabolizes the lactose. Most of the plasmid vectors carry a short segment of lacZ gene that contains coding sequence for the first 146 amino acids of β-galactosisdase. The host E. coli competent cells contains lacZΔM15 deletion mutation. When the plasmid vector is uptaken by such cells, due to α-complementation process, a functional β-galatosidase enzyme starts to be synthesized. The plasmid vectors are manipulated in a way that this α-complementation process serves as a marker for recombination. A multiple cloning site (MCS) is present within the lacZ sequence in the plasmid vector which can be nicked by restriction enzymes to insert the foreign DNA. When a plasmid vector containing foreign DNA takes entry within the host E. coli, the α-complementation is not occured, therefore, a functional β-galactosidase enzyme is not synthesized. If the foreign DNA is not inserted into the vector DNA or if it is inserted at a location other than MCS, the lacZ of the  plasmid vector complements the lacZ deletion mutation in the host E. coli therefore produces a functional enzyme.