Question

Construct a recombinant bacteria strain with a gene mutated. You need to design and suggest the steps. 1) cloning of gene 2) mutation (how to do it) 3) which homologous recombinant method you use 4) how to verify the strain EXPLAIN THE STEPS IN DETAIL.

Answer 1

First you need to isolate gene of your interest (without having introns if its irom eukaryotic cells). Insert this gene into plasmid forming recombinant plasmid. Transform the recombinant plasmid into bacterial cell.

There are various methods to introduce mutation in gene of interest. Such as site directed mutagenesis (in-vitro mutation), CRISPR-CAS9 mediated mutagenesis, transposon mediated mutagenesis etc.

Transposon mediated mutagenesis is based on homologous rrcombination. This can be used to introduce point Mutation in gene of interest.

methodology

You need to construct bacterial transposon containing Mutant copy of the gene (it can be done using site directed mutagenesis). the transposon can be moved to the recipient cell efficiently by transformation, transduction and conjugation. The transposon must have a selectable marker (antibiotic resistance gene for which bacteria must not having resistance previously) that functions in the recipient. The transposon must reside on a suicide vector so that the carrier is not retained in the recipient cell.

After entry of this recombinant transposon inside the cell, transposon will express transposase enzyme which will replace wild-type gene with Mutant gene in the plasmid. In addition, it will also insert new antibiotic resistance gene into plasmid so bacteria will get new antibiotic resistance.

It will help you to screen those bacteria who have Mutant gene. Colonies on plate containing that new antibiotics contains mutant gene. You can further confirm the Mutation by sequencing of that gene isolated from those colonies.