Question

How to carry out mutations on a gene of bacteria strain with lambda red recombinase and homologous recombination?

How to clone and verify the strains?

Please explain the steps in detail.

Answer 1

Lambda red system is based on homologous recombination and is a method of cloning to directly modify DNA within E. coli. This system is independent of restriction sites.

This system is derived from the lambda red bacteriophage and is frequently used for homologous recombination-mediated genetic engineering. The step by step process include –

a) Electroplating a linear donor DNA substrate i.e either dsDNA or ssDNA in the presence of lambda red enzymes in E.coli.

b) Lambda red enzymes the catalyzes homologous recombination of a) substrate and b) target DNA sequence

c) In vivo cloning

As compared to a regular cloning method, this method differs for following -

1. Design and generation of Substrate DNA –

dsDNA substrate is best for insertions or deletions of more than 20 nucleotides

ssDNA substrate is suitable for point mutations or mutation at few base pairs

1. Expression of lambda red recombination genes

The lambda red gene can be expressed in following ways-

a) From a plasmid

b) From mini-λ

c) From the lambda red phage itself

d) From a bacteria with integrated defective prophage

1. Electroporation of substrate DNA
2. Selection and confirmation of recombinant clones. - Colony PCR is used to detect positive clones while restriction enzyme digest is used for plasmids having appropriate mutations. Sequencing is used to confirm point mutation