Lambda Red Mediated Recombination Recombineering Notes

Improving Efficiency of Red Recombination

Three general approaches:

  • a) Engineering of genes related to DNA repair systems and DNA degradation.

    • MutS is a mismatch repair protein. Removal of MutS improves recombination efficiency.

    • MutS recognizes and binds to mismatched base pairs in DNA, initiating a repair process that can remove the newly introduced mutation. By removing MutS, the repair mechanism is disabled, allowing the desired mutation to be retained.

  • b) Inhibiting nucleases involved in oligonucleotide degradation

    • Nucleases degrade single-stranded DNA (ssDNA), which are intermediates in Red recombination. Inhibiting these nucleases increases the concentration of ssDNA, leading to higher recombination efficiency.

    • Examples of nucleases to inhibit include RecJ and ExoI. Specific inhibitors or mutations in these nucleases can be used.

  • c) Modified DNA to be introduced: Not recognized by nucleases

    • Modifying the DNA substrate to be introduced into the cell can protect it from degradation by nucleases. This can be achieved through chemical modifications or by using non-natural nucleotides.

Recombinase-Mediated Cassette Exchange/Removal

  • Site-specific recombinases:-

    • Catalyze reversible sequence-specific recombination events between two short, identical sequences.

    • Derived from prokaryotes, unicellular yeasts, and bacteriophages.

    • Mediate efficient “cut and paste”-type DNA exchange between recognition sites in the range of 30–40 bp or longer.

    • Two families: Tyrosine recombinase and Serine recombinase

    • Best studied are the Tyrosine-type Cre and Flp

    • Detailed Explanation of Site-Specific Recombinases:

    • Site-specific recombinases are enzymes that recognize and bind to specific DNA sequences (recognition sites) and catalyze the exchange of DNA strands between these sites.

    • This process is highly precise and efficient, making it valuable for genome engineering.

    • The reaction is reversible, allowing for both insertion and excision of DNA segments.

    • The recognition sites are typically short, ranging from 30 to 40 base pairs, and are unique to each recombinase system.

    • Two main families of site-specific recombinases are:

    • Tyrosine recombinases: These recombinases, such as Cre, form a covalent intermediate between a tyrosine residue in the enzyme and the DNA. The reaction involves two sequential single-strand cleavages and strand exchanges.

    • Serine recombinases: These recombinases, such as ΦC31 integrase, also form a covalent intermediate, but with a serine residue. They typically catalyze unidirectional recombination.

    • Cre/loxP and Flp/FRT are the most extensively used systems due to their