Lambda-red mediated recombination using ssDNA

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Lambda-red mediated recombination using ssDNA

Lambda-red mediated recombination using ssDNA can be used to introduce precise mutations into the genome of Streptomyces strains. These mutations are usually to introduce single nucleotide changes (e.g. to introduce codon changes to alter amino acids or introduce premature stop codons), or small insertions or deletions. This method relies on the ssDNA recombineering protocols developed for Escherichia coli, and subsequent introduction, and homologous recombination, of the mutated locus into the Streptomyces strain of interest.

ssDNA recombineering relies on the high levels of recombination seen using lambda-red mediated recombination with short oligonucleotides that introduce mutations but avoid repair by the methyl-directed mismatch repair (MMR) system [1]. This occurs at such a high frequency that selection for the desired mutation is not necessary: instead, a suitable screen (e.g. a diagnostic PCR) can be used to identify the recombinants.

Thus, using a suitable MMR-deficient strain that expresses the lambda recombinase functions, you can introduce unmarked mutations into a Streptomyces gene or DNA sequence of interest; these are then subsequently transferred into your Streptomyces strain of interest. For a comprehensive guide to ssDNA recombineering, see the helpful guides and protocols published by the Court lab.

Organisms

This protocol has been confirmed to work for the following-

Materials Needed

  • E. coli strains:
    • A suitable recombineering strain, such as HME68 (deficient in MMR and capable of inducible lambda red recombinase expression) - see Court lab strain list
    • DH5α or another strain suitable for cloning
    • ET12567/pUZ8002
  • Your Streptomyces strain(s) of choice
  • Oligonucleotides for introducing your desired mutation(s), screening for the mutant allele(s), and amplifying and sequencing the allele(s)

Workflow Overview

1. Identify desired mutation and design suitable oligonucleotides to create this mutation. Design a suitable screen that will allow you to distinguish between wild-type and recombinant alleles. Design primers that will allow you to sequence the allele.

2. Identify a suitable cosmid or plasmid carrying the Streptomyces DNA that you wish to mutate.

3. Transform this cosmid/plasmid into E. coli strain HME68 using an appropriate method.

4. Perform ssDNA-recombineering to introduce your desired mutation(s) into your cosmid/plasmid.

5. Screen for successful recombinants, and purify cosmid/plasmid by miniprep.

6. At this point, you have a mixed population of recombinant/wild-type cosmids, and need to isolate the cosmids that carry the mutant allele. To do this, transform recombinant cosmid/plasmids into DH5α, and screen again for recombinant cosmids.

7. Purify your recombinant cosmid/plasmid by miniprep.

8. Confirm the sequence of your gene/DNA sequence of interest: amplify it by PCR and use Sanger sequencing.

9. Transform finished construct into ET12567/pUZ8002 cells.

10. Use your ET12567/pUZ8002,cosmid strain to conjugate your mutant allele into your desired Streptomyces strain, and select for ex-conjugants.

11. Restreak ex-conjugants several times, which will allow the wild-type/mutant alleles to assort into single copy such that you have exconjugants with either the wild-type or the mutant allele.

12. Screen for exconjugants carrying the mutant allele, and confirm the correct sequence (and absence of the wild-type sequence) by PCR amplification of the gene and Sanger sequencing.


Detailed Protocols

Transform mutant plasmid/cosmid into ET12567/pUZ8002 cells using electroporation

1. Many Streptomyces strains contain a methyl-sensing restriction system therefore disrupted cosmids must initially be passaged through a non-methylating E. coli strain


Conjugate ET12567/pUZ8002,cosmid into Streptomyces

Also see Conjugation using ET12567/pUZ8002


References

[1] Costantino, N., & Court, D. L. (2003). Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proceedings of the National Academy of Sciences of the United States of America, 100(26), 15748–15753. doi:10.1073/pnas.2434959100


Protocol developed & written by Dr. Morgan Feeney, John Innes Centre, based on the Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect") and the Court lab protocols for ssDNA recombineering.