Lambda-red mediated recombination using ssDNA

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.

The ssDNA recombineering protocol relies on the high levels of recombination seen using lambda-red mediated recombination with short oligonucleotides in the absence of the methyl-directed mismatch repair (MMR) system^[1]. An oligonucleotide carrying the desired mutation is transformed into an MMR-deficient recombineering strain that carries the Streptomyces gene on a cosmid. This recombination using the oligonucleotide occurs at such a high frequency that selection for the desired mutation is not necessary: instead, a suitable screen (such as a diagnostic PCR) can be used to identify the recombinants.

Once the mutant allele(s) have been generated in E. coli, you can introduce the unmarked mutations into a Streptomyces strain of interest using intergeneric conjugation. Once a double cross-over has occurred, exconjugants will have either the wild-type or the mutant allele (in theory, if the mutated gene is near the centre of the cosmid, ~50% exconjugants should have the wild-type and ~50% should have the mutant allele). This process is essentially "scarless" genome editing as the resulting strains do not have any antibiotic resistance markers, or any other changes apart from the desired mutation(s).

For a comprehensive guide to ssDNA recombineering in E. coli, see the helpful guides and protocols published by the Court lab. For a guide to the Streptomyces "Redirect" method, see the helpful guides and protocols available on StrepDB.

Organisms

This protocol has been confirmed to work for the following-

• Streptomyces coelicolor
• Streptomyces venezuelae

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

Step 1. Mutant design

Step 2. ssDNA recombineering in E. coli

Step 3. Identify and isolate successful recombinants. Confirm correct recombination event.

Step 4. Introduce mutant allele(s) into your Streptomyces strains of interest.

Detailed Protocols

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 (usually electroporation).

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

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

6. At this point, the miniprep will contain a mixed population of recombinant/wild-type cosmids, and you will 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. (These minipreps should now contain a pure population with only the recombinant (mutant) cosmid/plasmid.)

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

9. Transform the 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.

Step 7: 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

Step 8: Introduction of mutant allele(s) into Streptomyces

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

Conjugate ET12567/pUZ8002,cosmid into Streptomyces

Also see Conjugation using ET12567/pUZ8002

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.

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.