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.

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, 2]. 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.[3]


Uses

  • Engineering single nucleotide changes at the native chromosomal locus
    • Amino acid changes, i.e. to study the function(s) of a particular amino acid
    • Nonsense mutations
  • Engineering small deletions/insertions (~20-30nt can be inserted[2]
  • Useful when working with essential genes that cannot be deleted using the standard "Redirect" method[4], or if the standard "Redirect" method may have polar effects on downstream genes


Figure 1. Illustration of two key steps in ssDNA recombineering in Streptomyces. Left, ssDNA recombineering in E. coli (Stage 2). A MMR-deficient strain of E. coli carrying the Streptomyces gene of interest, is transformed with a long oligonucleotide containing the desired mutation. Lambda-red mediated recombination occurs, generating the desired mutant allele at high frequency. Right, double cross-over in the Streptomyces strain of interest (Stage 4). The mutant allele generated in stage 2 is introduced into the chosen Streptomyces strain by intergeneric conjugation. After a double crossover occurs, the antibiotic resistance gene encoded on the cosmid is lost, and usually ~50% of the exconjugants should have the mutant allele.


Workflow Overview

Stage 1. Mutant design

Stage 2. ssDNA recombineering in E. coli

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

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

Organisms

This protocol has been confirmed to work for the following organisms:

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)
  • Reagents/equipment needed for DNA extraction, PCR, gel electrophoresis, electroporation, etc. - detailed in the steps below.

Detailed Protocols

Stage 1: Mutant design

In this stage, you will 1)Design your mutation(s) in silico: 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. and 2)Select a suitable cosmid/plasmid to use for your recombineering. Modify the cosmid/plasmid to contain an oriT for its eventual conjugal transfer into Streptomyces.

Step 1. in silico design of desired mutation(s)

Materials needed:

  • Sequence of the gene(s) you wish to mutate
  • DNA editing software, NEBCutter, the EXPASy translate tool, or similar tools
  • Codon usage chart for your organism


Instructions:

Design suitable oligonucleotides to create your mutation(s)

  1. Identify the point mutation(s) that you want to introduce onto the chromosome. From the desired amino acid change, work backwards to identify the DNA sequence that will encode it.
  2. You will want to pay attention to codon usage (I try to keep the same relative frequency of codon usage as the wild-type codon, if possible - i.e. a frequently used codon should be replaced by another frequently used codon).
  3. Your recombineering oligo will need to be about 70bp long, with the mutation in the center.
  4. Once you have designed your recombineering oligo, take its reverse complement. (Oligos 1 and 1' in Figure 2). The oligo corresponding to the lagging strand will be more efficient for recombineering, but if you don't know which strand is the lagging strand – usually the case for our cosmids – order oligos corresponding to both strands. If designing multiple changes for the same cosmid, it may be best to first determine for one such change, which is the most efficient oligo, and then use this sense oligo for all subsequent changes.)
Figure 2. Oligonucleotide design for ssDNA recombineering. Oligo 1 (and 1') are the two long (~70nt) recombineering oligos used to introduce the desired change(s) into your gene of interest (these change(s) should be near the middle of the oligo.) Oligos A and B are shorter (~20nt) oligos used to amplify the gene for diagnostic restriction digest and sequencing.


Design a suitable screen that will allow you to distinguish between wild-type and recombinant alleles. There are two basic approaches: diagnostic restriction digest and mismatch PCR (Figure 3).

  1. First, determine whether you can use a diagnostic restriction digest. You want to determine whether there are any restriction sites that are present in your mutant allele, but absent in the wild-type.
    1. Take ~500bp on either side of the mutation and copy it into a file (for both the wild-type and mutant alleles).
    2. Use NEBCutter or a similar tool to look for restriction fragment polymorphisms. (You can do this first with the 70bp wild-type sequence/mutant oligo, to simplify the restriction map, and make it easier to see the introduction of a new restriction site. Then, you should use NEBcutter with the entire ~500bp sequence, to make sure that your restriction site is unique in the amplified sequence, so that mutant allele will be easily identified by PCR/restriction digest on a gel.)
    3. If there are no suitable diagnostic restriction digests to distinguish between wild-type and mutant alleles, go back to step 1 and try using different codons for your desired amino acid change.
  2. If none of the codon changes change a restriction site, try designing PCR primers for a diagnostic mis-match PCR – this usually works if you have at least 2-3 base pairs mutated, but can be a bit fiddly in terms of determining the right PCR conditions for a successful diagnostic PCR.
Figure 3. Diagnostic restriction digests and mismatch PCRs to identify nt changes introduced by ssDNA recombineering. Option 1 (diagnostic restriction digests, top). The desired nucleotide change(s) in your gene also introduces a restriction site. The gene can be amplified by PCR using flanking primers (usually the same as oligos A and B in Figure 2), and then digested with the restriction enzyme. The digest is analysed by gel electrophoresis. Option 2 (mismatch PCR, bottom). The desired nucleotide changes are used to design primers for a mismatch PCR. The 3' end of the mismatch oligonucleotide anneals to the mutant allele, but not the wild-type, and therefore a PCR product can only be generated if the mutant allele is present.


Order the oligos you will need:

  • Both recombineering oligos (oligo 1 and oligo 1')
  • Flanking primers for diagnostic PCR/digest and sequencing (oligos A and B)
  • Diagnostic mismatch PCR primers, if needed
  • Oligo100, if needed (see below)

Step 2. cosmid/plasmid selection

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

  1. If working with S. coelicolor or S. venezuelae, use StrepDB to identify which cosmid(s) contain your gene of interest.
    1. If your organism of interest does not have a cosmid library, you can clone the gene and flanking sequences into an appropriate vector and use this for ssDNA recombineering. You want at least 1kb either side of your gene of interest (more is better) - the frequency with which you will observe recombination from your cosmid/plasmid onto the chromosome is dependent on the length of the sequence.
  2. If you have a choice between several different cosmids, select the one where your gene is closest to the centre of the cosmid.
  3. Check your cosmid for the sequence of Oligo 100, 5'-AAGTCGCGGTCGGAACCGTATTGCAGCAGCTTTATCATCTGCCGCTGGACGGCGCACAAATCGCGCTTAA-3' [1] – if there is not enough homology for the oligo to recombine with your cosmid, you can use this as a control in later steps (to confirm that your recombineering is working.)

Introduce an oriT into your cosmid/plasmid

An origin of transfer (oriT) is required for the conjugal transfer of the cosmid into your Streptomyces sp. (The standard cosmid libraries that we use do not already contain an oriT.) In the commonly used "Redirect" methodology, this oriT is generally part of the antibiotic resistance cassette.

Any strategy that introduces an oriT onto the cosmid should work here. The approach described below replaces the kanR marker on the cosmid backbone with an apraR-oriT cassette generated by PCR product pIJ794. (The resulting cosmids are therefore apraR and kanS.)

Materials needed:

  • Electrocompetent cells of an E. coli strain suitable for lambda-red recombineering (i.e. BW25113/pIJ790 or TB10) (see note 1)
  • Electrocompetent cells of a standard E. coli cloning strain (i.e. DH5α or similar)
  • Cosmid selected above
  • Solid and liquid LB medium
  • Antibiotic solutions (here, kanamycin, carbenicillin, and apramycin)
  • Electroporation cuvettes, SOC, other supplies needed for electroporation
  • Miniprep kit or reagents/supplies needed for cosmid DNA purification

Instructions:

  1. Transform your recombineering strain of choice (BW25113/pIJ790 or TB10) by electroporation.
  2. Plate the transformation on LB/kan/carb to select for the cosmid.
  3. Restreak transformant(s) on LB/kan/carb.
  4. Prepare competent cells of your recombineering/cosmid strain (i.e. TB10/cosmid).
  5. Transform your recombineering/cosmid strain with PCR product pIJ794.
  6. Plate the transformation mix on LB/apra or LB/apra/carb.
  7. Restreak transformant(s) on LB/apra or LB/apra/carb.
  8. Isolate candidate cosmid DNA from the transformant(s). This DNA may contain a mixture of the original cosmid (apraS, oriT-), and the recombinant cosmid (apraR, oriT+), so a further step to purify the recombinant cosmid is needed.
  9. Transform DH5α, or a similar cloning strain, with the mixed cosmid prep, and plate on LB/apra/carb (or LB/apra).
  10. Restreak transformants on LB/apra/carb and then screen them by patching onto LB/apra versus LB/kan. The transformants you want are those that are apraR, and kanS (These carry the oriT+ cosmids).

Notes:

  1. HME68 and BW25113/pIJ790 are both temperature-sensitive and should be grown at 30°C.

Stage 2: ssDNA-recombineering in E. coli

In this stage of the protocol, you will perform the ssDNA recombineering in E. coli: 3. Transform the recombineering strain with the cosmid/plasmid. 4. Perform recombineering using ssDNA to introduce the desired mutation(s) into the cosmid/plasmid.

See the brief ssDNA protocol (useful as a checklist) and the step-by-step ssDNA protocol, available from the Court lab website, for more detailed protocols and guidance on ssDNA recombineering.


Step 3. Transform the recombineering strain with the cosmid/plasmid

Materials needed:

  • A suitable E. coli strain deficient in MMR (i.e. HME68 (W3110 galKtyr145UAG ΔlacU169 [λ cI857 Δ(cro-bioA)] mutS<>cat))
  • The cosmid engineered to contain oriT in Step 2 above
  • Solid and liquid LB medium
  • Antibiotic solutions (here, apramycin and carbenicillin)
  • Electroporation cuvettes, SOC, other supplies needed for electroporation


Instructions:

  1. Transform your recombineering strain (i.e. HME68) with your apraRoriT cosmid.
  2. Plate the transformation mixture on LB/apra/carb (30°C).
  3. Restreak transformant(s) on LB/apra/carb (30°C). You have now generated the strain needed to perform the ssDNA recombineering.

Step 4. ssDNA recombineering

In this step, you will perform ssDNA-recombineering to introduce your desired mutation(s) into your cosmid/plasmid.

The recombineering strain HME68 has a missense mutation in the galK gene which results in white/colourless colonies on MacConkey/galactose medium. Oligo100 repairs the galK gene to its wild-type sequence, and can thus be used as a control for your recombineering experiments.

If the cosmid you are using does not contain the oligo100 sequence, you can co-transform your strain with oligo100 and your recombineering oligo, plating on MacConkey/galactose and screening for gal+ transformants. This helps you to identify colonies from cells that were expressing the lambda recombinase functions and were electrocompetent. A higher percentage of these colonies (up to ~50% of these) will contain cosmids carrying your mutation of interest.

However, the recombination with ssDNA occurs at a high enough frequency that such a screen is not necessary. If not using oligo100, simply plate on LB/apra and screen for your mutation (usually about 10-20% will be recombinants).

Materials needed:

  • The HME68/cosmid strain generated in step 3 above - see also note 1
  • The oligos designed in Step 1 above, and oligo100 (if using).
  • Solid and liquid LB medium - see note 2
  • MacConkey/1% galactose medium (if using oligo100)
  • Antibiotic solutions (here, apramycin and carbenicillin)
  • Electroporation cuvettes, SOC, other supplies needed for electroporation


Instructions:

  1. Prepare HME68/cosmid competent cells as follows:
    1. Grow a 50mL culture of your HME68/cosmid strain in LB/apra or LB/apra/carb at 30°C, until OD600 ~0.4-0.5.
    2. Heat-shock the cells for 15 minutes at 42°C (to induce the recombineering functions provided by the ts λ prophage).
    3. Chill the cells in an icewater-slurry for ~15 minutes.
    4. Prepare electrocompetent competent cells.
  2. Transform your cells with 1-2 μl of your recombineering oligo. (If you can use oligo100 (see step 5 in protocol above), you can cotransform the cells with your recombineering oligo and oligo100 - use 1 μl of each).
  3. Recover at 30°C for 30 minutes.
  4. Dilute the transformation mixture to plate. (A lot of cells will grow - there is no antibiotic selection), so I usually dilute the electroporation mixture 1:1000 and plate a few microlitres of this out. You may need to experiment with different dilutions to get single colonies.)
  5. Plate this dilution on Maconkey/1%galactose/apra medium, if using oligo100, or on LB/apra. In parallel, you should also plate out a control (a few microlitres of untransformed cells plated on the same medium.) Continue to grow the cells always at 30°C.

Notes:

  1. HME68 is temperature-sensitive and should be grown at 30°C. Growth at higher temperatures risks activation of the lambda prophage - this is useful for inducing the lambda recombinase functions, but prolonged growth at higher temperatures leads to cell lysis.
  2. High salt concentrations can reduce cell viability, choose an LB formulation with 5 g NaCl/L.
  3. Even if you are not cotransforming your HME68/cosmid strain with your oligo and oligo100, it may be useful to perform an oligo100 transformation in parallel to yours - this should give a good idea of whether your recombineering has been successful or not.

Stage 3: Identify and isolate recombinants. Confirm correct recombination event.

Screen for successful recombinants, i.e. cosmids carrying the desired mutation.(initially, because the cosmids are present in >1 copy/cell, these will exist in a mixed population of wild-type and mutant cosmids.) Isolate pure populations of mutant cosmids. Confirm the correct recombination event has occurred.

Step 5. Screen for successful recombinants

Materials needed:

  • The transformants generated in Step 4 above
  • The oligos designed in Step 1 above (for diagnostic restriction digest or mismatch PCR)
  • Solid and liquid LB medium
  • Antibiotic solutions (here, apramycin and carbenicillin)
  • PCR reagents and kit, restriction enzymes, etc. as needed

Instructions:

  • The details of your screen will vary depending on the design in Step 1 above.
  • If using oligo100, pick transformants that are dark red/have dark red sectors, to screen for your mutation(s). I usually find that screening ~7-15 transformants is sufficient.
  • Use the control plated above (untransformed HME68/cosmid strain) as a control for your diagnostic restriction digest/mismatch PCR; this strain carries the wild-type allele of your gene.
  • If using a diagnostic restriction digest, note that some enzymes are active in PCR buffer - see for example, Activity of Restriction Enzymes in PCR buffers
  • Select 2-4 positive candidates to proceed with step 6.


Step 6. Transform DH5a with your recombineered cosmid/plasmid

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 then screen again for recombinant cosmids.

Materials needed:

  • Electrocompetent cells of a standard E. coli cloning strain (i.e. DH5α or similar)
  • Cosmid candidates selected above
  • Solid and liquid LB medium
  • Antibiotic solutions (here, apramycin and carbenicillin)
  • Electroporation cuvettes, SOC, other supplies needed for electroporation
  • Miniprep kit or reagents/supplies needed for cosmid DNA purification

Instructions:

  1. Purify your recombinant cosmid/plasmid by cosmid prep/miniprep. (This will contain a mixture of wild-type and mutant cosmid/plasmid.)
  2. Transform DH5α or a similar cloning strain with your cosmid prep/miniprep.
  3. Plate transformation mixture on LB/apra or LB/apra/carb. (Cells can now be grown at 37°C.)
  4. Repeat the screen designed in step 1 and performed in step 5, to identify transformants that carry the mutant allele.
  5. Purify the cosmid/plasmid from these transformants. (These cosmid preps/minipreps should now contain a pure population with only the recombinant (mutant) allele.)

Step 7. Confirm the sequence of your gene/DNA sequence of interest

At this step, you will determine 1)if you have introduced the desired change(s) in your gene, and 2)if you have inadvertently introduced any undesired change(s).

Materials needed:

  • Cosmid candidates selected above
  • Oligos designed in step 1 (oligos A and B)
  • PCR reagents and kit, Nanodrop, etc. as needed
  • DNA analysis software

Instructions:

  1. Amplify the DNA sequence surrounding your your desired nucleotide change(s) by PCR (using oligos A and B designed in Step 1)
  2. Determine the sequence of this PCR fragment by Sanger sequencing. Examine the chromatograms carefully so that you can be certain whether you have a pure DNA prep containing only the mutant allele.

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

In this stage, you will (8-9) move the mutant allele(s) you constructed in E. coli, into your Streptomyces strain of interest and (10) identify exconjugants that have the mutant allele(s).

Many Streptomyces strains contain a methyl-sensing restriction system and therefore your cosmids/plasmids must initially be passaged through a non-methylating E. coli strain (such as ET12567/pUZ8002). If your Streptomyces strain does not contain such a restriction system, you do not need to use ET12567/pUZ8002 and may use another suitable strain (such as S17-1) instead.

Step 8. Transform ET12567/pUZ8002 with the mutant cosmid/plasmid.

Materials needed:

  • ET12567/pUZ8002 or another suitable strain for intergeneric conjugation
  • The cosmids carrying the mutant allele(s) (from step 7 above)
  • Solid and liquid LB medium - see note 2
  • Antibiotic solutions (here, apramycin and carbenicillin)
  • Electroporation cuvettes, SOC, other supplies needed for electroporation


Instructions:

  1. Transform ET12567/pUZ8002 with your cosmids using electroporation. Plate on LB/apra or LB/carb.
  2. Restreak transformants (on LB/apra or LB/carb)


Step 9. Conjugation into Streptomyces

Materials needed:

  • ET12567/pUZ8002,cosmid strain(s) from step 8
  • Antibiotics (here, apramycin and nalidixic acid) and reagents as detailed in the Conjugation using ET12567/pUZ8002 protocol
  • MS solid medium or another medium suitable for growth of your Streptomyces strain

Instructions:

  1. Follow the Conjugation using ET12567/pUZ8002 protocol to conjugate your mutant allele into your desired Streptomyces strain.
  2. Select for ex-conjugants using apra and nal.
  3. Pick single colonies and restreak once on medium containing apra and nal.
  4. Restreak ex-conjugants several times on non-selective medium (i.e. on MS for S. coelicolor). This will allow the wild-type/mutant alleles to assort into single copy such that you end up with exconjugants that carry only either the wild-type or the mutant allele. (Depending on the selective pressure for the wild-type/mutant alleles, you may need to restreak several times to see the wild-type allele completely eliminated from the exconjugants.)
  5. Screen for apraS exconjugants (those in which a double-crossover has occurred.)

Step 10. Identify mutant exconjugants.

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.

Materials needed:

  • The exconjugants generated in Step 9 above
  • The oligos designed in Step 1 above (for diagnostic restriction digest or mismatch PCR)
  • PCR reagents and kit, restriction enzymes, Nanodrop, etc. as needed
  • DNA analysis software

Instructions:

  • The details of your screen will vary depending on the design in Step 1 above.
  • If using oligo100, pick transformants that are dark red/have dark red sectors, to screen for your mutation(s). I usually find that screening ~7-15 transformants is sufficient.
  • Use the control plated above (untransformed HME68/cosmid strain) as a control for your diagnostic restriction digest/mismatch PCR; this strain carries the wild-type allele of your gene.
  • If using a diagnostic restriction digest, note that some enzymes are active in PCR buffer - see for example, Activity of Restriction Enzymes in PCR buffers
  • Select 2-4 positive candidates to proceed with step 6.

Instructions:

  1. Screen for exconjugants carrying the mutant allele (as in step 5 above).
    1. Follow the Streptomyces colony PCR protocol from the High GC PCR tips page, or extract genomic DNA from your exconjugants to use as a template for the PCRs.
    2. If the mutation(s) you introduced are close to the middle of the cosmid, and if there is no strong selective pressure for/against your mutant allele(s), you can expect to see ~50% of the exconjugants with the mutant allele. Therefore, it is not usually necessary to screen very many exconjugants.
  2. Confirm the sequence of the allele(s) by Sanger sequencing (as in step 7 above). Again, be sure to examine the chromatograms carefully so that you can be certain whether you have a pure DNA prep containing only the mutant allele.
  3. Perform further phenotypic analysis/characterization of your new strains.

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

[2]Sawitzke, J.A., Costantino, N., Li, X., Thomason, L.C., Bubunenko, M., Court, C., & Court, D.L. (2011). Probing Cellular Processes with Oligo-Mediated Recombination and Using the Knowledge Gained to Optimize Recombineering, Journal of Molecular Biology, 407(1), 45-59.

[3] Gust B., Challis G.L., Fowler K., Kieser T., Chater K.F.. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A. 2003;100(4):1541‐1546. doi:10.1073/pnas.0337542100

[4]Feeney, M.A., Chandra, G., Findlay, K.C., Paget, M.S.B., & Buttner, M.J. (2017). Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon. mBio, 8(3) e00815-17; DOI: 10.1128/mBio.00815-17

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.