Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella

Materials and Reagents

1. PCR tubes
2. 1.5 ml Eppendorf tubes
   
3. 15 ml test tubes
4. Plastic Petri dishes (9 cm diameter) for bacterial growth
5. Sterile loops (1 µl)
6. Pipette tips
7. 250 ml E-flask
8. 50 ml plastic tubes
9. Electroporation cuvettes, 1 mm gap (BTX, product number: 45-0124) 
10. Salmonella enterica serovar Typhimurium strain LT2 (Biosafety level 2)
11. PCR primers that bind outside of the cassette insertion site
12. Ampicillin (Sigma-Aldrich, catalog number: A9518)
13. Chloramphenicol (Sigma-Aldrich, catalog number: C0378)
14. Glucose (Sigma-Aldrich, catalog number: G7021)
15. Bacteriological agar (Oxoid, catalog number: LP0011)
16. Ethanol (95%)
17. PCR Master Mix (2x) (Thermo Fisher, catalog number: K0171)
18. Sodium chloride (Sigma-Aldrich, catalog number: S3014)
19. Sucrose (Sigma-Aldrich, catalog number: 84100)
20. Tryptone (Oxoid, catalog number: LP0042)
21. Yeast extract (Oxoid, catalog number: LP0021)
22. LB medium (see Recipes)
23. LA plates (see Recipes)
24. Salt-free LA + 5% sucrose (see Recipes)
    

Equipment

1. Test tube racks
2. Autoclave
3. Incubator at 37 °C
4. PCR machine
5. Pipettes (10 µl, 20 µl, 100 µl and 1,000 µl)
6. Shaking incubator at 37 °C
7. UV Transilluminator (e.g., 365 nm)
8. Shaking incubator at 30 °C
9. Shaking water bath at 43 °C
   
10. Centrifuge (temperature controlled) for 50 ml tubes (Thermo Scientific, model: Haraeus Megafuge 16R)
11. Centrifuge (temperature controlled) for Eppendorf tubes (Eppendorf, model: Centrifuge 5424R)
12. Electroporator (Bio-Rad, model: Gene Pulser Xcell)
    

Procedure

1. Insert the genetic cassette into the chromosomal location for which recombination rates should be determined
   Note: This step is specific for each location at which recombination rates will be measured. The details of the procedure will also vary depending on the Lambda-red recombination system that is used. For detailed instructions on Lambda-red recombineering see Yu et al., 2000. The following steps are a summary for pSIM6-based recombineering.
   
   1. Amplify the cat-sacB-yfp cassette by PCR and purify the product.
      Note: Primer design and amplification conditions are specific for the used selectable/counter-selectable cassette and insertion location and therefore not further described in this protocol.
   2. Start an overnight culture: Inoculate 2 ml sterile LB containing 100 mg/L ampicillin in a 15 ml test tube with cells from a single colony.
   3. Incubate in an incubator at 30 °C, shaking at 200 rpm for ~20 h.
   4. Mix 50 ml salt-free LB containing 100 mg/L ampicillin and 0.2% glucose with 500 µl o/n culture in a 250 ml E-flask.
   5. Incubate in an incubator at 30 °C, shaking at 200 rpm for ~2 h.
   6. To induce red expression, move the flask to a 43 °C shaking water bath for 15 min.
   7. Cool in an ice-water bath for at least 10 min. Swirl carefully to cool cells.
   8. Transfer the culture to a cold 50 ml plastic tube and pellet the cells (3,500 x g, 8 min, 4 °C).
   9. Remove as much as possible of the supernatant and resuspend the cells in 20 ml ice-cold sterile water. Do not vortex! Pellet the cells at 3,500 x g, 8 min, 4 °C.
   10. Resuspend the cells in 1 ml ice-cold water and transfer into a cold Eppendorf tube. Pellet the cells at 16,000 x g, 3 min, 4 °C and remove as much as possible of the supernatant.
   11. Resupend cells in 100 µl ice-cold water.
   12. Mix 50 µl cells with 5 µl purified PCR product, incubate on ice for 5 min and transfer into a cold electroporation cuvette. 
   13. Electroporate (1.8 kV, 200 Ω, 25 µF). Immediately, resuspend the cells in 1 ml pre-warmed (37 °C) LB and transfer back into the Eppendorf tube.
   14. Let the cells recover for at least 3 h or overnight at 37 °C.
       
   15. Plate 100 µl recovered culture on an LA plate containing 25 mg/L chloramphenicol, incubate the plate o/n at 37 °C.
   16. Restreak colonies on LA plates containing 25 mg/L chloramphenicol, incubate the plates o/n at 37 °C.
   17. Confirm the correct insertion by PCR size verification and test the phenotype of the recombinant cells (YFP fluorescence and sensitivity to growth on salt-free LA plates containing 5% sucrose).
   
   
2. Determine the number of colony-forming units per ml of overnight cultures
   
   1. Start three independent overnight cultures for each strain to be measured: Inoculate 2 ml sterile LB in a 15 ml test tube with cells from a single colony.
   2. Incubate in an incubator at 37 °C, shaking at 200 rpm for ~20 h.
   3. Make a 10-fold dilution series in a sterile 0.9% NaCl solution to a dilution factor of 10^-7.
   4. Plate 100 µl of dilutions 10^-5, 10^-6 and 10^-7 on LA plates.
      Note: Under these incubation conditions Salmonella cultures usually grow to density of approximately 4 x 10⁹ cfu/ml. Change the plated dilution factors for strains or conditions that form cultures with a different density.
   5. Incubate the LA plates in an incubator at 37 °C for ~20 h.
   6. Count the colonies per plate. Choose a dilution with 30-500 colonies per plate to calculate the original culture density according to formula 1 (see Data analysis). 
   
   
3. Determine the volume of overnight culture to be plated for assay
   Note: Steps B1-B3 are identical to “Procedure A” and the same dilutions can be used for both parts.
   
   1. Start three independent overnight cultures for each strain to be measured: Inoculate 2 ml sterile LB in a 15 ml test tube with cells from a single colony.
   2. Incubate in an incubator at 37 °C, shaking at 200 rpm for ~20 h.
   3. Make a 10-fold dilution series in a sterile 0.9% NaCl solution to a dilution factor of 10^-5.
   4. Plate 100 µl of each dilution (10⁰ to 10^-5) on salt-free LA plates containing 5% sucrose.
   5. Incubate in an incubator at 37 °C for ~20 h.
   6. Assess colonies under UV light for fluorescence. Re-streak non-fluorescent colonies onto salt-free LA plates containing 5% sucrose.
      Note: Use UV light with a wavelength that does not cause DNA damage (e.g., 365 nm), re-streak two colonies per colony morphology (different colony shapes or sizes) if more than 5 colonies are non-fluorescent.
   7. Incubate in an incubator at 37 °C for ~20 h.
   8. Confirm recombinational removal of the cat-sacB-yfp cassette by PCR size verification followed by sequencing of the insertion site.
   9. Estimate the dilution factor where approximately half of the cultures would yield a colony with recombinational removal of the cat-sacB-yfp cassette.
      Note: The estimated dilution does not need to be part of the 10-fold dilution series but can be any dilution.
   
   
4. Measure recombination rate
   
   1. Start twenty independent overnight cultures for each strain to be measured: Inoculate 2 ml sterile LB in a 15 ml test tube with cells from a single colony.
   2. Incubate in an incubator at 37 °C, shaking at 200 rpm for ~20 h.
   3. Make appropriate dilutions, as determined in “Procedure B”, in a sterile 0.9% NaCl solution.
   4. Plate 100 µl of each dilution on salt-free LA plates containing 5% sucrose.
   5. Incubate in an incubator at 37 °C for ~20 h.
   6. Assess colonies under UV light for fluorescence. Re-streak non-fluorescent colonies onto salt-free LA plates containing 5% sucrose.
      Note: Use UV light with a wavelength that does not cause DNA damage (e.g., 365 nm). Re-streak two colonies per colony morphology (different colony shapes or sizes) if more than 5 colonies are non-fluorescent.
   7. Incubate in an incubator at 37 °C for ~20 h.
   8. Confirm recombinational removal of the cat-sacB-yfp cassette by PCR size verification followed by sequencing of the insertion site.
   9. Calculate the recombination rate according to the formula 2 (see Data analysis). 

Data analysis

1. The culture density is calculated according to the formula:
   P = N/V*D                                                                     (1)
   where P is the population density in cfu/ml, N is the number of colonies on the plate, V is the volume plated in ml and D is the dilution factor.
2. The recombination rate is calculated according to the formula:
   µ = -ln(P₀)/N                                                                 (2)
   where µ is the mutation rate, P₀ is the proportion of cultures without recombinational removal of the cat-sacB-yfp cassette and N is the number of cells plated for the assay.
   

Notes

1. This protocol can be performed in any bacterial strain in which lambda-red recombineering can be used.
2. This protocol is written for the use of a cat-sacB-yfp cassette (available on request), but any selectable/counter-selectable cassette and screening marker can be used.
   

Recipes

1. LB medium
   Mix and autoclave:
   10 g Tryptone
   5 g Yeast extract
   10 g Sodium chloride (omit in salt-free LB)
   1 L water
2. LA plates
   Mix and autoclave:
   10 g Tryptone
   5 g Yeast extract
   10 g Sodium chloride
   15 g Agar
   1 L water
   Pour media into plastic Petri dishes and let solidify
3. Salt-free LA + 5% sucrose
   
   1. Mix and autoclave
      10 g Tryptone
      5 g Yeast extract
      15 g Agar
      700 ml water
   2. Mix and autoclave
      50 g Sucrose
      300 ml water
   3. Mix both solutions, pour media into plastic Petri dishes and let solidify

Acknowledgments

This work was supported by grants to DH from the Swedish Science Research Council (Vetenskapsrådet, grant numbers 2016-04449, 2017-03953) and the Carl Trygger Foundation, (grant numbers CTS16:194, CTS17:204).

Competing interests

The authors have no conflict of interest.

References

1. Abdulkarim, F. and Hughes, D. (1996). Homologous recombination between the tuf genes of Salmonella typhimurium. J Mol Biol 260(4): 506-522.
2. Anderson, R. P. and Roth, J. R. (1977). Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol 31: 473-505.
3. Brandis, G., Cao, S. and Hughes, D. (2018). Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella. Mol Microbiol 108(6): 697-710.
4. Gay, P., Le Coq, D., Steinmetz, M., Berkelman, T. and Kado, C. I. (1985). Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol 164(2): 918-921.
5. Hughes, D. (2000). Co-evolution of the tuf genes links gene conversion with the generation of chromosomal inversions. J Mol Biol 297(2): 355-364.
6. Segall, A.M. and Roth J.R. (1989). Recombination between homologies in direct and inverse orientation in the chromosome of Salmonella: intervals which are nonpermissive for inversion formation. Genetics 122(4): 737-747.
7. Thomas, C. M. and Nielsen, K. M. (2005). Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat Rev Microbiol 3(9): 711-721.
8. Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G. and Court, D. L. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97(11): 5978-5983.