Overview

Lab protocol for using the pSLTS plasmid method of scarless genome editing developed by the Copley lab.

If you use this protocol, you should cite:

Kim, J., Webb, A.M., Kershner, J.P., Blaskowski, S., Copley, S.D. (2014) A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica. BMC Biotechnol. 14:84. Link

Protocol

Materials

The following general materials should be located before starting:

• Plasmids pSLTS & pT2SK
• Primers MF & MR
• Primers pHAFor & pHARev

Mutation Cassete Construction

Amplified from genome

1. Design primers for the mutation cassette. A total of 4 primers must be designed to generate the following (standard primer purification should be sufficient):
   • 5' mutation cassette. A ~200bp fragment containing the mutation of interest in the 30-50bp at the 3' end of the fragment flanked by overhang sequences:
     • 5'-AGGCGTATCACGAGGCCCTTxxxxx where xxxxx represents 14-25bp of homologous DNA at the 5' end of the fragment
     • 5'-ACCGCTGCCACTCTTGAGATxxxxx where xxxxx represents 14-25bp of homologous DNA at the 3' end of the fragment
   • 3' mutation cassette. A ~200bp fragment containing the mutation of interest in the 30-50bp at the 5' end of the fragment flanked by overhang sequences:
     • 5'-GCAGGGCGGGGCGTAAxxxxx where xxxxx represents 14-25bp of homologous DNA at the 5' end of the fragment
     • 5'-CTCACATGTTCTTTCCTGCGxxxxx where xxxxx represents 14-25bp of homologous DNA at the 3' end of the fragment
2. PCR amplify the following:
   1. Plasmid backbone. (May be available as lab stock as this is not specific to any project)
      • Template: pT2SK
      • Primers: pHAFor & pHARev
      • Conditions:
      • Expected size: 1887
   2. Selection cassette. (May be available as lab stock as this is not specific to any project)
      • Template: pT2SK
      • Primers: MF & MR
      • Conditions:
      • Expected size: 1217
   3. 5' mutation cassette.
      • Template: Genomic DNA purified from strain of interest containing mutation you wish to introduce into new strain.
      • Primers: Primers designed in previous step for 5' mutation cassette
      • Conditions: Will vary.
      • Expected size: ~200bp depending on specific fragments
   4. 3' mutation cassette.
      • Template: Genomic DNA purified from strain of interest containing mutation you wish to introduce into new strain.
      • Primers: Primers designed in previous step for 5' mutation cassette
      • Conditions: Will vary.
      • Expected size: ~200bp depending on specific fragments
3. Gel purify each of the 4 PCR fragments using standard conditions.
4. Mutation Cassette generation using Gibson reaction. General Gibson Reaction Protocol
   1. Mix the following products at the given amount:
      • Plasmid Backbone: 25 fmol
      • Selection Cassette: 75 fmol
      • 5' Mutation Cassette: 125 fmol
      • 3' Mutation Cassette: 125 fmol
   2. Adjust the volume to 10µl with DNase-free water.
   3. Add 10µl of Gibson assembly master mix.
   4. Incubate 1 hr at 50°C.
   5. Use 2µl to transform.
   6. Outgrowth 45 minutes at 37°C.
   7. Plate overnight on LB crb kan plates.
   8. Verify correct mutation construct using pHA.seq.F and pHA.seq.R primers.

Strain Preparation

1. Obtain an electro-competent version of the strain you wish to edit. Electro competent protocol
2. Transform electro-competent strain with 100ng of pSLTS.
3. Plate on LB Carbenicillin plates, and grow overnight at 30°C
   • pSLTS has a temperature sensitive origin of replication, growth at 37°C will cause loss of plasmid.
4. Pick a single colony into 5mL LB with Carbenicillin, grow overnight at 30°C
   • pSLTS has a temperature sensitive origin of replication, growth at 37°C will cause loss of plasmid.
5. Inoculate 10mL LB with Carbenicillin, with 100µl of overnight culture.
6. Grow for 1 hour at 30C.
7. Add L-Arabinose to a final concentration of 2 mM to induce the expression of λ-Red recombinase.
   • The original paper uses both 1 mM and 2 mM of arabinose for induction in different E. coli strains. The lower concentration can be used in strains like BW25113 which has mutations disrupting arabinose utilization (Juhan Kim, personal communication).
8. Grow an additional 2-3 hours at 30C until the OD₆₀₀ reaches 0.7-0.9.
9. Harvest cells by centrifugation at 4500 x g and wash twice with ice-cold 10% glycerol.
10. Resuspend in 50-100 µl of 10% glycerol and use immediately or store at -70°C until use.

Genome Editing

1. Transform 50-100ng of mutation cassette into 50-100µl of electro competent cells expressing the λ-Red recombinase.
   • PCR amplify mutation cassette using forward primer of 5' mutation cassette and reverse primer of 3' mutation cassette
   • Remove template plasmid via DpnI digestion
   • Mutation cassette can be either gel or PCR purified and transformed into the recipient strain
2. Incubate 3 hours at 30°C with shaking for outgrowth.
3. Plate onto LB plates containing Carbenicillin and Kanamyocin.
4. Grow overnight at 30°C.
5. Streak 4 independent colonies onto fresh LB + Carbenicillin and Kanamyocin.
6. Grow overnight at 30°C.
7. Resuspend an entire colony in 500µl saline.
   • Original protocol calls for PBS not saline. Saline used instead as thought to be unlikely to effect anything, and makes use of existing stock solutions.
8. Plate 50-100µl of resuspended colony onto LB plates containing Carbenicillin and anhydrotetracycline (100ng/mL).
   • Frequency of Double stranded break repair reported at 1 in 10⁵. If no colonies are obtained, consider replating at higher density and/or checking the density of cells actually plated by plating on LB Carbenicillin plates without anhydrotetracycline.
   • If frequency appears lower than expected, efficency can be increased by a pre induction of the λ-Red recombinase. These are optional steps that should not be necessary.
     1. Resuspend a single colony containing the integrated mutation cassette in 50µl of DNase-free water.
     2. Confirm correct integration of mutation cassette by PCR.
        1. 20µl used as template
        2. 100°C incubation prior to amplification.
        3. Amplification should be done using forward primer of 5' mutation cassette and reverse primer of the 3' mutation cassette.
     3. The remaining 30µl was mixed with 120µl of saline. again, note PBS suggested in original protocol.
     4. 50µl plated on LB Carbenicillin plates with or without anhydrotetracycline (100ng/mL).
     5. Remaining 50µl used to inoculate 1mL LB + Carbenicillin media.
     6. Incubate 1hr 30°C with shaking.
     7. Add L-Arabinose to final concentration of 2mM. see note above about variability in amount of L-Arabinose used in induction.
     8. Incubate 2hr 30°C with shaking.
     9. 50µl plated on LB Carbenicillin plates with or without anhydrotetracycline (100ng/mL).
   • 5-10 colonies from each plate patched onto LB and LB + Kanamyocin plates.
9. Patch colonies on LB and LB+Kan plates, sequence verify clones that grow on LB and not on LB+Kan using specific primers for the target region