Overview

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

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 10l with DNase-free water.
    3. Add 10l of Gibson assembly master mix.
    4. Incubate 1 hr at 50C.
    5. Use 2l to transform.
    6. Outgrowth 45 minutes at 37C.
    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 30C
    • warning pSLTS has a temperature sensitive origin of replication, growth at 37C will cause loss of plasmid.
  4. Pick a single colony into 5mL LB with Carbenicillin, grow overnight at 30C
    • warning pSLTS has a temperature sensitive origin of replication, growth at 37C will cause loss of plasmid.
  5. Inoculate 10mL LB with Carbenicillin, with 100l 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.
    • help Original paper cites both 1mM and 2mM for induction concentration of different E. coli strains. No explanation given for the difference, 2 is given as amount to use as it is assumed that 1 was deemed sufficient in some cases but not others and that 2 would have worked in all cases.
  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 l of 10% glycerol and use immediately or store at -70C until use.

Genome Editing

  1. Transform 50-100ng of mutation cassette into 50-100l of electro competent cells expressing the λ-Red recombinase.
  2. Incubate 3 hours at 30C with shaking for outgrowth.
  3. Plate onto LB plates containing Carbenicillin and Kanamyocin.
  4. Grow overnight at 30C.
  5. Streak 4 independent colonies onto fresh LB + Carbenicillin and Kanamyocin.
  6. Grow overnight at 30C.
  7. Resuspend an entire colony in 500l saline.
    • TIP 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 50-100l 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 50l of DNase-free water.
      2. Confirm correct integration of mutation cassette by PCR.
        1. 20l used as template
        2. 100C 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 30l was mixed with 120l of saline. TIP again, note PBS suggested in original protocol.
      4. 50l plated on LB Carbenicillin plates with or without anhydrotetracycline (100ng/mL).
      5. Remaining 50l used to inoculate 1mL LB + Carbenicillin media.
      6. Incubate 1hr 30C with shaking.
      7. Add L-Arabinose to final concentration of 2mM. TIP see note above about variability in amount of L-Arabinose used in induction.
      8. Incubate 2hr 30C with shaking.
      9. 50l plated on LB Carbenicillin plates with or without anhydrotetracycline (100ng/mL).
    • 5-10 colonies from each plate patched onto LB and LB + Kanamyocin plates.

iDT "Gene-block" based

Reference

Kim et al BMC Biotechnol. 2014 Sep 25;14(1):84.

-- Main.DanielDeatherage - 20 Apr 2015

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Topic revision: r7 - 01 May 2015 - 20:18:43 - Main.DanielDeatherage
 
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