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 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
    • Warning, important 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
    • Warning, important 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.
    • 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 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.
  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.
    • 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.
    • Frequency of Double stranded break repair reported at 1 in 105

iDT "Gene-block" based

Reference

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

-- Main.DanielDeatherage - 20 Apr 2015

Edit | Attach | Watch | Print version | History: r10 | r8 < r7 < r6 < r5 | Backlinks | Raw View | More topic actions...

 Barrick Lab  >  ProtocolList  >  ProtocolsPsltsEditing

Contributors to this topic Edit topic DanielDeatherage, DaciaLeon, JeffreyBarrick
Topic revision: r6 - 2015-05-01 - 15:11:57 - Main.DanielDeatherage
 
This site is powered by the TWiki collaboration platform Powered by Perl This site is powered by the TWiki collaboration platformCopyright ©2024 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback