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 50C.
   5. Use 2µl 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

iDT "Gene-block" based

Reference

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

-- Main.DanielDeatherage - 20 Apr 2015