Difference: ProtocolsPsltsEditing (4 vs. 5)

Overview

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

Protocol

Materials

• 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.
   • 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.

iDT "Gene-block" based

Reference

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

-- DanielDeatherage - 20 Apr 2015