(r8) ProtocolsPsltsEditing < Lab

---++ 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
   1 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
      1 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
      1 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
      1 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
   1 Gel purify each of the 4 PCR fragments using standard conditions.
   1 Mutation Cassette generation using Gibson reaction. [[ProtocolsGibsonCloning][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
      1 Adjust the volume to 10µl with DNase-free water.
      1 Add 10µl of Gibson assembly master mix.
      1 Incubate 1 hr at 50°C.
      1 Use 2µl to transform.
      1 Outgrowth 45 minutes at 37°C.
      1 Plate overnight on LB crb kan plates.
      1 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. [[ProtocolsElectrocompetentCells][Electro competent protocol]]
   1 Transform electro-competent strain with 100ng of pSLTS.
   1 Plate on LB Carbenicillin plates, and grow overnight at 30°C
      * %ICON{"warning"}% pSLTS has a temperature sensitive origin of replication, growth at 37°C will cause loss of plasmid.
   1 Pick a single colony into 5mL LB with Carbenicillin, grow overnight at 30°C
      * %ICON{"warning"}% pSLTS has a temperature sensitive origin of replication, growth at 37°C will cause loss of plasmid.
   1 Inoculate 10mL LB with Carbenicillin, with 100µl of overnight culture. 
   1 Grow for 1 hour at 30C.
   1 Add L-Arabinose to a final concentration of 2 mM to induce the expression of &lambda;-Red recombinase.
      * %ICON{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.
   1 Grow an additional 2-3 hours at 30C until the OD<sub>600</sub> reaches 0.7-0.9.
   1 Harvest cells by centrifugation at 4500 x g and wash twice with ice-cold 10% glycerol.
   1 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 &lambda;-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
   1 Incubate 3 hours at 30°C with shaking for outgrowth.
   1 Plate onto LB plates containing Carbenicillin and Kanamyocin.
   1 Grow overnight at 30°C.
   1 Streak 4 independent colonies onto fresh LB + Carbenicillin and Kanamyocin.
   1 Grow overnight at 30°C.
   1 Resuspend an entire colony in 500µl saline.
      * %T% 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.
   1 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 10<sup>5</sup>. 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 &lambda;-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.
         1 Confirm correct integration of mutation cassette by PCR.
            1 20µl used as template
            1 100°C incubation prior to amplification.
            1 Amplification should be done using forward primer of 5' mutation cassette and reverse primer of the 3' mutation cassette.
         1 The remaining 30µl was mixed with 120µl of saline. %T% again, note PBS suggested in original protocol.
         1 50µl plated on LB Carbenicillin plates with or without anhydrotetracycline (100ng/mL).
         1 Remaining 50µl used to inoculate 1mL LB + Carbenicillin media.
         1 Incubate 1hr 30°C with shaking.
         1 Add L-Arabinose to final concentration of 2mM. %T% see note above about variability in amount of L-Arabinose used in induction.
         1 Incubate 2hr 30°C with shaking.
         1 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.
   1 Sequence verify clones using specific primers for the target region

---++++iDT "Gene-block" based

---++Reference
[[http://www.biomedcentral.com/1472-6750/14/84][Kim et al BMC Biotechnol. 2014 Sep 25;14(1):84.]]



-- Main.DanielDeatherage - 20 Apr 2015
Barrick Lab > ProtocolList > ProtocolsPsltsEditing
Topic revision: r8 - 2015-09-18 - 20:11:16 - Main.DaciaLeon