---++ 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 λ-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 λ-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 λ-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 Patch colonies on LB and LB+Kan plates, sequence verify clones that grow on LB and not on LB+Kan 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
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