Homologous Recombination

Aim

Preparation

Be careful, all recombination protocols should be done at 30°C unless indicated otherwise !

Strain Preparation

Transform the PTKred Plasmid

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with the strain of interest.
Start an over night culture with the strain transformed with PTKred at 30°C.
Dilute the overnight culture in a ratio of 1 mL/100 mL and add IPTG (isopropyl β-D-1-thiogalactopyranoside; final concentration of 2 mM) and Spectinomycin (60 mg/mL).
Grow up to OD600: 0.5 then make electrocompetent cells .

Integration

Prepare the PCR fragment to be integrated flanked by 50 bp genomic sequences.
Gel-purify the product and transform with the electrocompetent PTKred strain.
Recover in LB for 2 hours with IPTG.
After 2 hours incubation add the selective drug which should be inserted in the genom (for example kanamycin if you have kan R cassette inserted) and incubate overnight in liquid culture at 30°C.
Next day, culture should be very turbid or the recombination did not work. Plate streak 50 μL of the overnight culture on a plate containing selection drug which in this case is kanamycin and grow overnight at 30°C.
Next day pick a colony and make glycerol stocks

Elimination of PTKred plasmid

Inoculate the strain that you have made by Homologous Recombination in LB overnight with spectinomycin at 30°C.
Adding spectinomycin in this step ensures that the plasmid will not integrate in the genome in a rare occurrence.
Dilute 1/100 and grow at 42°C for 4 hours and then plate sreak 50 μL of the culture on LB plate at 42°C.
Next day, pick colonies and grid plate on LB then spectinomycin and grow at 37°C.

Flip Recombinase and Anti-biotic Resistance Gene

Transform a cured strain with PCP20 plasmid
Grow overnight in LB with Cam at 30°C overnight.
Dilute 1/100 and grow for 3 hours at 37°C.
Streak plate on LB plates and grow over night at 42°C.
Repeart the grid plating as in the following diagram:

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