3.7 Performing
Recombineering
and Counterselection
Counterselection vector and recombineering oligonucleotides are
concurrently electroporated into the conditionally recombinogenic
S. aureus strain. We recommended that counterselection experiments be performed in parallel with the transformation of untargeted pCAS9counter plasmid (i.e., vector which does not contain a
targeting oligo) or pCAS9counter targeted to a target absent from
the genome (such as GFP), in order to control for transformation
efficiency and to assess the success rate of counterselection (see
Subheading 3.9 and Note 5).
1. Precipitate and concentrate 1 μg counterselection plasmid
DNA using pellet paint as in Subheading 3.5, step 3, except
use 2 μL of 100 μM recombineering oligonucleotide (rather
than TE buffer) to resuspend the precipitated pellet.
2. Transform S. aureus by electroporation as in Subheading 3.5,
step 4, except incubate transformants 32
C for 2 h with
shaking at 250 rpm.
3. Plate aliquots of the electroporation mixtures onto LB media
containing 10 μg/mL chloramphenicol and 10 μg/mL erythromycin. We recommend plating four volumes of 250 μL each.
4. Incubate plates a 48–60 h at 32
C, until colonies appear.
3.8 Screening
for Engineered Strains
and Performing
Plasmid Curing
Efficiency of genome engineering may range considerably from
target to target (in our experience, from 5% to 50% of colonies
screened may contain the desired change), so an appropriate number of colonies should be screened.
1. For deletion mutants, it is sufficient to screen colonies using
colony PCR fragment length analysis, using primers flanking
the deleted region and appropriate amplification conditions (see
Subheading 3.6.2, step 6).
2. For point mutations, it is necessary to assess the success of
genome engineering using DNA sequencing. Design primers
flanking the intended change, perform colony PCR to amplify
the region using appropriate primers and amplification conditions (see Subheading 3.6.2, step 6), and subject the amplicon
to Sanger sequencing.
3. Once a successfully engineered colony is identified, the bacteria
may be cured of the temperature sensitive recombineering and
counterselection plasmids to produce an isogenic strain. Streak
the colony onto LB plates (lacking antibiotics) and incubate
overnight at 43
C. Verify loss of the plasmid in one or two
resulting colonies by restreaking them onto LB media containing 10 μg/mL chloramphenicol and LB media containing
10 μg/mL erythromycin: successfully cured strains will not
grow on either antibiotic.
Genome Editing in Staphylococcus aureus by Conditional. . .
139
Recombineering
and Counterselection
Counterselection vector and recombineering oligonucleotides are
concurrently electroporated into the conditionally recombinogenic
S. aureus strain. We recommended that counterselection experiments be performed in parallel with the transformation of untargeted pCAS9counter plasmid (i.e., vector which does not contain a
targeting oligo) or pCAS9counter targeted to a target absent from
the genome (such as GFP), in order to control for transformation
efficiency and to assess the success rate of counterselection (see
Subheading 3.9 and Note 5).
1. Precipitate and concentrate 1 μg counterselection plasmid
DNA using pellet paint as in Subheading 3.5, step 3, except
use 2 μL of 100 μM recombineering oligonucleotide (rather
than TE buffer) to resuspend the precipitated pellet.
2. Transform S. aureus by electroporation as in Subheading 3.5,
step 4, except incubate transformants 32
C for 2 h with
shaking at 250 rpm.
3. Plate aliquots of the electroporation mixtures onto LB media
containing 10 μg/mL chloramphenicol and 10 μg/mL erythromycin. We recommend plating four volumes of 250 μL each.
4. Incubate plates a 48–60 h at 32
C, until colonies appear.
3.8 Screening
for Engineered Strains
and Performing
Plasmid Curing
Efficiency of genome engineering may range considerably from
target to target (in our experience, from 5% to 50% of colonies
screened may contain the desired change), so an appropriate number of colonies should be screened.
1. For deletion mutants, it is sufficient to screen colonies using
colony PCR fragment length analysis, using primers flanking
the deleted region and appropriate amplification conditions (see
Subheading 3.6.2, step 6).
2. For point mutations, it is necessary to assess the success of
genome engineering using DNA sequencing. Design primers
flanking the intended change, perform colony PCR to amplify
the region using appropriate primers and amplification conditions (see Subheading 3.6.2, step 6), and subject the amplicon
to Sanger sequencing.
3. Once a successfully engineered colony is identified, the bacteria
may be cured of the temperature sensitive recombineering and
counterselection plasmids to produce an isogenic strain. Streak
the colony onto LB plates (lacking antibiotics) and incubate
overnight at 43
C. Verify loss of the plasmid in one or two
resulting colonies by restreaking them onto LB media containing 10 μg/mL chloramphenicol and LB media containing
10 μg/mL erythromycin: successfully cured strains will not
grow on either antibiotic.
Genome Editing in Staphylococcus aureus by Conditional. . .
139