mutations is not possible, for example, when a PAM overlaps a
proline or a glycine codon. In these situations, a conservative
amino acid substitution can be used. There are multiple amino
acid exchangeability matrices available in the literature which
are based on empiric data (e.g., [30]) that can be used to
inform the choice for the substituted amino acid. If this
approach must be taken, we recommend engineering a mutant
strain containing the PAM-ablating substitution alone, as well
as a mutant strain carrying that substitution in concert with the
desired engineered change so that unintended effects of the
substitution can be experimentally assessed. Measures must be
taken as above to escape MMR repair of any dedicated,
PAM-ablating mutations.
3.3 Preparation
of Electrocompetent
Cells
This basic protocol can be used to prepare electrocompetent cells
for either E. coli or S. aureus strains, as long as appropriate media are
used. The protocol essentially follows those of Monk et al. [7] and
Ausbuel et al. [31], with minor modifications.
1. Inoculate a single bacterial colony into 3 mL of the appropriate
broth. Incubate overnight at 37
C (or 32
for strains carrying
temperature sensitive vectors) with constant aeration.
(a) For growth of S. aureus the preferred media is B2 broth
[7], although LB or TSB can alternatively be used (see
Note 1). If the strain is carrying pEF2131-tet, the media
should contain 10 μg/mL chloramphenicol and a reduced
temperature should be used.
(b) For growth of E. coli, use LB media.
2. Add 1.5 mL of the overnight culture to 150 mL of the appropriate, fresh broth in a 1-L flask.
3. Incubate the cells with constant aeration (shaking at 250 rpm)
at the appropriate temperature (37
C or 32
) until OD
650 nm reaches 0.5 (typically 130–160 min).
4. Chill the culture on ice for 15 min to stop growth. From this
point on, keep cells cold at all times during processing. As the
culture chills, transfer ~37.5 mL aliquots into four 50 mL
conical centrifuges tubes, on ice.
5. Harvest the cells by centrifugation at 4600 Â g for 10 min at
4
C. Remove supernatant.
6. Add ~10 mL ice-cold sterile water to each tube, resuspend
pellets, then consolidate all aliquots into a single 50 mL tube.
Spin at 4600 Â g for 10 min at 4
C, then remove supernatant.
7. Add ~10 mL of ice-cold sterile water and resuspend. Mostly fill
tube with ice-cold sterile water (~30 mL). Centrifuge at
4600 Â g for 10 min at 4
C. Remove supernatant.
134
Kelsi Penewit and Stephen J. Salipante
proline or a glycine codon. In these situations, a conservative
amino acid substitution can be used. There are multiple amino
acid exchangeability matrices available in the literature which
are based on empiric data (e.g., [30]) that can be used to
inform the choice for the substituted amino acid. If this
approach must be taken, we recommend engineering a mutant
strain containing the PAM-ablating substitution alone, as well
as a mutant strain carrying that substitution in concert with the
desired engineered change so that unintended effects of the
substitution can be experimentally assessed. Measures must be
taken as above to escape MMR repair of any dedicated,
PAM-ablating mutations.
3.3 Preparation
of Electrocompetent
Cells
This basic protocol can be used to prepare electrocompetent cells
for either E. coli or S. aureus strains, as long as appropriate media are
used. The protocol essentially follows those of Monk et al. [7] and
Ausbuel et al. [31], with minor modifications.
1. Inoculate a single bacterial colony into 3 mL of the appropriate
broth. Incubate overnight at 37
C (or 32
for strains carrying
temperature sensitive vectors) with constant aeration.
(a) For growth of S. aureus the preferred media is B2 broth
[7], although LB or TSB can alternatively be used (see
Note 1). If the strain is carrying pEF2131-tet, the media
should contain 10 μg/mL chloramphenicol and a reduced
temperature should be used.
(b) For growth of E. coli, use LB media.
2. Add 1.5 mL of the overnight culture to 150 mL of the appropriate, fresh broth in a 1-L flask.
3. Incubate the cells with constant aeration (shaking at 250 rpm)
at the appropriate temperature (37
C or 32
) until OD
650 nm reaches 0.5 (typically 130–160 min).
4. Chill the culture on ice for 15 min to stop growth. From this
point on, keep cells cold at all times during processing. As the
culture chills, transfer ~37.5 mL aliquots into four 50 mL
conical centrifuges tubes, on ice.
5. Harvest the cells by centrifugation at 4600 Â g for 10 min at
4
C. Remove supernatant.
6. Add ~10 mL ice-cold sterile water to each tube, resuspend
pellets, then consolidate all aliquots into a single 50 mL tube.
Spin at 4600 Â g for 10 min at 4
C, then remove supernatant.
7. Add ~10 mL of ice-cold sterile water and resuspend. Mostly fill
tube with ice-cold sterile water (~30 mL). Centrifuge at
4600 Â g for 10 min at 4
C. Remove supernatant.
134
Kelsi Penewit and Stephen J. Salipante