Biology Reference
In-Depth Information
by PCR, patch onto nutrient agar plates with and without ampicillin to confirm
loss of pKD46.
5.
Make the new strain electrocompetent, transform with pCP20 and plate onto
nutrient agar containing 100
g/mL ampicillin and grow at 30
C overnight.
From a single colony, grow at 30
CtoanOD
600
of 0.5 in LB before inducing
FLP-recombinase expression by shifting the temperature to 43
C for 1 hour, then
plate onto nutrient agar plates (serial dilutions will be needed to ensure single
colonies are obtained). To confirm loss of antibiotic resistance and the loss of
pCP20, patch onto nutrient agar without any antibiotics, with ampicillin and with
chloramphenicol. Select a colony with no antibiotic resistance and check
microscopically for fluorescence.
m
2.3
Staphylococcus aureus
S
.
aureus
is one of the more recalcitrant bacterial species with respect to genetic mod-
ification due to the presence ofmultiple restriction andmodification systems.A recently
developed non-integrative low-copy plasmid system has been utilised for protein loca-
lisation studies and represents a major breakthrough in studying subcellular protein
dynamics in this important pathogen (
Liew
et al.
,2011
). This system uses a low-copy
number plasmid (pLow) with a tightly controlled IPTG-inducible promoter to provide
precise control of the level of protein fusion produced, and is currently themost efficient
way to fluorescently label proteins in this organism. pLow is a shuttle plasmid that can
be grown in
E
.
coli
for cloning and amplificationpurposes prior toelectroporation into
S
.
aureus
using the approach described by
Grkovic
et al.
(2003)
.
2.4
Acinetobacter spp.
Acinetobacter
spp. are fast emerging as very important clinical pathogens and are the
focus of increased research.
A
.
baylyi
ADP1 could be classified as a super-competent
organismandcanbe transformedwith recombinantDNAbysimplyadding it toanexpo-
nentially growing culture (
de Berardinis
et al.
,2008
).
A
.
baumannii
transformation can
be achieved by standard electroporation procedures such as that outlined below.
1.
From a single colony, grow overnight at 37
C in 2 mL of LB
2.
In the morning, make a 1:50 dilution in 10 mL of LB and grow to an OD
600
of 0.5
3.
Remove cells to a fresh 50-mL Falcon-type tube and chill on ice for 10 min
before pelleting cells
4.
Wash the pellet three times in 5 mL of chilled dH
2
O, before resuspending in
400
LofdH
2
O
5.
Mix 1 ng of DNA to be transformed with 50
m
L of cells in a 0.2-cm
electroporation cuvette and electroporate according to manufacturer's
instructions (settings for BioRad MicroPulser are 25 mFD, 200 W and 2.5 kV)
We have created C-terminal fluorescent gene fusions by single crossover with inte-
grative plasmids such as those designed for
B
.
subtilis
(
Table 4.1
). From our expe-
rience, at least 500 bp of homology is required to ensure efficient recombination.
m