Biology Reference
In-Depth Information
One of the more elegant approaches in strain construction is the adaptation of the
l
Red system originally developed to create markerless gene knockout strains in
E
.
coli
(
Datsenko and Wanner, 2000
). We have successfully used this system to cre-
ate markerless GFP and mCherry strains in
E
.
coli
, which contain gene fusions driven
by their wild-type promoters (
Doherty
et al.
, 2010
). Briefly, a PCR product contain-
ing the fluorescent tag and antibiotic resistance cassette flanked by homology to the
gene of interest is transformed into electrocompetent
E
.
coli
expressing the
Red
genes, and recombinants are selected for using the antibiotic resistance. This resis-
tance cassette is flanked by FLP-recombinase sequences (FRT), which allows
subsequent removal when transformed with pCP20, which encodes the FLP recom-
binase (
Table 4.1
). The protocol for creating fluorescently labelled
E
.
coli
strains
using the
l
Red system is outlined below and schematically in
Figure 4.3
. We illus-
trate the process using the chloramphenicol resistance gene from pKD3, but the
system works equally well using the kanamycin resistance gene from pKD4. (NB.
pKD46 and pCP20 are temperature sensitive plasmids and will be lost from the cells
when grown at 37
C without ampicillin selection.)
l
1.
PCR amplify the chloramphenicol resistance gene (
Figure 4.3
light grey box)
flanked by the FLP-recombinase sequences (
Figure 4.3
grey striped boxes) using
pKD3 as the template. Separately, amplify the desired fluorescent protein
(
Figure 4.3
dark grey box) from any available template. The forward primer used
for amplifying the antibiotic resistance gene and the reverse primer for amplifying
the fluorescent protein-encoding gene are engineeredwith the same restriction site
(or restriction sites with compatible ends) to allow ligation. Refer to
Table 4.1
for
primers used to create the
gfpmut3
-chloramphenicol resistance cassette.
2.
Clean up both the PCR products using a standard PCR cleanup kit, digest with
appropriate restriction enzyme(s) and ligate. Using the fluorescent protein
forward primer and the chloramphenicol acetyl transferase reverse primer,
perform PCR on the above ligation and gel purify the
1800-bp product. This
can be used as the template for creating any C-terminal protein fusion.
3.
Design primers that will amplify the above template that also contain the last
50 nt of the gene of interest (not including the stop codon) and 50 nt downstream
of the stop codon. This provides enough homology for the recombinase to
integrate into the chromosome.
4.
Transform the desired
E
.
coli
strain with pKD46, the
Red recombinase
expressing plasmid, and plate onto nutrient agar containing 100
l
g/mL
ampicillin and grow at 30
C overnight. From a single colony, grow at 30
Ctoan
OD
600
of 0.1 in LB containing 100
m
g/mL ampicillin before adding
L
-arabinose
to a final concentration of 10 mM to induce
m
Red recombinase expression. Grow
until an OD
600
of 0.4 is reached and then make the cells electrocompetent using
standard procedures. Now, electroporate the PCR construct into the pKD46-
induced electrocompetent cells and plate onto nutrient agar plates containing
chloramphenicol (20
l
g/mL) and incubate overnight at 37
C (see below for an
electroporation protocol). Once recombination in the strain has been confirmed
m
