- Pick five to ten colonies that grew on the selective plate and
screen them for the presence of the gene by PCR. Prepare 10μl
PCR reaction mixtures containing 200 nM of the oligonucleo-
tides T7 Pro primer and T7 Term primer. Using sterile pipet
tips or toothpicks, pick the single colony, streak a small amount
onto a clean LB agar plate supplemented with 100μg/ml
ampicillin, and dip the tips into the PCR reaction mixtures.
Incubate the plates at 37C for 18–24 h and amplify the
gene in a thermocycler using the following parameters: 98C
for 2 min followed by 30 cycles of 98C for 10 s, 55C
for 15 s, and 72C for 2 min, and a final extension of 72C
for 10 min. - After PCR amplification, add 2μlof6gel-loading dye to the
PCR reaction mixtures. Load the solutions into the solid 1%
(wt/vol) agarose gel. In a separate well load an appropriate
DNA ladder. Run the gel in a DNA electrophoresis apparatus
at 120 V for 35 min. - After electrophoresis, view the gel using a UV illuminator.
Correct PCR amplification should lead to a DNA band of
approximately 1000 bp. - If the molecular weight of the PCR band is correct, inoculate
the corresponding colony from the plate created instep 12
into 5 ml of LB broth supplemented with 100μg/ml ampicil-
lin. Incubate overnight at 37C with shaking. - Harvest the cells by centrifugation at 4000gfor 10 min at
4 C. Extract the plasmid from the resulting cell pellet by using
a commercial kit for plasmid DNA extraction from bacterial
cells. - Sequence the plasmid with the oligonucleotides described in
step 12to confirm the presence of thegklgene.
3.2 Overexpression
and Purification of the
GKL Lactonase
- Add 5μl of purified plasmid (prepared in Subheading3.1,step
16 ) into 50μl of chemically competentE. coliBL21 (DE3)
cells (Novagen). Incubate the cells on ice for 30 min. Trans-
form the cells using heat shock treatment. Heat the cells con-
taining the plasmid at 42C for 30 s. Incubate the cells on ice
for 2 min. Add 200μl of LB broth into the transformed cells.
Incubate and shake the cells at 37C for 1 h. Plate 100μl of the
transformed cells onto selective LB agar plates supplemented
with 100μg/ml ampicillin. Incubate the plates at 37C for
16–24 h. - Pick one colony that grew on the selective plate. Using a sterile
tip or inoculation loop, scrape cells from the plate and dip it
into 5 ml of LB broth supplemented with 100μg/ml ampicil-
lin. Close the tube and let it shake overnight at 37C.
316 Maybelle Kho Go et al.