Biology Reference
In-Depth Information
lyophilized form in pairs of 96-well blocks, the first containing the forward primers,
the second containing the reverse primers. Handling steps are performed with a
TECAN robot (Tecan Group Ltd., Switzerland). Oligonucleotides are resuspended
and diluted to 20 pmol/
l. PCRs to amplify DNA fragments are set up in 96-well
trays with well volumes of 0.2 ml. Each reaction contains 1
m
m
l each of the forward
and reverse primers, 48
buffer
and DNA polymerase) and 25 ng of chromosomal DNA. Having confirmed success-
ful amplification of DNA fragments of the correct size (
m
l of a master mix (containing dNTPs, MgSO
4
,10
400 bp) by gel electropho-
resis, DNA products are purified using the QIAGEN 96-well PCR clean up kit
(QIAGEN, Crawley, UK) with the steps carried out on the TECAN robot addition-
ally incorporating a vacuum manifold. The concentration of the purified DNA is
determined using a nanodrop spectrophotometer. LIC single-stranded DNA over-
hangs (
Figure 1.1
) are generated in a 96-well tray, adding to each well 0.2 pmol
of each PCR product, 2.5 units of LIC qualified T4 DNA polymerase enzyme
(Novagen), 2
m
lof10
T4 polymerase buffer (Novagen, Darmstadt, Germany),
m
m
1
l of 100 mM dithiothreitol, and 2
l of 25 mM dTTP in a final volume of
l. The plate is incubated at 22
C for 30 min and the reaction stopped by heat
treatment at 75
C for 20 min. Approximately 5 ng of each T4 DNA polymerase-
treated PCR product is mixed with
m
20
15 ng of linear pBaSysBioII with compatible
LIC ends (
Botella
et al.
,2010
). Plasmid and insert are annealed for a minimum of
10 min and transformed into appropriate
E. coli
strains. Twenty microlitres of the
transformation cell suspension from each of the 96 wells is plated onto 1 ml of solid
Luria Bertani (LB) agar medium with appropriate antibiotics, that is, four plates each
with 24 wells. Two colonies from each agar well are picked into 1 ml of liquid LB
medium in 96-well plates (well volume 2 ml) and grown at 37
C.
Plasmid DNA is isolated using a QIAGEN 96-well miniprep kit, in steps carried
out on the TECAN robot using the vacuum manifold, as specified in supplier's
instructions. The concentration of the purified plasmid is determined by nanospec-
trophotometry and inserts are verified by DNA sequencing. The success rate for clon-
ing inserts with correct sequences was typically 80% for a 96-well plate. Plasmids
encoding transcriptional fusions are transformed into
B. subtilis
by the procedure
of
Anagnostopoulos and Spizizen (1961)
.
7.4
Strain storage
The capability to generate large numbers of promoter fusion-containing strains
requires the establishment of a coordinated system to store, transfer, and grow them
and to determine promoter activity in a high-throughput manner. We have developed
a methodology to perform these tasks that gives highly reproducible growth and
expression profiles. We use a Boekel 96-pin replicator (Boekel Scientific, PA,
USA) and a multichannel pipette for rapid manipulation and growth of strains in
a 96-well format (
Figure 1.2
). Individual promoter fusion containing
B. subtilis
trans-
formants are picked using toothpicks and inoculated into 0.75 ml LB broth in 96-well
plates with a well capacity of 2 ml. Strains are not stored in the outer wells of the
