Biology Reference
In-Depth Information
To render Pseudomonas putida KT2440 electrocompetent, cells were grown in 50 ml
of EM-medium to OD600 of 0.6 to 1.0 and subsequently cooled on ice for 15 min.
The cells were centrifuged (4,000 g, 4°C) and washed twice with H
2
O
dist
. The cells
were washed twice in 0.3 M cold solution of sucrose and resuspended in 0.5-1.0 ml
of 0.3 M sucrose solution. The electrocompetent cell were used for transformation
by electroporation with Gene Pulser (BioRad, Munich, Germany) using the EZ:TN
<Kan-2> Tnp Transposome. The 20-40 μl of cells was mixed with 1-2 μl of DNA in
ice-cooled cuvette. The electroporation setting were 25 μF, 200 Ω, and 1.7 or 2.5 for
the gap size 0.1 and 0.2 cm, respectively. After 2 hr of incubation in SOC-medium,
transformants were selected on EM agar plates with 60 μg/ml of kanamycin. Selection
of auxotrophic mutants was performed on minimal medium with acetate as the sole
carbon source, by replica-plating
P. putida
KT2440::Tn5(Kan
r
) strains on the minimal
and EM media.
Identification of Flanking Sequences
The auxotrophic
P. putida
KT2440::Tn5(Kan
r
) mutants were genotyped by enrichment
of either flanking sequences of transposon insertions using PCR [85, 86]. Two rounds
of amplifications were performed using primers specific to the ends of transposons
and random primers that can anneal to the chromosome. In the first round of amplifi-
cation the Kan-2 RP1 (5′-GCAATGTAACATCAGAGATTTTGAG-3′) primer com-
plementary to the end of Tn5-element and the arbitrary primer ARB1 (5′-GGCCAC-
GCGTCGACTAGTACNNNNNNNNNNGATAT-3′) were used. A 1 μl of supernatant
from a
P. putida
KT2440 lysate was used as the DNA-template. The PCR-reaction
was performed in following mixture (H
2
O
dist
- 28.7 μl, incubation buffer(10×) - 5 μl,
dNTPs(5 μM) - 5 μl, primer(10 μM) - 2,5 μl, Taq DNA-polymerase (5U/μl) - 0.2 μl)
under following conditions: (i) 5 m at 95°C, (ii) 30 × (30 s at 30°C, 90 s at 72°C), (iii)
30 × (30 s at 95°C, 30 s at 45°C 120 s at 72°C). In the second round of amplification a
5 μl of product of the first PCR-reaction was used as the DNA-template, together with
the primers TnINT Rev (5′-GAGACACAATTCATCGATGGTTAGTG-3′) and ARB-
2 (5′-GGCCACGCGTCGACTAGTAC-3′). The reaction conditions were following:
30 × (30 s at 95°C, 30 s at 45°C, 120 s at 72°C). The PCR-products were purified
with “QIAquick- spin PCR Purification Kit” (Qiagen GmbH, Hilden, Germany) ac-
cording to manufacturer's instructions. Subsequently, the sequencing procedure was
performed. The 200-500 μg of dsDNA in normal sequencing vectors (pBlueskript,
pUC18, etc.) with 10 pmol of primer (TnINT Rev) and 6 μl of “Big Dye Terminator v.
2.0 Ready Reaction Mix” were mixed in total volume of 10 μl. The conditions of the
reaction were following: 25 × (30 s at 95°C, 30 s at 60°C, 4 m at 60°C]. After the cy-
cle sequencing the remaining dNTP were removed using “Dye Ex Spin Kit” (Qiagen
GmbH, Hilden, Germany) according to manufacturer's instructions. To the purified
product 50 μl sterile MiliQ-H2O was added and the DNA was precipitated wit 250 μl
Ethanol (100% v/v) for 30 min at 16,000 × g in the room temperature. The supernatant
was removed and the pellet washed with 250 μl of ethanol (100% v/v), precipitated
again by centrifugation (16,000 × g, RT, 10 min) and dried in vacuum-centrifuge. All
the DNA-pellets were stored in −20°C in 20 μl Hi-Di Formamide (PE Biosystems) un-
til sequencing. The sequencing was performed with ABI PRISM 377 sequencer [87].
