Purification and cloning of SOE fragments
The SOE products for use as templates were purified by electrophoresis
in agarose (0.8% agarose, Promega) in TAE buffer (40 mM Tris-acetate,
1 mM ethylenediaminetetraacetic acid) with 0.5 µg
mL–1 ethidium bromide. DNA was recovered from the gel
fragment using a DNA Purification Kit (GeneAll, Seoul, South Korea). The
final recombinant products were gel purified prior to cloning.
The SOE products were TA cloned into pGEM-T Easy (Promega) and sequenced
by Macrogen Services (Daejeon, South Korea). Error-free clones were
digested with Xho l and Sac l and ligated into the
corresponding positions in pBBR1MCS5 (Kovach et al ., 1995).
RT-PCR analysis of wild-type P. ananatis carotenoid cluster
genes
Wild-type P . ananatis PA13 was grown in LB medium to
exponential growth phase (12 h after inoculation); total RNA was
isolated using an RNeasy Mini Kit according to the supplier’s
instructions (Qiagen); and the RNA samples were treated with RQ1 DNase
(Promega) to remove any contaminating DNA. RT-PCR was performed
according to a previous report (Kim et al ., 2004) as follows.
Total RNA from P . ananatis PA13 was reverse transcribed
into cDNA using M-MLV reverse transcriptase as described by the
manufacturer (Promega) at 50°C for 1 h, followed by 5 min at 75°C. Next,
PCR was performed using a T100 Thermal Cycler (Bio-Rad) under the
following conditions: 96°C for 2 min, followed by 40 cycles of 96°C for
1 min, 50°C for 1 min, and 72°C for 1 min. The following primers were
used for RT reactions, RT1 (crtB ) and RT2 (crtZ ). The
following PCR primers were used: PCR1f and PCR1r; PCR2f and PCR2r; PCR3f
and PCR3r; PCR4f and PCR4r; and PCR5f and PCR5r (Supplementary Table
S3). Southern hybridisation and DNA sequencing were carried out to
confirm the RT-PCR products. As a positive control, pCOK218 DNA was
used. As a negative control, PCR reactions with the same primer sets
were performed using RNA samples that had not been reverse transcribed.