Oligonucleotides and PCR primers used in the study.

<p>Oligonucleotides and PCR primers used in the study.</p

Heterologous expression of <i>lux</i> operon from pBAV1K-T5-<i>luxABCDE</i>.

<p>Five bacterial species (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013244#pone-0013244-g002" target="_blank">Fig. 2</a>) were transformed with pBAV1K-T5-<i>lux</i>. The transformants were propagated in liquid culture (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013244#pone-0013244-t003" target="_blank">Table 3</a>). The luminescence from equal volumes of culture (100 microliters) was measured in a luminometer. The values (plotted on a log scale) represent the averages of three independent experiments.</p

Plasmid stability assays.

<p>(A) Plasmids pBAV1K-T5-<i>gfp</i>, pGK12, pLZ12-T5-<i>gfp</i>, pQBAV3Cm-T5-<i>gfp</i> and pIMBB-T5-<i>gfp</i> were separately propagated in <i>E. coli</i> for 80 generations without antibiotic selection. Plasmid stability was determined by replica plating onto selective media and presented as a percentage of cells that retain antibiotic resistance. (B) Plasmids pBAV1K-T5-<i>gfp</i>, pGK12, pLZ12-T5-<i>gfp</i>, were propagated in <i>B. subtilis</i> for 80 generations without antibiotic selection. Plasmid stability was determined by the plasmid content comparison in the total DNA pools between different generations. Error bars represent standard error.</p

Bacterial strains and transformation procedures used in the study.

<p>Bacterial strains and transformation procedures used in the study.</p

Plasmid pBAV1K-T5-<i>gfp</i> replicates to high copy number in <i>Escherichia coli</i>.

<p>(A) <i>E. coli</i> was transformed with plasmids pBAV1K-T5-<i>gfp</i>, pLZ12-T5-<i>gfp</i>, pGK12 (two other RCR plasmids), pQBAV3Cm-T5-<i>gfp</i> or pIMBB-T5-<i>gfp</i> (two ColE1 derived plasmids). The transformants were propagated in liquid LB cultures supplemented with the appropriate antibiotics. The plasmids were purified, and 2 microliters of each were analyzed on a 0.8% agarose gel. The higher yield and faster mobility of the pBAV1k relative to the larger pWV01 derivatives indicates supercoiling. (B) Five different species of bacteria (namely <i>Agrobacterium tumefaciens</i>, <i>Streptococcus pneumoniae</i>, <i>Bacillus subtilis</i>, <i>Acinetobacter baylyi</i> ADP1 and <i>E. coli</i>) were transformed with pBAV1K-T5-<i>luxABCDE</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013244#pone-0013244-t003" target="_blank">Table 3</a>). The APH(3′)-IIIa gene present on the plasmid was used as a target to estimate the copy number in reference to the chromosomal <i>relA</i>/<i>spoT</i> gene (or its homolog) by quantitative real-time PCR. Each bar represents the average of three replicates. Error bars represent standard error.</p

Probes and Primers for Real Time PCR used in the study.

<p>Probes and Primers for Real Time PCR used in the study.</p

Construction of pBAV1K-T5-<i>lux</i>, a very broad host range expression vector.

<p>The cryptic plasmid, pWV01, exhibits broad host range but is unstable in many species. The ORF D, and inverted repeats IV, V and VI were deleted from its plasmid origin; terminators t0 and T1 were inserted on opposite ends of the shortened origin (upper right). The selectable marker, the <i>Enterococcus</i> 3′,5″-aminoglycoside phosphotransferase type III, and a T5 promoter within a BioBrick multiple cloning site were cloned into the plasmid (top circle). The lux genes of <i>Photorhabdus luminescens</i> were individually PCR amplified, cloned, assembled with ribosome binding sites (middle) and cloned into the plasmid to create pBAV1k-T5-<i>luxABCDE</i> (bottom circle).</p

Deletion mutations can affect the growth of <i>Escherichia coli</i>, and the production of light from bacterial luciferase.

<p><i>E. coli</i> Keio strains, and the parental BW25113 strain, were transformed with a bacterial luciferase expression vector (pIMBB-T5-<i>lux</i>). The transformants were shaken in minimal medium at 37°C for 48 hours; the optical density at 600 nm (green or blue) and luminescence (red) were recorded every 30 minutes. Data from the (A) parental <i>lux</i>/BW25113 control, and three representative Keio transformants, (B) <i>lux</i>/Δ<i>ybhC</i>, (C) <i>lux</i>/Δ<i>lon</i> and (D) <i>lux</i>/Δ<i>fiu</i>, are presented here. The growth curves of the three Keio strains differ from that of the BW25113 control, but the luminescence curves are all similar in shape.</p

The <i>lux</i>/Δ<i>thrL</i> and <i>lux</i>/Δ<i>hyfC</i> strains exhibit improvements over the parental <i>lux</i>/BW25113.

<p>Each strain was propagated (N = 6) for 24 hours in M9 medium supplemented with ampicillin and IPTG in a Biotek Synergy2 plate reader (37°C, medium shaking); the OD₆₀₀ and luminescence were recorded from each culture every 15 minutes. The lux/Δ<i>thrL</i> strain (blue squares, A) grows more quickly and to higher cell density than does the lux/BW25113 (green squares), and produces more light (B). The lux/Δ<i>hyfC</i> transformant grows more slowly the parental control (C), but produces more light (D).</p

The maximum growth rate and luminescence per cell of 384 lux/BW25113 control replicates (A) or <i>lux</i>/Keio transformants (B) are shown in scatter plots.

<p>The distances between points (green squares, A) reflect the variations among isogenic <i>lux</i>/BW25113 cultures in a 384 well plate; the error bars represent the variations between repetitions of the experiment (N = 3). The same data (green squares, B) was plotted among those of the <i>lux</i>/Keio transformant (blue squares, B) to facilitate direct comparisons.</p