Development of expression vectors for Escherichia coli based on the pCR2 replicon

<p>Abstract</p> <p>Background</p> <p>Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility.</p> <p>Results</p> <p>We utilized the pCR2 replicon of <it>Corynebacterium renale</it>, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different <it>Escherichia coli </it>plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these <it>E.coli </it>plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the <it>lac </it>promoter in an expression plasmid pCR2GFP. Also high level production of human IFNγ was obtained by cloning the h-IFNγ under a T7 promoter in the expression plasmid pCR2-IFNγ and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNγ, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility.</p> <p>Conclusion</p> <p>We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.</p

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

AbstractStudies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis

Multidrug resistant enteric fever in South Asia: unmet medical needs and opportunities.

Investments in newer diagnostics and antimicrobial treatments are critical to improve management of enteric fever in South Asia, say Christopher Parry and colleague

Precise excision of the <i>vlsE</i> variable region during PCR amplification.

<p><b>A)</b> Schematic showing the variable, constant and 17 bp direct repeats of the <i>vlsE</i> gene. The location of PCR primers used for amplification are shown by arrows below the constant regions. <b>B)</b> An ethidium bromide-stained agarose gel showing amplification of a portion of <i>vlsE</i> with Phusion DNA polymerase using three different templates and two different primer sets. The templates used were <i>B. burgdorferi</i> B31 5A4 genomic DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Purser1" target="_blank">[29]</a> pMBL20, a plasmid carrying the <i>vlsE</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Lawrenz1" target="_blank">[51]</a>; the 776 bp or 935 bp PCR products resulting from PCR amplification. The asterisks indicate smaller discrete bands observed in lanes 1–3 and 4–6. M denotes a 100 bp molecular weight ladder marker. PCR samples were run on a 1.2% agarose gel in 1X TAE buffer at 80 V for 1.2 hours. <b>C)</b> Characterization of precise deletions in <i>vlsE</i>. The first entry on the left side of the panel shows the sequence obtained from direct sequencing of the PCR product obtained with either B248 and B249 or B1701 and 1702. The remainder of the lineup shows sequence generated with 10 randomly selected <i>E. coli</i> transformants. The transformants were generated by cloning the 223 bp truncated PCR product generated <i>in vitro</i> with primers B248 and B249. The fragment was gel-excised and cloned into pJET1.2/blunt vector (Fermentas). The alignment shows that all the sequenced <i>vlsE</i> inserts had a precise excision of the 570 bp variable region. <b>D)</b> Schematic showing the precise excision of the <i>vlsE</i> variable region that occurred during PCR amplification.</p

Distribution of G_3–5 runs on the linear plasmids carrying the <i>vls</i> loci in <i>B. burgdorferi</i> strains B31, N40 and JD1.

<p>G runs of 3–5 nucleotides in length were counted in the sequence of lp28-1 (Accession NC_001851 and FJ472338) from B31 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Zhang1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Casjens2" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Tourand1" target="_blank">[53]</a>, lp36 (Accession CP002230) from N40 and lp28-1 (Accession NC_017404) of JD1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Schutzer1" target="_blank">[36]</a>. The distribution of G_3–5 runs on both strands of <i>vls</i> and non-<i>vls</i> DNA was plotted. The coding strand is defined as the strand in which the silent cassettes code for the VlsE protein. The <i>vls</i> DNA includes only the silent cassettes, as the <i>vlsE</i> sequence is only known for B31.</p

Schematic of antigenic variation in <i>B. burgdorferi</i>.

<p><b>A)</b> Illustration showing the arrangement of the <i>vls</i> expression site (<i>vlsE</i>) and the contiguous array of 15 silent cassettes comprising the <i>vls</i> locus at the right end of the linear 28 kb plasmid, lp28-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Zhang1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Norris2" target="_blank">[13]</a>. <i>vlsE</i> is composed of a variable (blue) and two constant (yellow) regions separated by 17 bp direct repeats. The variable region is where DNA switching occurs. The 15 silent cassettes are displayed in different colors because they carry different variable regions. The silent cassettes are also separated by 17 bp direct repeats and are found in the opposite orientation of <i>vlsE.</i> Silent cassettes act as a source of DNA for nonreciprocal recombination events with the expression locus whereby segments within the <i>vlsE</i> variable region are replaced by sections of varied length from the silent cassettes. <b>B)</b> A comparison of the genes implicated in antigenic switching or its control at the <i>pilE</i> locus in <i>N. gonorrhoeae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Hill1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Cahoon1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Koomey1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Hill2" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Stohl1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Skaar1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Sechman1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Rohrer1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Mehr1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Mehr2" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Mehr3" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Kline1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Helm1" target="_blank">[26]</a>, and the <i>vlsE</i> locus in <i>B. burgdorferi </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Dresser1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Lin1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057792#pone.0057792-Liveris1" target="_blank">[39]</a>. A+sign denotes genes whose absence has a significant affect upon switching, while a – sign indicates genes that have no apparent involvement. The term “absent” indicates genes that are not present in the <i>B. burgdorferi</i> genome.</p

Methylation protection of the <i>vlsE</i> G4-forming sequence.

<p><b>A)</b> Autoradiogram of a 25% denaturing polyacrylamide gel of dimethylsulfate treated 14-mer and 17-mer oligonucleotides. ³²P-labeled oligonucleotides were annealed in 200 mM KCl and treated with 0.5% dimethylsulfate. The methylated oligos were then subjected to electrophoresis in a 20% native polyacrylamide gel to separate the single stranded (SS) oligos from the G4-DNA. The free and the G4 bands were treated with 1 M piperidine to induce strand breaks at the methylated guanine residues and the cleavage products were resolved on a 25% denaturing polyacrylamide gel. C represents the control oligo which was not treated with DMS. The arrows correspond to the DMS-protected guanine residues. <b><i>B)</i></b> Sequence of the oligonucleotides used in this experiment. The dots show the position of G residues. The smallest cleavage fragment was run off the bottom of the gel.</p

The affect of DNA strand and mutations in the G–motif on quadruplex formation.

<p><b>A,C)</b> Oligonucleotides corresponding to the wild-type 14-mer and 17-mer top (see <b>Fig. 4</b>) and bottom strands, and mutated top strands in which four successive G’s were substituted with TATA. <b>B,D)</b> Autoradiogram of a 20% native gel run to assess G4 formation, as described in <b>Fig. 4</b>.</p

Affect of mutations in the 17 bp direct repeats on precise excision of the <i>vlsE</i> variable region.

<p><b>A)</b> DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. <b>B)</b> Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. <b><i>C</i></b><b>)</b> An ethidium bromide-stained agarose gel showing amplification of a portion of <i>vlsE</i> with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in <b>Fig. 2</b>.</p