Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

<p>Abstract</p> <p>Background</p> <p>The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.</p> <p>Results</p> <p>Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning.</p> <p>Conclusion</p> <p>The residual <it>att </it>sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.</p

Development of a Method to Detect Mycobacterium paratuberculosis in the Blood of Farmed Deer Using Actiphage® Rapid

&lt;jats:p&gt;&lt;jats:italic&gt;Mycobacterium avium&lt;/jats:italic&gt; subsp paratuberculosis (MAP) is the causative agent of Johne's disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.&lt;/jats:p&gt

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-0

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p>; 4 sites) were grown in LB medium containing 0.5% (w/v) xylose at 37°C. Growth (OD 600 nm; open symbols) and luminescence (Relative Light Units; RLU closed symbols) were monitored over time. Reporter gene data are presented as RLU/ODto account for increasing cell number during the experiment

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-1

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p> ●; no sites) were grown to mid-log phase in Tris Minimal Succinate medium with 5 μg mlCm and then diluted 1/20 into fresh medium supplemented with 0.5% xylose. Triplicate samples were placed in a 96 well microtitre plate and incubated at 37°C in a Tecan Genios Pro. Panel A: fluorescence (solid symbols, Relative Fluorescence Units; RFU) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3004 (□, ■; 4 sites), pSB3002 (□, ▲; 2 sites) and pSB3000 (○, ●; no sites). Panel B. luminescence (solid symbols, Relative Light Units; RLU)) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3014 (□, ■; 4 sites), pSB3012 (△, ▲; 2 sites) and pSB3010 (○, ●; no sites). Data is presented as % maximal signal to allow direct comparison of expression kinetics despite the fact that light levels from each construct were different

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-3

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p>operon under the control of the Ppromoter (see Table 2). Cells were grown in MWB at 37°C and luminescence (closed symbols, Relative Light Units; RLU)) and growth measurements (open symbols) were taken at intervals. Reporter gene data are presented as RLU/ODto account for increasing cell number during the experiment

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-2

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p> ●; no sites) were grown to mid-log phase in Tris Minimal Succinate medium with 5 μg mlCm and 0.5% xylose. Bacteria were washed and resuspended in an equal volume of medium containing 5 μg mlCm and 1% glucose. Aliquots were placed in a 96 well microtitre plate and incubated at 37°C in a Tecan Genios Pro. Panel A: fluorescence (solid symbols, (Relative Fluorescence Units; RFU) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3004 (□, ■; 4 sites), pSB3002 (□, ▲; 2 sites) and pSB3000 (○, ●; no sites). Panel B: luminescence (solid symbols, Relative Light Units; RLU) and absorbance (open symbols) were measured at 10 minute intervals. Plasmids used were pSB3014 (□, ■; 4 sites), pSB3012 (□, ▲; 2 sites) and pSB3010 (○, ●; no sites). Data is presented as % maximal signal to allow direct comparison of repression kinetics despite the fact that light levels from each construct were different