Reporter gene systems are useful for studying bacterial molecular biology, including

Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the gene is a useful reporter gene in and (especially and have all been developed into useful cell factories for producing recombinant proteins [8]. However, the known LAB protein expression systems usually confer only a low level of recombinant protein expression. In order to test the efficiency of different LAB expression systems, a suitable reporter gene system is required. Several genes are used as reporter genes in LAB bacterial genetics, such as those encoding chloramphenicol acetyltransferase (from and from -amylase [11], the luciferase [12], the -glucuronidase [13], the -galactosidase [14], and the nuclease [15]. Moreover, green fluorescent protein ([16] has been exploited as a reporter and used successfully in a variety of bacteria including different LAB organisms [17C19]. Here, we investigated the usefulness of a known reporter gene, the -glucuronidase gene, and another heterologous gene, the -1,4-mannanase gene, in using the expression vector pELX1 derived from the naturally occurring pMC11 plasmid [20]. Furthermore, the transmission peptide for the secreted protein Usp45 from [21] and the full-length SlpA protein from your S-layer of NCFM [22] were tested for their capacity to drive the secretion of heterologous proteins in strains were produced in MRS broth (OXOID, Hampshire, England) at 30C without shaking. JM109 was propagated in Luria-Bertani medium (Thermo Scientific Molecular Biology, Waltham, MA, USA) at 37C with moderate shaking. Antibiotics were used as follows: ampicillin (100 gml-1, Sigma, St. Louis, MO, USA) for strains and erythromycin (10 gml-1, Sigma) for strains. Table 1 Bacterial strains and plasmids utilized in this study. DNA Manipulations Standard molecular cloning techniques were performed [23]. Total DNA was extracted from cells according to a previously explained method [20]. Plasmid DNA was isolated from using an Omega Plasmid Mini-Preparation Kit buy 26833-85-2 (OMEGA bio-tek, Norcross, GA, USA), and PCR products were purified with an Omega Cycle-Pure Kit (OMEGA bio-tek, Norcross, GA, USA). Pfu and Taq polymerases were purchased from Transgen (Beijing Transgen Co. Ltd., Beijing, China), and DNA restriction enzymes and T4 DNA ligase were purchased from Fermentas (Thermo Scientific Molecular Biology). All enzymes were used according to the manufacturers instructions. Construction of plasmids for recombinant protein expression DNA fragment encoding the mature peptide of was PCR amplified with the primers as template. Rabbit Polyclonal to TF3C3 The PCR product was digested with the restriction enzymes (REs) was obtained by PCR, and the producing PCR product was cloned into pELX1 to buy 26833-85-2 generate the other expression plasmid, pELX1-GusA. Table 2 Primers utilized in this study. For protein secretion, the DNA fragment encoding the transmission peptide of secreted protein Usp45 in was amplified using the primers SOE-SPUsp45-F and XhoI-SPUsp45-R. The promoter fragment of the S-layer protein precursor-encoding gene (Pgene with native promoter, was amplified from NCFM genomic DNA using the primers gene was cloned into the vectors pELSH and pELWH using the same strategy as for pELX1-ManB, yielding pELSH-ManB and pELWH-ManB, respectively. All constructs were verified by restriction analysis and sequencing. Electroporation transformation Electroporation transformation of was performed according to previously reported [20] using a Multiporator (Eppendorf, Hamburg, Germany) and the transformants were screened on MRS agar plates made up of erythromycin. Detection of target proteins in lactobacilli cells For intracellular expression, 500 ml MRS broth was inoculated with a transformant culture grown overnight (1%, buy 26833-85-2 v/v), 20 ml fractions of the culture were taken every 1 hours to measure the OD600 (optical density at 600 nm), and cells were then collected by centrifugation at 12000 rpm for 5 min. Whole cell proteins.