oneidensis MR-1 genomic DNA as template. The PCR product was purified from an agarose gel, restriction digested with HindIII and XbaI and ligated into a HindIII and XbaI restriction digested pProbe NT vector yielding
pJM6. All reporter constructs were introduced www.selleckchem.com/products/XL184.html into E. coli S17-λ pir by standard procedures. Plasmid was then prepared from positive clones and introduced into S. oneidensis MR-1 wild type or mutant strains by electroporation. Quantitative cell aggregation assay S. oneidensis MR-1 wild type and mutant cells were grown in test tubes on a roller drum to exponential (OD600 = 0.3) and stationary phase (OD600 = 2.0) in minimal medium amended with 50 mM sodium lactate. Immediately after removing test tubes from the roller drum, one milliliter samples were taken and OD600 www.selleckchem.com/products/elacridar-gf120918.html was determined. Further samples were taken after 15 minutes and 30 minutes. After measuring the optical density, cells were vigorously vortexed for 20 seconds and the optical density measurement was repeated. The ratio of OD600 before and OD600 after dispersion was calculated and used as an approximation to estimate the extend of cell aggregation
in the different strains. Construction of gene deletions S. oneidensis MR-1 in-frame deletions were constructed by homologous recombination. The deletion constructs were created by amplifying the regions flanking the target gene. The fragment length was optimized to about 750 bp. The primers for the 5’- end fragment were 5-O (outside) and 5-I (inside) and the primers for the 3’- end fragment were 3-I (inside) and 3-O (outside). Subsequent to amplification, the flanking regions were
fused via a complementary Fenbendazole tag that was added to the 5’- end of each inner primer. The fusion product was inserted into the cloning vector pDS3.1 and the mobilizing strain E. coli S17-λ pir  was transformed with this sucicide vector. Functionality of the sacB gene was verified before STAT inhibitor transferring the deletion vector by conjugation into the S. oneidensis MR-1 target strain. Single crossover events were selected for on LB plates containing gentamycine and confirmed by using two primer combinations: 1) primer X-F and primer 3-O and 2) primer X-R and primer 5-O, whereas primer X-F and primer X-R will bind upstream and downstream of the flanking regions, respectively. The functionality of the sacB gene was verified in S. oneidensis MR-1 strains that tested positive for a single crossover event. Resolution of the integrated vector by a second crossover event was performed with a positive strain. This strain was grown in LB medium without selection and plated onto solid LB medium containing 10% sucrose. Deletion events were verified by PCR using primer X-F and primer X-R, where a successful deletion resulted in a PCR product with a size of the wild type product minus the size of the target gene.