Oligonucleotide primers derived from annotated 50 kb contig of C. defragrans 65Phen (Acc. no. FR669447.2) . a wild type; b C. defragrans Δldi, c C. defragransΔgeoA. Ligation and transformation of plasmid constructs Fludarabine Subcloning of PCR products into pCR4-TOPO® vector (Invitrogen, Darmstadt,
Germany) was performed corresponding to manufacturer’s instructions. PCR products with GDC-0994 price inserted restriction sites and purified plasmids were digested with the appropriate restriction enzymes and separated by gel electrophoresis. Both digested plasmids and PCR products were gel excised and purified. For ligation reactions, an insert-vector ratio of 1:1, 3:1 or 10:1 was chosen. To this mixture, T4-ligase buffer (1x), Adriamycin purchase ATP (25 μM) and T4-ligase (2.5 U) were added. Incubation was for 12–16 h at 12°C. Transformation of 5 or 10
μL of the ligation reaction to chemical competent E. coli strains S17-1 or Top10 was performed as described . Single colonies growing on selective solid medium were picked and screened for the correct insert size by PCR applying M13 or T7 primers. Plasmids of positive tested clones were purified and served as sequencing templates. Construction of suicide plasmids The 5`- and 3`-flanking regions of ldi or geoA and the start and stop codons of the deleted gene separated by an appropriate specific restriction site were inserted into the suicide vector pK19mobsacB . Oligonucleotide sequences are listed in Table 4. Initially,
the flanking regions were amplified from genomic C. defragrans 65Phen DNA with primers adding restriction enzyme sites to the PCR-product. The 5`-flanking region to the ldi was obtained with the primer ADAM7 pair ORF25_EcoRI_F and ORF25_XhoIATG_R. During amplification of the 3`-flanking region with primer pairs ORF27_XhoI_TAA_F and ORF27_HindIII_R difficulties occurred due to a terminator structure in the genome sequence that was solved with a nested PCR approach. A 2.2 kb amplicon comprising ORF 27 was obtained with the primer pair p27plus_F and p27plus_R that served as template for the initial named primer with an increased initial denaturation time (from 4 min to 10 min). Sequencing of the 763 bp amplicon revealed a base exchange at position 373 from guanine to adenine causing an amino acid replacement from proline to threonine. This shift was revoked by a site directed mutagenesis approach using primer p27_mismatch_F and p27_mismatch_R in combination with ORF27_XhoI_TAA_F and ORF27_HindIII_R, respectively . The particular amplicons were bond to each other in another reaction with the exterior primer pair. The 5`-flanking region of the geoA was obtained with the primer pair ORF2930_XbaI_F & ORF2930_XhoI_R and the geoA 3`-flanking region ORF32_XhoI_F & ORF32_HindIII_R. The obtained products were subcloned into pCR4-TOPO (Invitrogen, Darmstadt, Germany) and yielded pCR4-ORF25, pCR4-ORF27, pCR4-ORF2930 and pCR4-ORF32.