Gene Cloning and Standard Molecular Biology Procedures

Gene cloning and standard molecular biology procedures:

Coding regions of selected genes were amplified using primers 832f and 832r (for gene Bbr_0832), 1756f and 1756r (for gene Bbr_1756), 1804f and 1804r (for gene Bbr_1804), 1805f and 1805r (for gene Bbr_1805), 1826f and 1826r (for gene Bbr_1826), 1827f and 1827r (for gene Bbr_1827), 406f and 406r, (for gene Bbr_0406) and 407f and 407r (for gene Bbr_0407) (supplementary table 1). In those cases where cloning in the NcoI site of pNZ8048 was not possible without changing an amino acid from the translated protein, alternative cloning was made into the blunt-ended ScaI site of pNZ8150. Thus, amplified regions comprising Bbr_0832, Bbr_1756 and Bbr_1826 were digested with SmaI and XbaI, and ligated into pNZ8150, which had been digested with ScaI and XbaI, yielding pNZ8150-832, pNZ8150-1756 and pNZ8150-1826, respectively. Amplified regions comprising genes to be cloned into pNZ8048 were digested with NcoI, or another restriction enzyme that would generate NcoI-compatible ends (BspHI or PciI). Thus, amplified regions containing Bbr_1805, and Bbr_0406, were digested with BspHIand XbaI, and then ligated into pNZ8048 previously digested with NcoI and XbaI, yielding pNZ8048-1805, and pNZ8048-406, respectively.

For Bbr_0838 gene, as its internal sequence contained a BspHI restriction site located 986 bp downstream from the start codon, cloning was performed in two steps. First, a region comprising the Bbr_0838 gene was amplified using primers 838f and 838r, which was then digested with BspHI, and the resulting DNA was ligated into pNZ8048 which had been digested with NcoI, yielding pNZ8048-838A. In the second step, the 3’ end of the Bbr_0838 coding region was amplified from chromosomal DNA using primer 838-IIf, which is located 20 bp upstream from an internal KasI site, and primer 838r. Amplified DNA was digested with KasI and XbaI, and ligated into similarly digested pNZ8048-838A, yielding pNZ8048-838.

Finally, constructs containing multiple genes were made. Thus, DNA regions comprising both Bbr_1804 and Bbr_1805, or Bbr_1826 and Bbr_1827, were amplified using primers 1804f and 1805r (to encompass Bbr_1804 and Bbr_1805) and 1827f and 1826r (to encompass Bbr_1826 and Bbr_1827), respectively. The two amplicons were digested with PciI and XbaI, and ligated into pNZ8048, which had been digested with NcoIand XbaI, yielding pNZ8048-1804-1805 and pNZ8048-1826-1827, respectively. Our inability to clone Bbr_0406 and Bbr_0407 together in a single construct, forced us to clone the individual genes each in a different vector. In this way, Bbr_0407 was amplified using 407f and 407r, and the amplified DNA fragment was digested with BspHI and XbaI, and ligated into pNZE8048 previously digested with NcoI and XbaI, yielding pNZE8048-407. This last construct was then transformed into competent cells of L. lactis carrying pNZ8048-406, thus generating a clone carrying both pNZ8048-406 and pNZE8048-407 (which could be stably maintained due to the different antibiotic markers present on each plasmid).

Electrocompetent cells of L. lactis and transformation were performed according to previously described procedures (P.G. de Ruyter et al., Appl. Environ. Microbiol. 62:3662-3667, 1996). The absence of PCR-introduced DNA mutations was verified in the final constructs through sequencing both DNA strands on an ABI Prism sequencer (Applied Biosystems). Every construct was first introduced into L. lactis NZ9000 and once the fidelity of its DNA sequence was verified, it was isolated from NZ9000 clones and transformed into competent cells of L. lactis ΔlmrCD.