Mutated triplets selleck chemicals are underlined. The start codon of inlA is in italics. Production of electrocompetent Lactococcus lactis The protocol of Holo and Nes [19] was adapted

for the transformation of L. lactis MG1363 derivative NZ9000. A GM17 overnight culture of NZ9000 was diluted 1:100 into 5 ml of GM17 containing 500 mM sucrose and 2.5% glycine (GS-GM17). This culture was inoculated into 50 ml of fresh GS-GM17 and grown overnight. The 50 ml culture was inoculated into 400 ml of fresh GS-GM17, grown to OD600 of 0.3 and cells were subsequently harvested by centrifugation at 4,000 × g for 20 min at 4°C. The pellet was resuspended in 200 ml of ice cold SGB (500 mM sucrose and 10% (w/v)

glucose – filter sterilized), centrifuged, resuspended in 100 ml SGB and left on ice for 15 min. The cells were centrifuged, resuspended in 50 ml SGB and left on ice for 15 min Selleckchem IWR1 before a final centrifugation and re-suspension with 2 ml SGB. Cells were frozen at -80°C in 40 μl aliquots. To electroporate, cells were thawed on ice, mixed with 4 ul of pellet paint (Novagen) precipitated DNA and transferred to a 1 mm electroporation cuvette (Biorad). Cells were pulsed at 20 kV/cm, 200 Ω and 25 μF, regenerated in 1 ml GM17 containing 2 mM CaCl2/20 mM MgCl2 for 1.5 h and then plated onto GM17 agar containing 5 μg/ml chloramphenicol. An efficiency of 1 × 107 cfu/μg was routinely obtained with pNZ8048. Cloning of InlA into pNZB The unique BglII site up stream oxyclozanide of the nisA promoter in pNZ8048 was removed by linearization of the vector with BglII and ends blunted with T4 DNA polymerase. The vector was religated to

generate pNZB. The inlA gene was PCR amplified (primers IM194 and IM188) as described previously [20], digested with NcoI/PstI and ligated into the complementary digested pNZB. Ligations were directly electroporated into NZ9000 as described above and the sequence of the inlA gene was verified by DNA sequencing. QuikChange mutagenesis in L. lactis Primers for site directed mutagenesis (SDM) (Table 1) were designed according to the Quikchange SDM manual (Stratagene). All plasmid template isolated from NZ9000 strains was methylated with Dam methylase following manufacturer recommendations (New England Biolabs). The PCR thermocycling conditions were conducted as described previously [21]. Separate 50 μl KOD hotstart high fidelity polymerase PCR reactions were preformed with each primer for 10 cycles and an extension time of 5 min 30 sec.