lactis selleck inhibitor Bu2-60 derivative carrying pRE25 tagged with tet(M) flanked by two 23- and 11-bp random sequences in its chromosome. Next, pRE25* was transferred to E. faecalis CG110/gfp via filter mating and transconjugants were selected on KF Streptococcus Agar (Becton Dickinson) supplemented with chloramphenicol
(10 μg mL−1). The resulting strain was designated E. faecalis CG110/gfp/pRE25*, harboring a chromosomal gfp and pRE25* (Fig. 1). The presence of the gfp gene was confirmed by PCR using primers gfp_F and gfp_R (Table 2). Sequencing of the two regions overlapping the random sequence was performed by Microsynth using primer pairs seq1_fw/vr and seq2_fw/rv (Table 2) and confirmed that the random sequences flanking tet(M) were integrated downstream the ermB gene. Overnight cultures of donor
and recipient were mixed 1 : 3 and passed through a sterile 0.45-μm nitrocellulose filter (Millipore AG, Zug, Switzerland). The filter was incubated overnight cell-side up on nonselective plates under optimal conditions for the recipient. The filter was then washed by vortexing for 1 min in 2 mL of sterile dilution solution [0.85% NaCl, 0.1% selleckchem peptone from casein (VWR), pH 8.0] and transconjugants were isolated by plating appropriate dilutions on selective medium. Correct plasmid transfer was confirmed by PCR using primer pairs pRE25*_F/R and pRE25_F/R (Table 2). Listeria transconjugants were verified using the primer pairs lmoF/R and linF2/R2. Leuconostoc mesenteroides transconjugants were identified by the absence of a tufA gene according to a negative filipin PCR using primer pair tufA_fw/rv (Table 2). Marker stability in E. faecalis CG110/gfp/pRE25* was examined by cultivating the cells serially in BHI broth at 37 °C for at least 200 generations. The presence of pRE25* was confirmed by plating daily on BHI agar supplemented with chloramphenicol
(10 μg mL−1). The stability of gfp was verified by PCR with primers gfp_F and gfp_R. All reactions were performed in a reaction volume of 25 μL. For real-time PCR using the SYBR Green method, 12.5 μL of 2 × SYBR® Green PCR Master Mix (Applied Biosystems, Zug, Switzerland) and each primer at a concentration of 200 nM was used. In the TaqMan-based method, 12.5 μL of qPCR MasterMix Plus Low ROX w/o UNG (Eurogentech, Seraing, Belgium), each primer at a concentration of 300 nM and the TaqMan probe at a concentration of 200 nM was used. Amplification was performed in a 7500 Fast Real-time PCR System (Applied Biosystems) and data were analyzed using the 7500 fast sds software (Applied Biosystems). Total gene copy numbers were quantified using a DNA calibration curve obtained by plotting Ct values from serial dilutions of the corresponding target, obtained in the same qPCR run. To test whether pRE25* and gfp copy numbers can be quantified by qPCR in a complex ecosystem, fresh overnight cultures of E. faecalis CG110/gfp/pRE25*, Listeria monocytogenes 10403S, and L.