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2.2.2.1 Genomic DNA purification from Lactobacillus rhamnosus

For PCR and Southern blot, genomic DNA was purified from L. rhamnosus strains using

a modified method described by Jankovic et al. (2007) [291]. Briefly, bacteria were

grown in 20 ml of MRS broth at 37Ԩ overnight. Cells were pelleted by centrifugation at 5,500xg for 10 minutes (min) at room temperature and subsequently resuspended in 20 ml of fresh MRS broth. The cultures were incubated at 37Ԩ for 2 hours (h). The cells were then harvested by centrifugation and washed twice in Buffer A (30 mM Tris pH 8.0, 5 mM EDTA and 50 mM NaCl) and the final cell pellet was resuspended in 0.5 ml of Buffer B [the lysis buffer; 50 mM Tris pH 8.0, 1 mM EDTA, 25% (w/v) sucrose, 20 mg/ml lysozyme and 20 μg/ml mutanolysin (Sigma-Aldrich)]. The resuspended cells were incubated in the Buffer B at 37Ԩ for 1 h to weaken the bacterial cell wall, followed by addition of 0.5 ml of 0.25 M EDTA and incubation at room temperature for 5 min. Next, 200 μl of 20% (w/v) SDS was added to solubilise the membranes and the mixture was incubated at 65Ԩ for 1 h. Proteinase K (10 μl of 20 mg/ml solution; Roche Applied Sciences) was added to the mixture and the reaction was incubated at 65Ԩ for 15 min to degrade the proteins. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) solution was added and mixed thoroughly. After centrifugation at 5000xg for 10 min, the DNA-containing aqueous phase was carefully transferred to a new tube and this purification step was repeated. DNase-free RNase was added to the aqueous fraction harvested after the second extraction at a final concentration of 100μg/ml and the reaction was incubated at 37Ԩ for 30 min followed by two phenol/chloroform/isoamyl alcohol extractions as described above until the white particulate protein layer at the interface was minimal. The DNA was precipitated by adding two volumes of cold 95% (v/v) ethanol

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and incubation on ice for 30 min. The DNA pellet was obtained by centrifugation at 14,000xg at 4Ԩ for 10 min then washed in 70% (v/v) ethanol. Finally, the DNA pellet was air-dried and resuspended in 100 μl of 10 mM Tris-HCl pH 8.0.

2.2.2.2 Plasmid purification

Small scale (up to 5 ml of bacterial culture) plasmid purification from E. coli was carried

out using a High Pure Plasmid Isolation Miniprep Kit (Roche) according to the

manufacturer’s instructions. For large scale plasmid purification from E. coli (25 ml of

bacterial culture), a PureLink® HiPure Plasmid Filter Midiprep Kit (Life Technologies)

was used according to the manufacturer’s instructions.

Plasmid purification from L. lactis MG1363 was carried out using the same kits as

described above, including additional steps to allow the lysis of these Gram-positive bacterial cell wall. Briefly, mid-log-phase (OD600 ≈ 0.6) MG1363 culture (up to 5 ml for

miniprep/100 ml for midiprep) was centrifuged at 5,500xg for 10 min to harvest the cells. The cell pellet was resuspended in Solution A [6.7% (w/v) sucrose, 50 mM Tris-HCl pH 8.0 and 1 mM EDTA] (379 μl for miniprep/6 ml for midiprep). Next, lysozyme (Sigma- Aldrich) solution (10 mg/ml in 25 mM Tris-HCl pH 8.0) was added to weaken the bacterial cell wall (97 μl for miniprep/1.5 ml for midiprep) followed by incubation at 37Ԩ

for 10 min. The Solution B (50 mM Tris-HCl pH 8.0 and 0.25 M EDTA) was added (48.2

μl for miniprep/0.75 ml for midiprep) to stop the reaction. The cells were then collected by centrifugation at 1,500xg for 3 min and processed for plasmid purification by following the instructions described in the kit manuals.

2.2.2.3 Polymerase Chain Reaction (PCR)

The PCR reactions for cloning and sequencing were carried out using the PrimeSTAR GXL DNA polymerase (Takara Bio). For diagnostic PCR reactions used to confirm the presence of correct DNA fragments, SpeedSTAR HS DNA polymerase (Takara Bio) was used. The Whatman Biometra thermal cycler (UK) was used for all PCR reactions.

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2.2.2.4 Southern blot

AmershamTM _{ECL Direct Labelling and Detection System (GE Health Life Sciences) and}

DIG Labelling and Detection Kit (Roche Applied Sciences) were used in the Southern blot experiments for confirmation of L. rhamnosus knock-out mutants, according to the

manufacturers’ instructions. Briefly, 3 μg purified genomic DNA of an L. rhamnosus

strain was digested by either 30 unit HindIII (for confirmation of HN001ΔspcA) or PstI (for confirmation of HN001ΔspcB, GGΔspcA and GGΔspcB) at 37Ԩ overnight. Next, digested DNA fragments were resolved based on the size using 0.8% (w/v) agarose gel electrophoresis, transferred to positively charged nylon membrane, and hybridized with one of the labelled DNA probes (Probe-U1 and Probe-D for HN001ΔspcA; Probe-U2 and Probe-D for HN001ΔspcB, GGΔspcA and GGΔspcB). When using the ECL kit, horse radish peroxidase (HRP) was directly cross-linked to the probes using glutaraldehyde. After hybridization at 42Ԩ overnight and stringent washing, the membrane was incubated in the substrate solution provided in the kit. The chemiluminescence was detected by exposure of an X-ray film. When using the DIG kit, the probes were firstly labelled with digoxigenin using the random priming method. After hybridization at 42Ԩ overnight and stringent washing, the membrane was incubated in alkaline phosphatase conjugated anti- digoxigenin antibody solution. The excess unbound antibodies were removed by washing, followed by incubation in the chromogenic substrate solution provided in the kit to obtain visible bands on the membrane.

2.2.3 Preparation and transformation of Escherichia coli TG1