Midae SY9Smr V. midae SY9::Tn10.52 E. coli SM10lpir E. coli JM109 Plasmid: pLOFKmgfp a r r Genotype/Relevant characteristica Reference Isolated from the Madrasin site digestive tract of H. midae, South Africa Streptomycin resistant strain of V. midae SY9; Smr V. midae SY9Smr::mini-Tn10-gfp-kan, Smr, Kmr thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Kmr recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiD F’ pLOFKm with cloned promoterless gfp, Kmr, Ampr r This study This study Sm, streptomycin resistant; Km, kanamycin resistant; Amp, ampicillin resistant. doi:10.1371/journal.pone.0086623.t001 2 Probiotic and Protease Localisation in Abalone Gut Chromosomal DNA was isolated according to the method described by Ausubel et al.. PCR amplification of the gfp gene using the oligonucleotide primers gfp-F and gfp-R was used to screen for the presence of the integrated gfp gene in order to 115103-85-0 web identify gfp-tagged V. midae SY9::Tn10.52 strains for further 22948146 analysis. labelled using a random-primed DNA labelling kit according to the manufacturer’s instructions and hybridised to equal amounts of V. midae SY9 and V. midae SY9::Tn10.52 chromosomal DNA. The Southern hybridization procedure was performed according to Church and Gilbert. Probe Design and Labelling Three oligonucleotide probes GFP001, GFP002 and GFP003 were designed to hybridize to segments of the gfp gene of pLOFKmgfp. The 16S rRNA gene eubacterial probe EUB338 was employed as an in situ hybridization control. The probe 25837696 ECJ109 was used as a negative control of the in situ hybridization experiments. All of the oligonucleotide probes were 59-DIG labelled under weak alkaline conditions in a sodium borate buffer/dimethyl formamide mixture at 22uC overnight. Southern Hybridization The plasmid pLOFKmgfp was purified from E. coli SM10lpir using the Qiagen Midi Prep kit according the manufacturer’s instructions. A 717 bp DNA fragment of the gfp gene was PCR amplified from pLOFKmgfp using the synthetic oligonucleotide primers gfp-F and gfp-R. The 717 bp PCR amplicon was resolved on a 1% TAE agarose gel and purified using the BioSpin Gel Extraction Kit according to the manufacturer’s instructions. The purified 717 bp DNA fragment was radioactively Preparation of V. midae SY9::Tn10.52-supplemented Feed The basal diet consisted of ABFEEDH S34 weaning chips as supplied by Marifeed Ltd., Hermanus, South Africa. The gfp chromosomally-tagged probiotic strain V. midae SY9::Tn10.52 was incorporated into ABFEEDH S34 weaning chips by vacuum infusion, to a final concentration of at least 1.06108 culturable cells g-1 feed. Briefly, V. midae SY9::Tn10.52 was cultivated for 24 hrs in 3 litres of P-MBM supplemented with 120 mg/ml Sm and 400 mg/ml Kan on an orbital shaker at 22uC. Thereafter, the bacterial cells were harvested by centrifugation, washed with one volume of artificial seawater and resuspended in 100 ml of ASW. Approximately 200 g of the ABFEEDH S34 weaning chips were sealed inside a glass vacuum jar and a vacuum drawn within the jar to approximately 80 kPa. Approximately 20 ml of the V. midae SY9::Tn10.52 suspension was drawn into the vacuum jar and thoroughly mixed with the feed. During the process of drawing the bacterial suspension into the chamber, the vacuum was decreased to 50 kPa and maintained at this pressure for 5 minutes. Thereafter, the vacuum was slowly released, the impregnated ABFEEDH S34 weaning chips removed from the jar and dried at 22uC overnight, before being sealed into clean plastic bags and stored at 4u.Midae SY9Smr V. midae SY9::Tn10.52 E. coli SM10lpir E. coli JM109 Plasmid: pLOFKmgfp a r r Genotype/Relevant characteristica Reference Isolated from the digestive tract of H. midae, South Africa Streptomycin resistant strain of V. midae SY9; Smr V. midae SY9Smr::mini-Tn10-gfp-kan, Smr, Kmr thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Kmr recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiD F’ pLOFKm with cloned promoterless gfp, Kmr, Ampr r This study This study Sm, streptomycin resistant; Km, kanamycin resistant; Amp, ampicillin resistant. doi:10.1371/journal.pone.0086623.t001 2 Probiotic and Protease Localisation in Abalone Gut Chromosomal DNA was isolated according to the method described by Ausubel et al.. PCR amplification of the gfp gene using the oligonucleotide primers gfp-F and gfp-R was used to screen for the presence of the integrated gfp gene in order to identify gfp-tagged V. midae SY9::Tn10.52 strains for further 22948146 analysis. labelled using a random-primed DNA labelling kit according to the manufacturer’s instructions and hybridised to equal amounts of V. midae SY9 and V. midae SY9::Tn10.52 chromosomal DNA. The Southern hybridization procedure was performed according to Church and Gilbert. Probe Design and Labelling Three oligonucleotide probes GFP001, GFP002 and GFP003 were designed to hybridize to segments of the gfp gene of pLOFKmgfp. The 16S rRNA gene eubacterial probe EUB338 was employed as an in situ hybridization control. The probe 25837696 ECJ109 was used as a negative control of the in situ hybridization experiments. All of the oligonucleotide probes were 59-DIG labelled under weak alkaline conditions in a sodium borate buffer/dimethyl formamide mixture at 22uC overnight. Southern Hybridization The plasmid pLOFKmgfp was purified from E. coli SM10lpir using the Qiagen Midi Prep kit according the manufacturer’s instructions. A 717 bp DNA fragment of the gfp gene was PCR amplified from pLOFKmgfp using the synthetic oligonucleotide primers gfp-F and gfp-R. The 717 bp PCR amplicon was resolved on a 1% TAE agarose gel and purified using the BioSpin Gel Extraction Kit according to the manufacturer’s instructions. The purified 717 bp DNA fragment was radioactively Preparation of V. midae SY9::Tn10.52-supplemented Feed The basal diet consisted of ABFEEDH S34 weaning chips as supplied by Marifeed Ltd., Hermanus, South Africa. The gfp chromosomally-tagged probiotic strain V. midae SY9::Tn10.52 was incorporated into ABFEEDH S34 weaning chips by vacuum infusion, to a final concentration of at least 1.06108 culturable cells g-1 feed. Briefly, V. midae SY9::Tn10.52 was cultivated for 24 hrs in 3 litres of P-MBM supplemented with 120 mg/ml Sm and 400 mg/ml Kan on an orbital shaker at 22uC. Thereafter, the bacterial cells were harvested by centrifugation, washed with one volume of artificial seawater and resuspended in 100 ml of ASW. Approximately 200 g of the ABFEEDH S34 weaning chips were sealed inside a glass vacuum jar and a vacuum drawn within the jar to approximately 80 kPa. Approximately 20 ml of the V. midae SY9::Tn10.52 suspension was drawn into the vacuum jar and thoroughly mixed with the feed. During the process of drawing the bacterial suspension into the chamber, the vacuum was decreased to 50 kPa and maintained at this pressure for 5 minutes. Thereafter, the vacuum was slowly released, the impregnated ABFEEDH S34 weaning chips removed from the jar and dried at 22uC overnight, before being sealed into clean plastic bags and stored at 4u.
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