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Lactobacillus Delbrueckii subsp. Bulgaricus Streptococcus Salivarius subsp. Thermophilus

LACTOBACILLUS TRANSFORMATION Estimated time for procedure: 3-4 days CELL CULTURE ELECTRICAL PULSE Inoculate serial dilutions of fresh bacterial culture into 100 ml of Mix 80 µl of cell suspension with 0.3 to 2 µg of plasmid DNA MRS containing 0.1% glycine and incubate at 42°C overnight. Subject sample to a 1-kV, 800-Ω, 25-µF electric pulse in a 0.2-cm Harvest 10 ml of culture cells at beginning of stationary phase cuvette by using a Gene Pulser and a Pulse Controller apparatus. (optical density at 600 nm, 1.7) by centrifugation (need ~2.3mL of culture per electroporation) EXPRESSION Immediately add 2 milliliters of milk medium (0.2 M sucrose, 5% WASH BUFFER skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM `Wash bacteria once with 100 ml of cold electroporation buffer MgCl2) (EB) (0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6) Wash bacteria twice with 30 ml of cold EB PLATING, SELECTION Incubate cells for 3h at 37°C before plating on MRS agar THERMAL SHOCK supplemented with antibiotics. Add antibiotic erythromycin at concentration 7.5 µg/ml and chloramphenicol at concentration Resuspend cells in EB to an optical density at 600 nm of about 50 7.5 µg/ml depending on plasmid resistance gene Incubate cell suspension at 45°C for 20 min then keep on ice for Incubate plates at 37°C for 2 to 3 days under anaerobic 10 min conditions in jars containing GasPak

CELL CULTURE

INCUBATE

CENTRIFUGE

WASH BUFFER THERMAL SHOCK ELECTROPORATE

INCUBATE UNDER ANAEROBIC CONDITIONS

APPLICATIONS VS IMPLICATIONS

biological interactions

local water

London biotopes

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACAT TAAAGAGGAGAAAATGCCTCTTCCGCTGTACCTGCTCGCCGTGGCCGTCTGCG CCATGGGCACCTCGGAGTTCATGCTCGCCGGTCTCGTGCCGGACATCGCCTCG GATCTCGGCGTCACCGTCGGGACCGCAGGCACGCTCACCTCGGCCTTCGCGA TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACAT CCGGCATGATCGTCGGCGCTCCCCTCGTGGCGGCGCTGGCCCGCACCTGGCC TAAAGAGGAGAAAATGAGTATTCAACATTTTCGTGTCGCCCTTATTCCCTTTTTTGC CAGGCGTTCCAGCCTCCTCGGATTCATCCTCGCCTTCGCGGCGGCACACGCCG GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGAT TGGGAGCCGGCACCACGAGCTTCCCCGTCCTGGTGGCCTGCCGGGTCGTGG GCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG CCGCGCTCGCGAACGCGGGATTCCTCGCGGTCGCACTGACGACTGCCGCCG CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACT CACTGGTCCCTGCCGACAAGAAGGGACGCGCGCTGGCCGTGCTGCTGTCCG TTTAAAGTTCTGCTATGTGGTGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGC GCACGACGGTGGCCACGGTCGCCGGCGTCCCCGGCGGGTCACTCCTCGGCA AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT CATGGCTCGGCTGGCGGGCCACGTTCTGGGCCGTCGCCGTCTGCTGCCTGCC CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTG CGCGGCGTTCGGCGTGCTGAAGGCAATCCCCGCCGGACGTGCGACGGCAG CCATAACCATGAGTGATAACACTGCTGCCAACTTACTTCTGACAACGATCGGAG CGGCGACCGGTGGGCCGCCGCTGCGAGTCGAGCTCGCCGCGCTCAAGACC GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGC CCCCGGTTGCTGCTGGCGATGCTGCTGGGCGCGCTGGTGAACGCGGCAACCT CTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTG TCGCGAGCTTCACCTTCCTGGCCCCCGTCGTGACCGACACCGCAGGGCTGGG ACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGC CGACCTGTGGATCTCTGTCGCCCTGGTGCTCTTCGGCGCCGGTTCCTTCGCCGG GAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGAT CGTCACCGTCGCCGGACGACTGTCCGACCGACGCCCCGCCCAGGTGCTCGCC AAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT GTCGCCGGTCCGCTGCTGCTCGTCGGCTGGCCCGCGCTGGCGATGCTGGCCG GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG ACCGGCCGGTCGCCCTGCTGACCCTCGTGTTCGTCCAAGGCGCACTGTCGTTC GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGG GCGCTGGGCAGCACGCTGATCACGCGGGTCCTCTACGAGGCGGCGGGAGC CAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTA ACCCACCATGGCCGGTTCGTACGCGACCGCCGCCCTCAACGTGGGCGCCGC AGCATTGGTAAATTAAAGAGGAGAAAATGGCTTCCTCCGAAGACGTTATCAAA GGCCGGACCGCTCGTCGCCGCGACCACTCTCGGCCACACGACCGGCAACCT GAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCG CGGGCCGCTGTGGGCGAGCGGGCTCCTGGTCGCCGTCGCGCTGCTCGTCGC AAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAA GTTCCCCTTCCGCACGGTGATCACGACGGCCGCACCCGCCGACGCGACCCG ACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCC GTGAATTAAAGAGGAGAAAATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTG GCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGG TCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG ACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTT GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCA CGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTG CTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGT AGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGG GTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGA TTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCG GTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACG GAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACG GGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATA GTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTT GAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAA CAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAA ATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAA CGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCA GAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGT CCGGTGCTTAATAATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTG TCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTT TGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCC TTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACG AGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGG AAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACAC ATGGCATGGATGAACTATACAAATAATAATATAAACGCAGAAAGGCCCACCCG AAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATA AAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGG

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Interesting Tuur

by meneertuur on Sep 19, 2009

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