YOGHURT

Lactobacillus Delbrueckii subsp. Bulgaricus Streptococcus Salivarius subsp. Thermophilus

LACTOBACILLUS TRANSFORMATION

Estimated time for procedure: 3-4 days

CELL CULTURE

Inoculate serial dilutions of fresh bacterial culture into 100 ml of MRS containing 0.1% glycine and incubate at 42°C overnight.

Harvest 10 ml of culture cells at beginning of stationary phase (optical density at 600 nm, 1.7) by centrifugation (need ~2.3mL of culture per electroporation)

WASH BUFFER

`Wash bacteria once with 100 ml of cold electroporation buffer (EB) (0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6)

Wash bacteria twice with 30 ml of cold EB

THERMAL SHOCK

Resuspend cells in EB to an optical density at 600 nm of about 50

Incubate cell suspension at 45°C for 20 min then keep on ice for 10 min

ELECTRICAL PULSE

Mix 80 µl of cell suspension with 0.3 to 2 µg of plasmid DNA

Subject sample to a 1-kV, 800-Ω, 25-µF electric pulse in a 0.2-cm cuvette by using a Gene Pulser and a Pulse Controller apparatus.

EXPRESSION

Immediately add 2 milliliters of milk medium (0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM MgCl2)

PLATING, SELECTION

Incubate cells for 3h at 37°C before plating on MRS agar supplemented with antibiotics. Add antibiotic erythromycin at concentration 7.5 µg/ml and chloramphenicol at concentration 7.5 µg/ml depending on plasmid resistance gene

Incubate plates at 37°C for 2 to 3 days under anaerobic conditions in jars containing GasPak

CELL CULTURE

INCUBATE

CENTRIFUGE

WASH BUFFERTHERMAL SHOCKELECTROPORATE

INCUBATE UNDER ANAEROBIC CONDITIONS

APPLICATIONS VS IMPLICATIONS

biological interactions

local water

London biotopes

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATTAAAGAGGAGAAAATGAGTATTCAACATTTTCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGTGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCTGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAATTAAAGAGGAGAAAATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGG

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATTAAAGAGGAGAAAATGCCTCTTCCGCTGTACCTGCTCGCCGTGGCCGTCTGCGCCATGGGCACCTCGGAGTTCATGCTCGCCGGTCTCGTGCCGGACATCGCCTCGGATCTCGGCGTCACCGTCGGGACCGCAGGCACGCTCACCTCGGCCTTCGCGACCGGCATGATCGTCGGCGCTCCCCTCGTGGCGGCGCTGGCCCGCACCTGGCCCAGGCGTTCCAGCCTCCTCGGATTCATCCTCGCCTTCGCGGCGGCACACGCCGTGGGAGCCGGCACCACGAGCTTCCCCGTCCTGGTGGCCTGCCGGGTCGTGGCCGCGCTCGCGAACGCGGGATTCCTCGCGGTCGCACTGACGACTGCCGCCGCACTGGTCCCTGCCGACAAGAAGGGACGCGCGCTGGCCGTGCTGCTGTCCGGCACGACGGTGGCCACGGTCGCCGGCGTCCCCGGCGGGTCACTCCTCGGCACATGGCTCGGCTGGCGGGCCACGTTCTGGGCCGTCGCCGTCTGCTGCCTGCCCGCGGCGTTCGGCGTGCTGAAGGCAATCCCCGCCGGACGTGCGACGGCAGCGGCGACCGGTGGGCCGCCGCTGCGAGTCGAGCTCGCCGCGCTCAAGACCCCCCGGTTGCTGCTGGCGATGCTGCTGGGCGCGCTGGTGAACGCGGCAACCTTCGCGAGCTTCACCTTCCTGGCCCCCGTCGTGACCGACACCGCAGGGCTGGGCGACCTGTGGATCTCTGTCGCCCTGGTGCTCTTCGGCGCCGGTTCCTTCGCCGGCGTCACCGTCGCCGGACGACTGTCCGACCGACGCCCCGCCCAGGTGCTCGCCGTCGCCGGTCCGCTGCTGCTCGTCGGCTGGCCCGCGCTGGCGATGCTGGCCGACCGGCCGGTCGCCCTGCTGACCCTCGTGTTCGTCCAAGGCGCACTGTCGTTCGCGCTGGGCAGCACGCTGATCACGCGGGTCCTCTACGAGGCGGCGGGAGCACCCACCATGGCCGGTTCGTACGCGACCGCCGCCCTCAACGTGGGCGCCGCGGCCGGACCGCTCGTCGCCGCGACCACTCTCGGCCACACGACCGGCAACCTCGGGCCGCTGTGGGCGAGCGGGCTCCTGGTCGCCGTCGCGCTGCTCGTCGCGTTCCCCTTCCGCACGGTGATCACGACGGCCGCACCCGCCGACGCGACCCGGTGAATTAAAGAGGAGAAAATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGG

HARVEST

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