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Lactobacillus delbrueckii subsp. bulgaricus- Information and Protocols

General Info

  • Official name is Lactobacillus delbrueckii subs. bulgaricus
  • L. Bulgaricus is a gram-positive bacteria
  • Feeds on milk and produces lactic acid, and it is only able to break down lactose
  • When fermenting milk, produces acetaldehyde which gives the yogurt a fruity flavor
  • Transformation methods for each L. delbrueckii strains vary – we are using the optimized method described in the paper on the brainstorming page (Serror et al)

L. bulgaricus Bacterial Strain and its Compatible Plasmid(s)(# transformants produced (µg)) for Electrotransformation

  • VI104 strain- pLEM415 plasmid (derived from E. coli-L. reuteri) (10^3-10^4)
    • - pX3 plasmid (from L. delbrueckii) (10^3)
    • - pJK650 plasmid (from L. delbrueckii) (10^3)
    • - pULP8 plasmid (heterologous plasmid) (10^2) (low copy plasmid)
    • - pNZ12 plasmid (heterologous plasmid) (10^2) (low copy plasmid)
    • - pG+ host 4 plasmid (heterologous plasmid) (10^2) (low copy plasmid)
    • - pGB305 plasmid (heterologous plasmid) (10^2) (low copy plasmid)
    • - pGT633 plasmid(from L. reuteri) (10) (low copy plasmid)
    • - pCU1882 plasmid(from L. curvatus) (10) (low copy plasmid)
  • ATCC 11842 strain – pJK650 plasmid (2 x 10^3)

Optimized Electrotransformation Procedure

Estimated time for procedure: 3-4 days

    1. Inoculate serial dilutions of fresh bacterial culture into 100 ml of MRS containing 0.1% glycine and incubate at 42°C overnight.
    2. Harvest 10 ml of culture cells at beginning of stationary phase (optical density at 600 nm, 1.7) by centrifugation
    1. `Wash bacteria once with 100 ml of cold electroporation buffer (EB) (0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6)
    2. Wash bacteria twice with 30 ml of cold EB
    1. Resuspend cells in EB to an optical density at 600 nm of about 50
    2. Incubate cell suspension at 45°C for 20 min then keep on ice for 10 min
    1. Mix 80 µl of cell suspension with 0.3 to 2 µg of plasmid DNA
    2. 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.
    1. Immediately add 2 milliliters of milk medium (0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM MgCl2)
    1. 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
    2. Incubate plates at 37°C for 2 to 3 days under anaerobic conditions in jars containing GasPak

Materials Needed for Electrotransformation

  • MRS + glycine
  • Sucrose, MgCl2, Kh2PO4 for the EB
  • Gene Pulser and Pulse Controller apparatus for electrophoration
  • Sucrose, skim milk, yeast extract, casamino acids for the milk medium
  • Erythromycin, chloramphenicol antibiotics
  • Jars containing gaspak
  • Strain of L. Bulgaricus
  • approprate plasmid that is compatible with L. Bulgaricus strain we use
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