Team:MIT/Lactobacillus

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##add milk medium (.15M raffinose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2)
#PLATING, SELECTION
#PLATING, SELECTION
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##Skim milk agar + antibiotic
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====L. acidophilus electrotransformation procedure====
====L. acidophilus electrotransformation procedure====

Revision as of 19:16, 16 July 2008


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Contents

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. CELL CULTURE
    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 (need ~2.3mL of culture per electroporation)
  2. WASH BUFFER
    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
  3. THERMAL SHOCK
    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
  4. ELECTRICAL PULSE
    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.
  5. EXPRESSION
    1. Immediately add 2 milliliters of milk medium (0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM MgCl2)
  6. PLATING, SELECTION
    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 used

Original Electroporation Procedure

  • Procedure by Sasaki et al, 4th Symp. Lactic Acid Bacteria
  • used pX3- works in ATCC 11842 and VI104
  1. CELL CULTURE
    1. centrifuge 10 ml cells from overnight culture (OD600 >2)
    2. Suspend cells in 100 ml of MRS at pH 5.5
    3. Incubate for 2 h at 42C
  2. WASH BUFFER
    1. wash with Tris buffer (20 mM, pH 7.0)
  3. THERMAL SHOCK
    1. EB (.3 M raffinose, 1 mM MgCl2, 1 mM KH2PO4) (pH 7)
  4. ELECTRICAL PULSE
    1. 1.5 kV, 200ohm, 25 uf)
  5. EXPRESSION
    1. add milk medium (.15M raffinose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2)
  6. PLATING, SELECTION
    1. Skim milk agar + antibiotic

L. acidophilus electrotransformation procedure

  • From 2004 paper by Kim et al (Journal of App. Microbio. 2005, 99, 167-174)
  • used plasmid pNZ123, works with L. acidophilus strains 43121, 4356, NCFM, 30SC, A4, 107A, GP4A; L. helveticus KU107; L. brevis 3102
  1. Prepare Electrocompetent cells
    1. Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene
    2. Harvest at early-log phase (OD660 0.2-0.3)
    3. chill on ice for 10 minutes
    4. wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)
    5. Use cells within 30 minutes
  2. Electroporation
    1. add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
    2. electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
    3. dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
    4. plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
    5. incubate under anaerobic conditions











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