Team:MIT/Lactobacillus

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(Difference between revisions)
(→Original Electroporation Procedure)
(→Original Electroporation Procedure)
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##Suspend cells in 100 ml of MRS at pH 5.5
##Suspend cells in 100 ml of MRS at pH 5.5
##Incubate for 2 h at 42C  
##Incubate for 2 h at 42C  
-
 
#WASH BUFFER
#WASH BUFFER
##wash with Tris buffer (20 mM, pH 7.0)
##wash with Tris buffer (20 mM, pH 7.0)
-
 
#THERMAL SHOCK
#THERMAL SHOCK
##EB (.3 M raffinose, 1 mM MgCl2, 1 mM KH2PO4) (pH 7)
##EB (.3 M raffinose, 1 mM MgCl2, 1 mM KH2PO4) (pH 7)
-
 
#ELECTRICAL PULSE
#ELECTRICAL PULSE
##1.5 kV, 200ohm, 25 uf)
##1.5 kV, 200ohm, 25 uf)
-
 
#EXPRESSION
#EXPRESSION
##add milk medium (.15 raffinose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2)
##add milk medium (.15 raffinose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2)
-
 
#PLATING, SELECTION
#PLATING, SELECTION
##Skim milk agar + antibiotic
##Skim milk agar + antibiotic

Revision as of 18:55, 15 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 (.15 raffinose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2)
  6. PLATING, SELECTION
    1. Skim milk agar + antibiotic












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