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Team:MIT/Lactobacillus
From 2008.igem.org
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* Jars containing gaspak | * Jars containing gaspak | ||
*Strain of L. Bulgaricus | *Strain of L. Bulgaricus | ||
| - | *approprate plasmid that is compatible with L. Bulgaricus strain we | + | *approprate plasmid that is compatible with L. Bulgaricus strain we used |
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| + | ====Original Electroporation Procedure==== | ||
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| + | *Procedure by Sasaki et al, 4th Symp. Lactic Acid Bacteria | ||
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Revision as of 18:36, 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
- 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
- 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
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