Team:MIT/Lactobacillus

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
 * 2) Inoculate serial dilutions of fresh bacterial culture into 100 ml of MRS containing 0.1% glycine and incubate at 42°C overnight.
 * 3) 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)
 * 4) 	WASH BUFFER
 * 5) `Wash bacteria once with 100 ml of cold electroporation buffer (EB) (0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6)
 * 6) 	Wash bacteria twice with 30 ml of cold EB
 * 7) 	THERMAL SHOCK
 * 8) 	Resuspend cells in EB to an optical density at 600 nm of about 50
 * 9) 	Incubate cell suspension at 45°C for 20 min then keep on ice for 10 min
 * 10) 	ELECTRICAL PULSE
 * 11) Mix 80 µl of cell suspension with 0.3 to 2 µg of plasmid DNA
 * 12) 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.
 * 13) EXPRESSION
 * 14) 	Immediately add 2 milliliters of milk medium (0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM MgCl2)
 * 15) 	PLATING, SELECTION
 * 16) 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
 * 17) 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
 * 2) centrifuge 10 ml cells from overnight culture (OD600 >2)
 * 3) Suspend cells in 100 ml of MRS at pH 5.5
 * 4) Incubate for 2 h at 42C
 * 5) WASH BUFFER
 * 6) wash with Tris buffer (20 mM, pH 7.0)
 * 7) THERMAL SHOCK
 * 8) EB (.3 M raffinose, 1 mM MgCl2, 1 mM KH2PO4) (pH 7)
 * 9) incubate at 45C for ~20 minutes
 * 10) ELECTRICAL PULSE
 * 11) add in .3 to 2ug of plasmid DNA
 * 12) 1.5 kV, 200ohm, 25 uf)
 * 13) EXPRESSION
 * 14) add milk medium (.15M raffinose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2)
 * 15) PLATING, SELECTION
 * 16) 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
 * 2) Inoculate overnight culture at 10^6 CFU/ml in MRS containing 1% glycene
 * 3) Harvest at early-log phase (OD660 0.2-0.3)
 * 4) chill on ice for 10 minutes
 * 5) wash twice in cold washing buffer (5 mmol 1^-1 sucrose, 3 mmol 1^-1 MgCl2, pH 7.4)
 * 6) Use cells within 30 minutes
 * 7) Electroporation
 * 8) add 1 ul of plasmid DNA to 50 ul (w/ 10^8 CFU/ml) of ice-cold cell suspension in a .2 cm cuvette
 * 9) electroporate - 12.5 kV/cm, pulse number of 10, puse interval of 500 ms, plasmid DNA concentration of 25 ng/ul
 * 10) dilute cell suspension to 1 ml in MRS broth and incubate at 37C for 3 h
 * 11) plate bacteria onto MRS agar plates with 3 ug ml^-1 chloramphenicol
 * 12) incubate under anaerobic conditions