In vitro modification of DNA for L. plantarum

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(Preparation of the Extract)
(Preparation of the Extract)
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7.  Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).<br>
7.  Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).<br>
8.  Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).<br>
8.  Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).<br>
-
9.  Add 1.5mL 100% glycerol and 30μL BSA solution (10mg/mL)to the decanted cell extract.<br>
+
9.  Add 1.5mL 100% glycerol and 30μL BSA solution (10mg/mL) to the decanted cell extract.<br>
10. Separate the extract into 25μL aliquots and store at -20°C until use.<br>
10. Separate the extract into 25μL aliquots and store at -20°C until use.<br>

Revision as of 10:33, 6 April 2010

Contents

Overview

The following is a procedure for the in vitro modification of DNA before electrotransformation into Lactobacillus plantarum developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including Lactobacillus plantarum is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium.

Materials

  • Stock solution of 1.0 mM AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride)
  • Overnight culture of L. plantarum, OD=1.5-2.0
  • 1 mg ml BSA
  • Wash Buffer
    • 10 mM potassium phosphate
    • 10 mM EDTA
    • 50 mM NaCl
    • 0.2 mM AEBSF
  • Prepared extract
    • 50 mM Tris
    • 50 mM NaCl
    • 10 mM EDTA
    • 0.8 mM S-adenosylmethionine
    • 1 mg ml BSA
  • Prepared Plasmid DNA
  • Filtered glycerol

Procedure

Preparation of the Extract

1. Prepare a stock solution of AEBSF in water at 0.2 mM and store in a 4°C fridge.
2. Grow up 45 ml of L. plantarum cells in MRS overnight and wait until OD600 is between 1.5 and 2.0.
3. Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).
4. Resuspend pellet in 10 ml of wash buffer and centrifuge again.
5. Resuspend in 2 ml of wash buffer and put cells on ice.

    • Keep cells chilled (on ice) during the remainder of the procedure

6. Sonicate cells at 12 pulses of 30s with 60s intervals, using a micro tip at 60W.
7. Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).
8. Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).
9. Add 1.5mL 100% glycerol and 30μL BSA solution (10mg/mL) to the decanted cell extract.
10. Separate the extract into 25μL aliquots and store at -20°C until use.

DNA Modification

1. Add the following to a 25μL aliquot of cell extract:

    • 50μL Tris, NaCl and EDTA Solution
    • 10μL of S-adenosylmethionine
    • 1μL BSA (10mg/ml)
    • 10μL of plasmid DNA.

2. Incubate the mixture at 30°C for 16 hours.
3. Extract the mixture with a phenol/chloroform extraction.
4. Precipitate using ethanol.

Notes

All questions, input and feedback are welcome!

  1. AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.
  2. AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.
  3. There is a helpful protocol for phenol extraction posted[1] and a protocol for ethanol precipitation posted[2].

References

  1. Alegre et al. (FEMS Microbiology Letters 241 (2004), 73-77)
  2. Matsushima et al. (Microbiology 140 (1994), 139-143)

Contact

  • morto077@uottawa.ca

or instead, discuss this protocol. -->

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