In vitro modification of DNA for L. plantarum

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Materials)
(DNA Modification)
Line 38: Line 38:
===DNA Modification===
===DNA Modification===
1. Add the following to a 25μL aliquot of cell extract:
1. Add the following to a 25μL aliquot of cell extract:
-
** 50μL TNE Solution
+
** 50μL TNE Buffer
-
** 10μL of S-adenosylmethionine
+
** 10μL of S-adenosylmethionine Stock Solution
** 1μL BSA (10mg/ml)
** 1μL BSA (10mg/ml)
** 10μL of plasmid DNA.
** 10μL of plasmid DNA.

Revision as of 11:11, 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

  • AEBSF Stock Solution (1mM) (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride)
  • S-adenosylmethionine Stock Solution (0.8mM)
  • 10 mg ml BSA
  • Filtered glycerol
  • Prepared Plasmid DNA
  • Wash Buffer (15mL)
    • 21mg monopotassium phosphate (10mM)
    • 44mg EDTA (10mM)
    • 44mg NaCl (50mM)
    • 12mL Deionized Water
    • 3mL AEBSF Stock Solution (0.2mM)
    • Store at 4°C
  • TNE buffer (5mL)
    • 30mg Tris (50mM)
    • 15mg NaCl (50mM)
    • 15mg EDTA (10mM)

Procedure

Preparation of the Extract

1. Grow up 45 ml of L. plantarum cells in MRS overnight and wait until OD600 is between 1.5 and 2.0.
2. Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).
3. Resuspend pellet in 10 ml of wash buffer and centrifuge again.
4. Resuspend in 2 ml of wash buffer and put cells on ice.

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

5. Sonicate cells at 12 pulses of 30s with 60s intervals, using a micro tip at 60W.
6. Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).
7. Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).
8. Add 1.5mL 100% glycerol and 30μL BSA solution (10mg/mL) to the decanted cell extract.
9. 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 TNE Buffer
    • 10μL of S-adenosylmethionine Stock Solution
    • 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. -->

Personal tools