In vitro modification of DNA for L. plantarum

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(Preparation of the Extract)
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(Notes)
 
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==Overview==
==Overview==
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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.
+
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.  See notes for an alternative method.
==Materials==
==Materials==
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*Stock solution of 1.0 mM AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride)
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*AEBSF Stock Solution (1mM) (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride)
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*Overnight culture of L. plantarum, OD=1.5-2.0
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*S-adenosylmethionine Stock Solution (0.8mM)
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*1 mg ml BSA
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*10 mg ml BSA
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*Wash Buffer
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-
**10 mM potassium phosphate
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**10 mM EDTA
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**50 mM NaCl
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**0.2 mM AEBSF
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*Prepared extract
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**50 mM Tris
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**50 mM NaCl
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**10 mM EDTA
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**0.8 mM S-adenosylmethionine
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**1 mg ml BSA
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*Prepared Plasmid DNA
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*Filtered glycerol
*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)
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**'''Store at 4°C'''
 +
 +
*TNE buffer (5mL)
 +
**30mg Tris (50mM)
 +
**15mg NaCl (50mM)
 +
**15mg EDTA (10mM)
==Procedure==
==Procedure==
===Preparation of the Extract===
===Preparation of the Extract===
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1.  Prepare a stock solution of AEBSF in water at 0.2 mM and store in a 4°C fridge.<br>
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1.  Grow up 45 ml of L. plantarum cells in MRS overnight and wait until OD<sub>600</sub> is between 1.5 and 2.0.<br>
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2.  Grow up 45 ml of L. plantarum cells in MRS overnight and wait until OD<sub>600</sub> is between 1.5 and 2.0.<br>
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2.  Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).<br>
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3.  Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).<br>
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3.  Resuspend pellet in 10 ml of wash buffer and centrifuge again.<br>
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4.  Resuspend pellet in 10 ml of wash buffer and centrifuge again.<br>
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4.  Resuspend in 2 ml of wash buffer and put cells on ice.<br>
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5.  Resuspend in 2 ml of wash buffer and put cells on ice.<br>
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**'''Keep cells chilled (on ice) during the remainder of the procedure'''
**'''Keep cells chilled (on ice) during the remainder of the procedure'''
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6.  Sonicate cells at 12 pulses of 30s with 60s intervals, using a micro tip at 60 W.<br>
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5.  Sonicate cells at 12 pulses of 30s with 60s intervals, using a micro tip at 60W.<br>
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7.  Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).<br>
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6.  Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).<br>
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8.  Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).<br>
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7.  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>
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8.  Add 1.5mL 100% glycerol and 30μL BSA solution (10mg/mL) to the decanted cell extract.<br>
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10. Separate the extract into 25μL aliquots and store at -20°C until use.<br>
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9. Separate the extract into 25μL aliquots and store at -20°C until use.<br>
===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:
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** 50μL Tris, NaCl and EDTA Solution
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** 50μL TNE Buffer
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** 10μL of S-adenosylmethionine
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** 10μL of S-adenosylmethionine Stock Solution
** 1μL BSA (10mg/ml)
** 1μL BSA (10mg/ml)
** 10μL of plasmid DNA.
** 10μL of plasmid DNA.
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==Notes==
==Notes==
All questions, input and feedback are welcome!
All questions, input and feedback are welcome!
-
#AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.
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*AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.
-
#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.
+
*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.
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#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
+
*There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].
 +
*An alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species.  The procedure takes just as much ''linear'' time, but much less actual time; and is much easier.  The process goes as follows. 
 +
::#Miniprep the desired shuttle vector from ''E. coli''.
 +
::#Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.
 +
::#Let cells recover in 25ml GM17 media for one hour.
 +
::#Add the appropriate antibiotic to the media.
 +
::#Grow overnight at 30°C.
 +
::#Miniprep ''L. lactis'' culture.
 +
::#Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.
 +
::#Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!
 +
 
==References==
==References==
#Alegre et al. (FEMS Microbiology Letters 241 (2004), 73-77)
#Alegre et al. (FEMS Microbiology Letters 241 (2004), 73-77)

Current revision

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. See notes for an alternative method.

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!

  • AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.
  • 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.
  • There is a helpful protocol for phenol extraction posted[1] and a protocol for ethanol precipitation posted[2].
  • An alternative to this protocol is to use a lab strain of Lactococcus lactis (we use strain MG1363) as a shuttle species. The procedure takes just as much linear time, but much less actual time; and is much easier. The process goes as follows.
  1. Miniprep the desired shuttle vector from E. coli.
  2. Electroporate into L. lactis electro-comptent cells at 10,000kv/cm.
  3. Let cells recover in 25ml GM17 media for one hour.
  4. Add the appropriate antibiotic to the media.
  5. Grow overnight at 30°C.
  6. Miniprep L. lactis culture.
  7. Transform L. plantarum electro-competent cells at 10,000kv/cm.
  8. Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!

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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