Lactobacillus chromosomal integration: Difference between revisions

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==Procedure==
==Procedure==
===Preparing the Plasmid===
1. Culture the desired plasmid in ''E. coli''
1. Culture the desired plasmid in ''E. coli''
2. Miniprep the plasmid and digest with XbaI  
2. Miniprep the plasmid and digest with XbaI  
Line 19: Line 20:
5. Sequence or colony PCR to check for positive insertion
5. Sequence or colony PCR to check for positive insertion


===Preparation of the Extract===
===Transforming the Cells===
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>
   
2.  Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).<br>
===Selecting for Recombinants===
3.  Resuspend pellet in 10 ml of wash buffer and centrifuge again.<br>
4.  Resuspend in 2 ml of wash buffer and put cells on ice.<br>
**'''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.<br>
6.  Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).<br>
7.  Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).<br>
8.  Add 1.5mL 100% glycerol and 30μL BSA solution (10mg/mL) to the decanted cell extract.<br>
9.  Separate the extract into 25μL aliquots and store at -20°C until use.<br>
 
===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.<br>
3. Extract the mixture with a phenol/chloroform extraction.<br>
4. Precipitate using ethanol.


==Notes==
==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[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)
 
#Matsushima et al. (Microbiology 140 (1994), 139-143)


==Contact==
==Contact==
*morto077@uottawa.ca
*[[User:Michael A. Speer|Mike Speer]]


or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
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Revision as of 16:50, 24 March 2011

Overview

This procedure is used to integrate a desired DNA cassette into the chromosome of Lactobacillus plantarum at specific locations. This protocol can also be modified to perform gene knockouts and other specific chrosomal modifications. The two plasmids used for this procedure are pGIP73 and pP7B6 which integrate into the conjugated bile-acid hydrolase (cbh) sequence and the P7B6 prophage sequence respectively. These plasmids both operate based on homologous recombination between the plasmid and the chromosome. The desired cassette is inserted in a unique XbaI site in the middle of both the CBH and P7B6 sequences.

Materials

  • Integration plasmid DNA (either pGIP73 or pP7B6)
  • XbaI plus
  • MRS media
  • Erythromycin
  • Lactobacillus plantarum
  • E. coli

Procedure

Preparing the Plasmid

1. Culture the desired plasmid in E. coli 2. Miniprep the plasmid and digest with XbaI

  • Phosphatase helps to prevent self ligation).

3. Digest your desired insert with Enzymes to form compatible ends with XbaI (a SpeI + XbaI double digest works here). 4. Ligate and transform into E. coli 5. Sequence or colony PCR to check for positive insertion

Transforming the Cells

Selecting for Recombinants

Notes

References

Contact

or instead, discuss this protocol. -->

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