Lactobacillus chromosomal integration

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(Performing the Integration)
 
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==Overview==
==Overview==
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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.
+
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 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 are non-replicative in Lactobacillus spp. and 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==
==Materials==
*Integration plasmid DNA (either pGIP73 or pP7B6)
*Integration plasmid DNA (either pGIP73 or pP7B6)
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*XbaI plus
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*XbaI  
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*Antarctic Phosphatase
*MRS media
*MRS media
*Erythromycin
*Erythromycin
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==Procedure==
==Procedure==
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1. Culture the desired plasmid in ''E. coli''
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===Preparing the Plasmid===
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2. Miniprep the plasmid and digest with XbaI  
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1. Culture the desired plasmid in ''E. coli'' in SOB with erythromycin ad a concentration of 300μg/mL.<br>
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::*Phosphatase helps to prevent self ligation).
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2. Miniprep the plasmid and digest with XbaI.<br>
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3. Digest your desired insert with Enzymes to form compatible ends with XbaI (a SpeI + XbaI double digest works here).
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3. After two hours digest add Antarctic phosphatase (and the requisite buffer) and digest for one additional hour.
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4. Ligate and transform into ''E. coli''
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::*Phosphatasing the digest will help prevent self ligation.<br>
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5. Sequence or colony PCR to check for positive insertion
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4. Digest your desired insert to form compatible ends with XbaI (a SpeI + XbaI double digest works here).<br>
 +
::*Gel extracting the insert will increase ligation efficiency.
 +
5. Ligate and transform into ''E. coli''.<br>
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6. Sequence or colony PCR to check successful insertion.<br>
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===Preparation of the Extract===
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===Performing the Integration===
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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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1. Transform the Lactobacillus cells according to [[Preparation of Lactobacillus Competent Cells|one of these protocols]].<br>
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2. Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).<br>
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2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at 1μg/mL) and grow overnight.<br>
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3.  Resuspend pellet in 10 ml of wash buffer and centrifuge again.<br>
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3.  The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.<br>
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4.  Resuspend in 2 ml of wash buffer and put cells on ice.<br>
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:*Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 2.5μg/mL erythromycin.<br>
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**'''Keep cells chilled (on ice) during the remainder of the procedure'''
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4. Screen for your gene of interest to ensure successful integration.<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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:*A plate screen is really good here.<br><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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7. Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).<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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9.  Separate the extract into 25μL aliquots and store at -20°C until use.<br>
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===DNA Modification===
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<center>'''If you don't want a food grade microorganism then you can stop here.'''<br>
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1. Add the following to a 25μL aliquot of cell extract:
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'''If you want to remove the erythromycin resistance then continue.'''</center><br><br>
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** 50μL TNE Buffer
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** 10μL of S-adenosylmethionine Stock Solution
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5. Grow your integrated culture overnight in 5ml of MRS broth with NO ERYTHROMYCIN!!!<br>
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** 1μL BSA (10mg/ml)
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6. Plate a dilution (try 1 to 100,000) of this culture onto MRS plates with NO ERYTHROMYCIN!!!<br>
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** 10μL of plasmid DNA.
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7. Select ~10 colonies and spot plate them onto two plates: one containing erythromycin and one not.<br>
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2. Incubate the mixture at 30°C for 16 hours.<br>
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:*Be sure to label the spots to be sure which colony corresponds to which.<br>
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3. Extract the mixture with a phenol/chloroform extraction.<br>  
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8. Select the colonies that grow on the MRS but not on the MRS+Erythromycin.<br>
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4. Precipitate using ethanol.
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9. Screen these colonies for your insert activity.<br>
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:*~50% of colonies will have lost your insert.<br>
==Notes==
==Notes==
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All questions, input and feedback are welcome!
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Be sure to do a MIC experiment on your erythromycin every time you get a new batch.  The potency of various products can be startlingly different.  
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*AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.
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*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].
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*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. 
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::#Miniprep the desired shuttle vector from ''E. coli''.
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::#Electroporate into ''L. lactis'' electro-comptent cells at 10,000kv/cm.
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::#Let cells recover in 25ml GM17 media for one hour.
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::#Add the appropriate antibiotic to the media.
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::#Grow overnight at 30°C.
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::#Miniprep ''L. lactis'' culture.
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::#Transform ''L. plantarum'' electro-competent cells at 10,000kv/cm.
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::#Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!
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==References==
==References==
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#Alegre et al. (FEMS Microbiology Letters 241 (2004), 73-77)
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*Hols, P., T. Ferain, et al. (1994). "Use of homologous expression secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha amylase and levanase expression." Applied and Environmental Microbiology 60(5): 1401-1413.
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#Matsushima et al. (Microbiology 140 (1994), 139-143)
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*Rossi, F., A. Capodaglio, et al. (2008). "Genetic modification of Lactobacillus plantarum by heterologous gene integration in a not functional region of the chromosome." Applied Microbiology and Biotechnology 80(1): 79-86.
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==Contact==
==Contact==
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*morto077@uottawa.ca
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*[[User:Michael A. Speer|Mike Speer]]
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
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<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
 

Current revision

Contents

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 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 are non-replicative in Lactobacillus spp. and 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
  • Antarctic Phosphatase
  • MRS media
  • Erythromycin
  • Lactobacillus plantarum
  • E. coli

Procedure

Preparing the Plasmid

1. Culture the desired plasmid in E. coli in SOB with erythromycin ad a concentration of 300μg/mL.
2. Miniprep the plasmid and digest with XbaI.
3. After two hours digest add Antarctic phosphatase (and the requisite buffer) and digest for one additional hour.

  • Phosphatasing the digest will help prevent self ligation.

4. Digest your desired insert to form compatible ends with XbaI (a SpeI + XbaI double digest works here).

  • Gel extracting the insert will increase ligation efficiency.

5. Ligate and transform into E. coli.
6. Sequence or colony PCR to check successful insertion.

Performing the Integration

1. Transform the Lactobacillus cells according to one of these protocols.
2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at 1μg/mL) and grow overnight.
3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.

  • Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 2.5μg/mL erythromycin.

4. Screen for your gene of interest to ensure successful integration.

  • A plate screen is really good here.

If you don't want a food grade microorganism then you can stop here.
If you want to remove the erythromycin resistance then continue.


5. Grow your integrated culture overnight in 5ml of MRS broth with NO ERYTHROMYCIN!!!
6. Plate a dilution (try 1 to 100,000) of this culture onto MRS plates with NO ERYTHROMYCIN!!!
7. Select ~10 colonies and spot plate them onto two plates: one containing erythromycin and one not.

  • Be sure to label the spots to be sure which colony corresponds to which.

8. Select the colonies that grow on the MRS but not on the MRS+Erythromycin.
9. Screen these colonies for your insert activity.

  • ~50% of colonies will have lost your insert.

Notes

Be sure to do a MIC experiment on your erythromycin every time you get a new batch. The potency of various products can be startlingly different.

References

  • Hols, P., T. Ferain, et al. (1994). "Use of homologous expression secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha amylase and levanase expression." Applied and Environmental Microbiology 60(5): 1401-1413.
  • Rossi, F., A. Capodaglio, et al. (2008). "Genetic modification of Lactobacillus plantarum by heterologous gene integration in a not functional region of the chromosome." Applied Microbiology and Biotechnology 80(1): 79-86.

Contact

or instead, discuss this protocol. -->

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