Lactobacillus chromosomal integration

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

• Mike Speer

or instead, discuss this protocol. -->