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 plus
• 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.

• Phosphatasing the digest will help prevent self ligation.
  

3. Digest your desired insert 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 successful insertion.

Transforming the Cells

1. Transform the Lactobacillus cells according to one of these protocols. 2. Instead of plating, place 100μL of the recovered cells into 5ml MRS broth (erythromycin at 1μg/mL) and culture overnight.

Contact

• Mike Speer

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