Lactobacillus chromosomal integration
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.
- Integration plasmid DNA (either pGIP73 or pP7B6)
- XbaI plus
- MRS media
- Lactobacillus plantarum
- E. coli
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
or instead, discuss this protocol. -->