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Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

Yang P, Wang J, Qi Q - Microb. Cell Fact. (2015)

Bottom Line: Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods.We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, People's Republic of China. fwjt63298@126.com.

ABSTRACT

Background: Lactobacillus plantarum is a food-grade microorganism with industrial and medical relevance belonging to the group of lactic acid bacteria (LAB). Traditional strategies for obtaining gene deletion variants in this organism are mainly vector-based double-crossover methods, which are inefficient and laborious. A feasible possibility to solve this problem is the recombineering, which greatly expands the possibilities for engineering DNA molecules in vivo in various organisms.

Results: In this work, a double-stranded DNA (dsDNA) recombineering system was established in L. plantarum. An exonuclease encoded by lp_0642 and a potential host-nuclease inhibitor encoded by lp_0640 involved in dsDNA recombination were identified from a prophage P1 locus in L. plantarum WCFS1. These two proteins, combined with the previously characterized single strand annealing protein encoded by lp_0641, can perform homologous recombination between a heterologous dsDNA substrate and host genomic DNA. Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.

Conclusions: This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods. We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

No MeSH data available.


Related in: MedlinePlus

A general procedure for Lp_0640-41-42-mediated genome engineering. (1) A mutagenesis vector containing a lox66-cat-lox71 cassette flanked by 1-kb homologies was constructed. Homology arm A and Homology arm B were generated by PCR of the upstream and downstream of the target locus in the genome. DNA fragments sharing terminal sequence overlaps (indicated as colored-squares) were assembled via Gibson method. (2) dsDNA substrate was generated from the mutagenesis vector by PCR and subjected to DpnI digestion to eliminate the plasmid template. (3) Lp_0640-41-42-mediated recombination was performed to edit the genome of strains and correct mutants could be screened out by antibiotic selection and PCR test. (4) The recombinase-expressing plasmid was eliminated by culturing a correct mutant derived in procedure 3 in the absence of erythromycin selection for 24 h. (5) The Cre helper plasmid was used to excise the cat marker, and a lox72 site (34 bp) that is poorly recognized by Cre was formed. (6) The Cre plasmid was eliminated by culturing a correct mutant derived in procedure 5 in the absence of erythromycin selection for 24 h. The resultant strain was free of any plasmid or genetic selection marker
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Fig5: A general procedure for Lp_0640-41-42-mediated genome engineering. (1) A mutagenesis vector containing a lox66-cat-lox71 cassette flanked by 1-kb homologies was constructed. Homology arm A and Homology arm B were generated by PCR of the upstream and downstream of the target locus in the genome. DNA fragments sharing terminal sequence overlaps (indicated as colored-squares) were assembled via Gibson method. (2) dsDNA substrate was generated from the mutagenesis vector by PCR and subjected to DpnI digestion to eliminate the plasmid template. (3) Lp_0640-41-42-mediated recombination was performed to edit the genome of strains and correct mutants could be screened out by antibiotic selection and PCR test. (4) The recombinase-expressing plasmid was eliminated by culturing a correct mutant derived in procedure 3 in the absence of erythromycin selection for 24 h. (5) The Cre helper plasmid was used to excise the cat marker, and a lox72 site (34 bp) that is poorly recognized by Cre was formed. (6) The Cre plasmid was eliminated by culturing a correct mutant derived in procedure 5 in the absence of erythromycin selection for 24 h. The resultant strain was free of any plasmid or genetic selection marker

Mentions: Thus, a marker-free method combining recombineering and the loxP/Cre system for genetic manipulation in L. plantarum was developed (Fig. 5). By changing the lox66-cat-lox71 cassette, this method may also find application in gene insertion, promoter swapping and other genetic manipulations.Fig. 5


Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

Yang P, Wang J, Qi Q - Microb. Cell Fact. (2015)

A general procedure for Lp_0640-41-42-mediated genome engineering. (1) A mutagenesis vector containing a lox66-cat-lox71 cassette flanked by 1-kb homologies was constructed. Homology arm A and Homology arm B were generated by PCR of the upstream and downstream of the target locus in the genome. DNA fragments sharing terminal sequence overlaps (indicated as colored-squares) were assembled via Gibson method. (2) dsDNA substrate was generated from the mutagenesis vector by PCR and subjected to DpnI digestion to eliminate the plasmid template. (3) Lp_0640-41-42-mediated recombination was performed to edit the genome of strains and correct mutants could be screened out by antibiotic selection and PCR test. (4) The recombinase-expressing plasmid was eliminated by culturing a correct mutant derived in procedure 3 in the absence of erythromycin selection for 24 h. (5) The Cre helper plasmid was used to excise the cat marker, and a lox72 site (34 bp) that is poorly recognized by Cre was formed. (6) The Cre plasmid was eliminated by culturing a correct mutant derived in procedure 5 in the absence of erythromycin selection for 24 h. The resultant strain was free of any plasmid or genetic selection marker
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595204&req=5

Fig5: A general procedure for Lp_0640-41-42-mediated genome engineering. (1) A mutagenesis vector containing a lox66-cat-lox71 cassette flanked by 1-kb homologies was constructed. Homology arm A and Homology arm B were generated by PCR of the upstream and downstream of the target locus in the genome. DNA fragments sharing terminal sequence overlaps (indicated as colored-squares) were assembled via Gibson method. (2) dsDNA substrate was generated from the mutagenesis vector by PCR and subjected to DpnI digestion to eliminate the plasmid template. (3) Lp_0640-41-42-mediated recombination was performed to edit the genome of strains and correct mutants could be screened out by antibiotic selection and PCR test. (4) The recombinase-expressing plasmid was eliminated by culturing a correct mutant derived in procedure 3 in the absence of erythromycin selection for 24 h. (5) The Cre helper plasmid was used to excise the cat marker, and a lox72 site (34 bp) that is poorly recognized by Cre was formed. (6) The Cre plasmid was eliminated by culturing a correct mutant derived in procedure 5 in the absence of erythromycin selection for 24 h. The resultant strain was free of any plasmid or genetic selection marker
Mentions: Thus, a marker-free method combining recombineering and the loxP/Cre system for genetic manipulation in L. plantarum was developed (Fig. 5). By changing the lox66-cat-lox71 cassette, this method may also find application in gene insertion, promoter swapping and other genetic manipulations.Fig. 5

Bottom Line: Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods.We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, People's Republic of China. fwjt63298@126.com.

ABSTRACT

Background: Lactobacillus plantarum is a food-grade microorganism with industrial and medical relevance belonging to the group of lactic acid bacteria (LAB). Traditional strategies for obtaining gene deletion variants in this organism are mainly vector-based double-crossover methods, which are inefficient and laborious. A feasible possibility to solve this problem is the recombineering, which greatly expands the possibilities for engineering DNA molecules in vivo in various organisms.

Results: In this work, a double-stranded DNA (dsDNA) recombineering system was established in L. plantarum. An exonuclease encoded by lp_0642 and a potential host-nuclease inhibitor encoded by lp_0640 involved in dsDNA recombination were identified from a prophage P1 locus in L. plantarum WCFS1. These two proteins, combined with the previously characterized single strand annealing protein encoded by lp_0641, can perform homologous recombination between a heterologous dsDNA substrate and host genomic DNA. Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.

Conclusions: This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods. We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

No MeSH data available.


Related in: MedlinePlus