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"Direct cloning in Lactobacillus plantarum: electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete".

Spath K, Heinl S, Grabherr R - Microb. Cell Fact. (2012)

Bottom Line: Also this approach was successful and yielded a sufficient number of recombinant clones.A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete.The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Christian-Doppler-Laboratory for Genetically Engineered Lactic Acid Bacteria, Vienna Institute of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, Vienna 1190, Austria.

ABSTRACT

Background: Lactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species.

Results: Best transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 10(9) colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones.

Conclusions: Transformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.

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Map of pCD256, pCD256ΔEc and the synthetic hTFF1 expression cassette. A: Map of pCD256 containing the minimal replicon (miniori) from plasmid p256[33] for replication in LAB, pMB1 origin for replication in E. coli, a chloramphenicol resistance gene (CAT) for selection in LAB and an ampicillin resistance gene (Amp) for selection in E. coli. The dam/dcm-methylation sites are indicated. B: Map of the minimal plasmid pCD256ΔEc consisting exclusively of the LAB-minimal origin from plasmid p256 and the chloramphenicol resistance gene for selection in LAB. C: Map of the synthesised hTFF1 expression cassette consisting of the optimized hTFF1-gene, N-terminally fused to the L. plantarum CD032 plnI double glycine leader sequence (GG), promoter P2083 and terminator Tldh.
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Figure 1: Map of pCD256, pCD256ΔEc and the synthetic hTFF1 expression cassette. A: Map of pCD256 containing the minimal replicon (miniori) from plasmid p256[33] for replication in LAB, pMB1 origin for replication in E. coli, a chloramphenicol resistance gene (CAT) for selection in LAB and an ampicillin resistance gene (Amp) for selection in E. coli. The dam/dcm-methylation sites are indicated. B: Map of the minimal plasmid pCD256ΔEc consisting exclusively of the LAB-minimal origin from plasmid p256 and the chloramphenicol resistance gene for selection in LAB. C: Map of the synthesised hTFF1 expression cassette consisting of the optimized hTFF1-gene, N-terminally fused to the L. plantarum CD032 plnI double glycine leader sequence (GG), promoter P2083 and terminator Tldh.

Mentions: In order to see whether a transformation efficiency of up to almost 109 cfu would be sufficient for direct cloning, a ligation reaction consisting of the vector backbone and a target gene was directly transformed into L. plantarum CD033 by electroporation. First, the vector pCD256 was propagated in E. coli C2925 (dam-/dcm-), purified and digested. The gene for the human trefoil factor 1 (hTFF1) served as the model target gene, as it is of small size (213 bps) and encodes a protein that being expressed in a food grade organism would be of high benefit. The codon optimized, synthetic hTFF1 gene was amplified by PCR, cleaved and ligated into pCD256 to be expressed under control of the P2083 promoter. The ligation mix was directly transformed into L. plantarum CD033 by electroporation resulting in 10 colonies. PCR screening revealed that all clones contained the hTFF1 gene. Thus, the step of propagating the LAB expression plasmid first in E. coli could be dismissed, making the requirement for an E. coli/L. plantarum shuttle vector obsolete. In order to proof this, the E. coli specific elements were deleted by PCR amplification maintaining only the Lactobacillus specific sequences of pCD256, resulting in a 1690 bp DNA fragment. Self-ligation of this DNA fragment resulted in plasmid pCD256ΔEc, which now was 3100 bp smaller than pCD256 (Figure1). Transformation into L. plantarum CD033 yielded 2,5 x 105 cfu/250 ng vector DNA used in ligation mix. Given the fact that the intramolecular ligation reaction of linear fragments is very efficient, this high number of cfu is not surprising. Now, in order to test the feasibility of this approach also for direct cloning of a target gene, we linearized pCD256ΔEc by PCR and after digestion performed ligation with the synthetic hTFF1 expression cassette (Figure1C) digested with the same enzymes. Direct transformation of the ligation reaction into L. plantarum CD033 yielded 5380 cfu/ligation reaction. This is a markedly higher number than when a vector backbone containing E. coli specific elements such as the origin of replication and the ß-lactamase gene was used. Identity of the plasmid and its structural integrity were confirmed by PCR amplification of overlapping plasmid fragments and subsequent DNA sequencing. So, it was shown that L. plantarum CD033 is the most suitable strain of the as yet tested LAB strains for direct cloning of recombinant expression vectors, circumventing the intricate design of shuttle vectors that are compatible with E. coli. Thus, plasmids become smaller, are devoid of unwanted sequences and occasional mutagenesis during E. coli propagation is excluded.


"Direct cloning in Lactobacillus plantarum: electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete".

Spath K, Heinl S, Grabherr R - Microb. Cell Fact. (2012)

Map of pCD256, pCD256ΔEc and the synthetic hTFF1 expression cassette. A: Map of pCD256 containing the minimal replicon (miniori) from plasmid p256[33] for replication in LAB, pMB1 origin for replication in E. coli, a chloramphenicol resistance gene (CAT) for selection in LAB and an ampicillin resistance gene (Amp) for selection in E. coli. The dam/dcm-methylation sites are indicated. B: Map of the minimal plasmid pCD256ΔEc consisting exclusively of the LAB-minimal origin from plasmid p256 and the chloramphenicol resistance gene for selection in LAB. C: Map of the synthesised hTFF1 expression cassette consisting of the optimized hTFF1-gene, N-terminally fused to the L. plantarum CD032 plnI double glycine leader sequence (GG), promoter P2083 and terminator Tldh.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526553&req=5

Figure 1: Map of pCD256, pCD256ΔEc and the synthetic hTFF1 expression cassette. A: Map of pCD256 containing the minimal replicon (miniori) from plasmid p256[33] for replication in LAB, pMB1 origin for replication in E. coli, a chloramphenicol resistance gene (CAT) for selection in LAB and an ampicillin resistance gene (Amp) for selection in E. coli. The dam/dcm-methylation sites are indicated. B: Map of the minimal plasmid pCD256ΔEc consisting exclusively of the LAB-minimal origin from plasmid p256 and the chloramphenicol resistance gene for selection in LAB. C: Map of the synthesised hTFF1 expression cassette consisting of the optimized hTFF1-gene, N-terminally fused to the L. plantarum CD032 plnI double glycine leader sequence (GG), promoter P2083 and terminator Tldh.
Mentions: In order to see whether a transformation efficiency of up to almost 109 cfu would be sufficient for direct cloning, a ligation reaction consisting of the vector backbone and a target gene was directly transformed into L. plantarum CD033 by electroporation. First, the vector pCD256 was propagated in E. coli C2925 (dam-/dcm-), purified and digested. The gene for the human trefoil factor 1 (hTFF1) served as the model target gene, as it is of small size (213 bps) and encodes a protein that being expressed in a food grade organism would be of high benefit. The codon optimized, synthetic hTFF1 gene was amplified by PCR, cleaved and ligated into pCD256 to be expressed under control of the P2083 promoter. The ligation mix was directly transformed into L. plantarum CD033 by electroporation resulting in 10 colonies. PCR screening revealed that all clones contained the hTFF1 gene. Thus, the step of propagating the LAB expression plasmid first in E. coli could be dismissed, making the requirement for an E. coli/L. plantarum shuttle vector obsolete. In order to proof this, the E. coli specific elements were deleted by PCR amplification maintaining only the Lactobacillus specific sequences of pCD256, resulting in a 1690 bp DNA fragment. Self-ligation of this DNA fragment resulted in plasmid pCD256ΔEc, which now was 3100 bp smaller than pCD256 (Figure1). Transformation into L. plantarum CD033 yielded 2,5 x 105 cfu/250 ng vector DNA used in ligation mix. Given the fact that the intramolecular ligation reaction of linear fragments is very efficient, this high number of cfu is not surprising. Now, in order to test the feasibility of this approach also for direct cloning of a target gene, we linearized pCD256ΔEc by PCR and after digestion performed ligation with the synthetic hTFF1 expression cassette (Figure1C) digested with the same enzymes. Direct transformation of the ligation reaction into L. plantarum CD033 yielded 5380 cfu/ligation reaction. This is a markedly higher number than when a vector backbone containing E. coli specific elements such as the origin of replication and the ß-lactamase gene was used. Identity of the plasmid and its structural integrity were confirmed by PCR amplification of overlapping plasmid fragments and subsequent DNA sequencing. So, it was shown that L. plantarum CD033 is the most suitable strain of the as yet tested LAB strains for direct cloning of recombinant expression vectors, circumventing the intricate design of shuttle vectors that are compatible with E. coli. Thus, plasmids become smaller, are devoid of unwanted sequences and occasional mutagenesis during E. coli propagation is excluded.

Bottom Line: Also this approach was successful and yielded a sufficient number of recombinant clones.A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete.The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Christian-Doppler-Laboratory for Genetically Engineered Lactic Acid Bacteria, Vienna Institute of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, Vienna 1190, Austria.

ABSTRACT

Background: Lactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species.

Results: Best transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 10(9) colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones.

Conclusions: Transformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.

Show MeSH
Related in: MedlinePlus