High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitol
<p>Abstract</p> <p>Background</p> <p>A goal for the food industry has always been to improve strains of <it>Lactococcus lactis </it>and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while el...
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| Format: | Article |
| Language: | English |
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BMC
2007-03-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/7/15 |
| _version_ | 1818019177572597760 |
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| author | Filioussis George Avramidis Nicholaos Papagianni Maria |
| author_facet | Filioussis George Avramidis Nicholaos Papagianni Maria |
| author_sort | Filioussis George |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>A goal for the food industry has always been to improve strains of <it>Lactococcus lactis </it>and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to <it>L. lactis </it>spp. <it>lactis </it>cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into <it>L. lactis</it>). Two strains of <it>L. lactis </it>spp. <it>lactis </it>were used, <it>L. lactis </it>spp. <it>lactis </it>LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with <it>L. lactis </it>or other bacteria.</p> <p>Results</p> <p>Electrotransformation efficiencies of up to 10<sup>5 </sup>transformants per μg DNA have been reported in the literature for <it>L. lactis </it>spp.<it>lactis </it>LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 ± 52.5 × 10<sup>7 </sup>transformants per μg DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 ± 0.5 × 10<sup>5 </sup>transformants per μg DNA. Results of the same trend were obtained with <it>L. lactis </it>ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 10<sup>10 </sup>cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient.</p> <p>Conclusion</p> <p>This investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for <it>L. lactis </it>spp. <it>lactis</it>. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries.</p> |
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| publishDate | 2007-03-01 |
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| spelling | doaj.art-61cdf8a6e8c64a379f5148da1fd2aa612022-12-22T02:05:16ZengBMCBMC Biotechnology1472-67502007-03-01711510.1186/1472-6750-7-15High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitolFilioussis GeorgeAvramidis NicholaosPapagianni Maria<p>Abstract</p> <p>Background</p> <p>A goal for the food industry has always been to improve strains of <it>Lactococcus lactis </it>and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to <it>L. lactis </it>spp. <it>lactis </it>cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into <it>L. lactis</it>). Two strains of <it>L. lactis </it>spp. <it>lactis </it>were used, <it>L. lactis </it>spp. <it>lactis </it>LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with <it>L. lactis </it>or other bacteria.</p> <p>Results</p> <p>Electrotransformation efficiencies of up to 10<sup>5 </sup>transformants per μg DNA have been reported in the literature for <it>L. lactis </it>spp.<it>lactis </it>LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 ± 52.5 × 10<sup>7 </sup>transformants per μg DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 ± 0.5 × 10<sup>5 </sup>transformants per μg DNA. Results of the same trend were obtained with <it>L. lactis </it>ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 10<sup>10 </sup>cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient.</p> <p>Conclusion</p> <p>This investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for <it>L. lactis </it>spp. <it>lactis</it>. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries.</p>http://www.biomedcentral.com/1472-6750/7/15 |
| spellingShingle | Filioussis George Avramidis Nicholaos Papagianni Maria High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitol BMC Biotechnology |
| title | High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitol |
| title_full | High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitol |
| title_fullStr | High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitol |
| title_full_unstemmed | High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitol |
| title_short | High efficiency electrotransformation of <it>Lactococcus lactis </it>spp. <it>lactis </it>cells pretreated with lithium acetate and dithiothreitol |
| title_sort | high efficiency electrotransformation of it lactococcus lactis it spp it lactis it cells pretreated with lithium acetate and dithiothreitol |
| url | http://www.biomedcentral.com/1472-6750/7/15 |
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