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      New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria


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          New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivo assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium.

          Method summary

          New plasmid vectors were designed to streamline allelic exchange in gram-negative bacteria by allowing for blue/white screening and restriction digest-independent cloning techniques as well as different antibiotic resistance genes to expand their utility to different bacteria.

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          Most cited references 9

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          Enzymatic assembly of DNA molecules up to several hundred kilobases.

          We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
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            Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension

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              Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector.

              The ability to attach to epithelial cells, efface the microvillus surface, and disrupt the underlying cytoskeleton is characteristic of enteropathogenic Escherichia coli (EPEC). Recently, eae, a gene necessary for this phenomenon, was described (A. E. Jerse, J. Yu, B. D. Tall, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 87:7839-7843, 1990). We report the use of a novel suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis to construct an eae deletion mutant of EPEC. This system enables positive selection for the loss of vector sequences. The resulting mutant, CVD206, is indistinguishable from the wild-type strain except for the loss of a 94-kDa outer membrane protein and attaching and effacing ability. Both the 94-kDa outer membrane protein and attaching and effacing ability are restored upon reintroduction of the eae gene on a plasmid. These results confirm the role of the eae gene in the attaching and effacing activity of EPEC and establish the utility of a new system for the construction of deletion mutations.

                Author and article information

                Future Science Ltd (London, UK )
                25 January 2021
                February 2021
                : 70
                : 2
                : 116-119
                1South Texas Center for Emerging Infectious Diseases & Department of Biology, University of Texas San Antonio, San Antonio, TX 78249, USA
                Author notes
                [* ]Author for correspondence: karl.klose@ 123456utsa.edu
                © 2021 Karl E. Klose

                This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

                Page count
                Pages: 4
                Self URI (journal page): https://www.biotechniques.com/
                Funded by: The Brown Foundation


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