Flp-recombinase based strategy for inactivation of multiple genes in Borrelia burgdorferi. Sai Lakshmi Rajasekhar Karna

ISBN: 9780549682868

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NOOKstudy eTextbook

57 pages


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Flp-recombinase based strategy for inactivation of multiple genes in Borrelia burgdorferi.  by  Sai Lakshmi Rajasekhar Karna

Flp-recombinase based strategy for inactivation of multiple genes in Borrelia burgdorferi. by Sai Lakshmi Rajasekhar Karna
| NOOKstudy eTextbook | PDF, EPUB, FB2, DjVu, audiobook, mp3, ZIP | 57 pages | ISBN: 9780549682868 | 5.25 Mb

The causative agent of Lyme disease, Borrelia burgdorferi , has a complex segmented genome with one linear chromosome and multiple circular and linear plasmids. Several borrelial proteins with similar properties are distributed between the chromosomeMoreThe causative agent of Lyme disease, Borrelia burgdorferi , has a complex segmented genome with one linear chromosome and multiple circular and linear plasmids.

Several borrelial proteins with similar properties are distributed between the chromosome and plasmids necessitating a strategy to delete multiple, functionally related genes to observe phenotypic differences that are significantly different between the mutants and the control strains.

We modified our customized Tn7-based transposons encoding streptomycin (Str R) and kanamycin (KanR) resistance cassettes under the control of constitutive borrelial promoter PflgB to incorporate f&barbelow-lp-recombinase r&barbelow-ecognition t&barbelow-arget (FRT) sites. One of these customized transposons were used to insertional inactivate p66 using a Tn7-based in vitro mutagenesis system. Engineered restriction enzyme sites flanking the customized transposons facilitated in the targeted deletion of dbpB/A, bbk32 and bba34.

The gene encoding for Flp-recombinase, under the control of the IPTG-inducible borrelial promoter PflaC, has been cloned into borrelial shuttle vectors that encode for the LacI gene capable of driving the expression from PflaC. These borrelial shuttle vectors with KanR, StrR or GentR resistance cassettes will facilitate selection of transformants in mutants generated with various antibiotic resistance markers. The ability to induce flp-recombinase in these borrelial shuttle vectors will facilitate resolution of intervening sequences between the FRT sites resulting in mutants generated due to the presence of the FRT lesions in targeted genes.

Validation of these constructs in Escherichia coli indicated that these constructs, generated using borrelial promoters, will mediate resolution of DNA sequences between FRT sites facilitating recycling of a limited set of antibiotic resistance markers used for generating mutants in B. burgdorferi or for deletion of large intervening sequences between these sites. These novel genetic tools will facilitate inactivation of multiple genes located either in the chromosome or in various plasmids of B.

burgdorferi.



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