TransFLP - метод генетической модификации<em> Холерный вибрион</em> На основе природных преобразований и FLP-рекомбинации

Summary

Быстрый способ изменить геном<em> V. вибрион</em> Описано. Эти модификации включают в себя удаление отдельных генов, генных кластеров и геномные острова, а также интеграции коротких последовательностей (например, промоутер элементов или сродство-теги последовательности). Метод основан на естественной трансформации и FLP-рекомбинации.

Abstract

Several methods are available to manipulate bacterial chromosomes^1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons^1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene⁴. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)⁵ (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism⁶ and other representative of the genus Vibrio such as V. fischeri⁷. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region⁸. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.

This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences⁵. Several optimization steps concerning the initial protocol of chitin-induced natural transformation⁶ are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes⁸, the donation of PCR-derived DNA as transforming material⁹, and the addition of FLP-recombination target sites (FRT)⁵. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase¹⁰.

Protocol

Метод TransFLP (рис. 1) на примере трех различных подходов: I) удаление двух соседних генов, кодирующих вирулентности детерминанты В. вибрион (например, ΔctxAB); II) удаление островка патогенности (например, ΔVPI-1) и III) интеграция полимеразы T7-зависимой РНК-последовательность про…

Representative Results

Представитель результаты трех примерах показаны на рисунках 4 до 6. Первый подход, направленный на удаление соседних генов ctxA и ctxB. Вместе они кодируют основной фактор вирулентности V. вибрион, холерный токсин. Мы разработали олигонуклеотидов для метода …

Discussion

Метод TransFLP описано выше, и в других местах ⁵ была широко используется в нашей лаборатории. Такого рода генетических манипуляций, которые возможно включать, среди прочего: удаление отдельных генов и генных кластеров, удаление геномной островах (например, VPI-1), вставки последов?…

Materials

Name of the reagent	Company	Catalogue number	Comments
Chitin flakes	Sigma	C9213	Autoclaving required
OPTIONAL: Micropulser (in case antibiotic cassette should be excised by Flp recombinase encoded on pBR-flp)	Biorad	165-2100	Or comparable electroporators