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一种通过自然感受态实现的简单且经济高效的转化系统。

A simple and cost-effective transformation system for via natural competence.

作者信息

Abe Kimihiro, Yahara Hiroko, Nakao Ryoma, Yamaguchi Takehiro, Akeda Yukihiro

机构信息

Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.

Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.

出版信息

Front Microbiol. 2024 Oct 21;15:1476171. doi: 10.3389/fmicb.2024.1476171. eCollection 2024.

Abstract

is a major oral bacterial pathogen responsible for severe periodontal diseases. Numerous studies have used genetic approaches to elucidate the molecular mechanisms underlying its pathogenicity. Typically, electroporation and conjugation are utilized for mutagenesis of ; however, these techniques require specialized equipment such as high-voltage electroporators, conjugative plasmids and donor strains. In this study, we present a simple, cost-effective transformation method for without any special equipment by exploiting its natural DNA competence. ATCC 33277 was grown to the early-exponential phase and mixed with a donor DNA cassette. This mixture was then spotted onto a BHI-HM blood-agar plate and incubated for one day to promote colony biofilm formation. The resulting colony biofilm was suspended in a liquid medium and spread onto antibiotic-containing agar plates. Transformants appeared within 4 to 5 days, achieving a maximum efficiency of 7.7 × 10 CFU/μg. Although we optimized the transformation conditions using a representative strain ATCC 33277, but the method was also effective for other strains, W83 and TDC60. Additionally, we discovered that deletion of or , encoding putative ComEA and ComEC, abolished competency, indicating that these gene products are essential for the natural competence.

摘要

是导致严重牙周疾病的主要口腔细菌病原体。众多研究已采用遗传学方法来阐明其致病性背后的分子机制。通常,电穿孔和接合用于其诱变;然而,这些技术需要诸如高压电穿孔仪、接合质粒和供体菌株等专门设备。在本研究中,我们通过利用其天然DNA感受态,提出了一种无需任何特殊设备的简单、经济高效的转化方法。将ATCC 33277培养至早期指数期并与供体DNA盒混合。然后将该混合物点涂在BHI - HM血琼脂平板上并孵育一天以促进菌落生物膜形成。将所得菌落生物膜悬浮于液体培养基中并铺展在含抗生素的琼脂平板上。转化子在4至5天内出现,最大效率达到7.7×10 CFU/μg。尽管我们使用代表性菌株ATCC 33277优化了转化条件,但该方法对其他菌株W83和TDC60也有效。此外,我们发现编码假定的ComEA和ComEC的基因缺失会消除感受态,表明这些基因产物对于天然感受态至关重要。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/90eb/11532111/916fca86f927/fmicb-15-1476171-g001.jpg

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