摘要：枯草芽孢杆菌由于自身具有优良的安全性以及可以自发形成感受态的特性，同时便于基因工程改造，作为宿主常用于原核表达载体的构建。随着分子生物学技术的发展以及枯草芽孢杆菌的全基因组测序的完成，原核表达系统得到了进一步发展与完善。一般采样水浴加热的物理方法就能从样品中粗分离出多种芽孢杆菌。再通过进一步的16S rRNA基因以及其他特异性序列测序来鉴定，以确保是枯草芽孢杆菌。为了能够从自然界获得高转化效率的枯草芽孢杆菌，从南京、扬州两地采取大量泥土作为分离目标菌株的样品。同时，枯草芽孢杆菌作为益生菌，在牛奶以及酸奶中也存在。实验室中常用的转化方法有原生质体转化法、Spizizen转化法、电转化法。传统的Spizizen转化法能够较为方便并出色的完成枯草芽孢杆菌的转化实验。本研究也是通过Spizizen转化法从自然界得到具有高效转化率的枯草芽孢杆菌，所得菌株中最大转化效率为60 CFU/μg DNA，为日后构建优良原核重组载体提供可能性。82300

毕业论文关键词：枯草芽孢杆菌；16S rRNA；comK基因；Spizizen转化法

Isolated and identity Bacillus subtilis which can form natural competent cells

Abstract: Bacillus subtilis are often used for prokaryotic expression vector construction and genetic engineering, because of its own safety and charecteristics of spontaneous formation of competent cells。 With the development of molecular biology technology and the completion of whole genome sequencing of Bacillus subtilis, the development and improvement of prokaryotic expression system has been promoted。 Bacillus subtilis have advantages of wide distribution, easy separation and so on。 Different kinds of Bacillus can be isolated from sample by general physical methods of water bath heating。 This strains need to be further identified to ensure their spieces。 According to its own 16S rRNA gene sequencing and other specific gene sequence, those strains can be identified。 In order to obtain high efficiency of transformation of Bacillus subtilis from nature, a large amount of soil was taken from Nanjing and Yangzhou as samples for isolating target strains。 At the same time, Bacillus subtilis as probiotics, maybe exist in milk and yogurt as well。 Laboratory transformation methods commonly contain protoplast transformation method, Spizizen transformation method, electrical transformation method。 The traditional Spizizen transformation method can be more convenient and complete Bacillus subtilis transformation experiment very well。 In this study, Bacillus subtilis with high efficient transformation would be obtained by Spizizen transformation method, the most efficiency of transformation is 60 CFU/μg DNA, which provided the possibility of constructing perfect prokaryotic recombinant vector in the future。

Key Words: Bacillus subtilis ；16S rRNA；comK gene；Spizizen transformation method

目录

摘要 1

关键词 1

Abstract 1

Key Words 1

引言 2

1 材料与方法 2

1。1 样品 2

1。2 培养基制备 2

1。2。1 LB培养基 2

1。2。2 GMI与GMII培养基 3

1。3目标菌株分离与纯化 3

1。3。1 预处理以及梯度稀释 3

1。3。2 筛选与保存 3

1。4 目标菌株鉴定