一种用质粒DNA转化大肠杆菌感受态细胞的实用操作技巧

目的是建立一种简化、实用的用质粒DNA转化大肠杆菌的操作方法.采用氯化钙法制备大肠杆菌感受态细胞.以质粒pUC18,pCSN44,pAN52-1Not,pETts,pANth和植物双元表达栽体pCAMBIA1301分别转化用于质粒扩增与保存的常用大肠杆菌菌株Top10和DH5α以及用于原核表达的常用大肠杆菌菌株BL21(DE3)和TB1.质粒与感受态细胞的混合液置冰上作用一定时间后,直接涂布含有筛选抗生素的LB平板,于37℃培养12～16h.结果表明,用不同大小的质粒DNA转化不同的大肠杆菌菌株,都可以获得满足实验要求,转化效率可高达103～4阳性克隆/μg.该方法较标准的转化流程更加简便、省时、实用.     

细菌自然转化的机制及影响因素

水平基因转移对耐药基因传播、编码毒素基因质粒的扩散和毒力岛的转移等过程具有重要的生物学意义。自然转化是指具有感受态的细菌从外界摄取并整合裸露DNA,是水平基因转移的方式之一。细菌发生自然转化极大地促进了耐药基因在不同细菌间的播散,导致细菌对抗生素耐药,给临床治疗带来极大的困难。许多细菌具备自然转化能力,但不同细菌自然转化过程存在着差异。细菌自然感受及转化的诱发及效率亦受到多种因素的影响。文中着重于阐述不同细菌的自然转化机制及其影响因素。     

细菌自然转化的分子机制及生物学功能研究进展

基因的水平转移在细菌的进化中起着非常重要的作用。自然界中的细菌之间主要通过3种机制进行基因水平转移:由噬菌体介导的转导、接合转移和自然转化。自然转化是指自然感受态的细菌能够自发地从外界环境中摄取DNA分子并整合到自身基因组上的过程。该现象首先发现于肺炎链球菌,目前至少有83种细菌被发现具有发生自然转化的能力,其中革兰氏阳性菌以肺炎链球菌(Streptococcus pneumoniae,S. pneumoniae)为代表,革兰氏阴性菌以奈瑟氏菌(Neisseria)为代表,对其自然转化机制的研究和认识较为清楚,但不同细菌之间自然转化的机制有所差异。自然转化的生物学功能一直以来有以下几种推测:获取营养、修复DNA损伤、生物进化,而近年来对此认识争论不休。本文将详细描述细菌自然转化的分子机制,并对其主要的生物学功能争论焦点进行评述,以期对细菌自然转化有更深入的理解和认识。     

质粒pBR322在recA基因缺陷的大肠杆菌细胞内的遗传重组

使用琼脂糖凝胶电泳、DNA限制性内切酶水解以及电子显微镜等分析手段,证明在两株recA~-的大肠杆菌质粒pBR322转化子中,pBR 322 DNA的多倍周长环形寡聚物被大量合成。这个事实说明质粒pBR322 DNA在大肠杆菌细胞中的遗传重组似有独立于rec A基因的途径。本文介绍一个改进的大肠杆菌“清亮裂解液”制备法。按照这个方法制备的细菌清亮裂解液可排除染色体DNA的污染。     

胁迫诱导抗性基因转移导致细菌耐药的分子机制研究进展

抗性基因转移是细菌形成耐药性的重要原因.近年来的研究表明胁迫因子可通过多种机制诱导抗性基因转移.DNA损伤可导致细菌产生SOS应激反应,进而诱导接合DNA介导的抗性基因转移.在一些缺乏SOS系统的细菌中,抗生素胁迫可诱导细菌建立自然转化感受态.此外,作者最近的研究表明普通胁迫应答因子RpoS调控一种由双链质粒DNA介导的固体基质表面的抗性基因转移方式.本文在总结SOS依赖和非依赖型胁迫因子诱导细菌接合和转化介导的DNA转移以及RpoS调控固体基质表面双链质粒DNA转移的基础上,提出今后需重点研究胁迫因子如何激活关键调控蛋白以及这些调控蛋白如何影响DNA转移相关基因表达等关键问题.解决上述问题将为寻找合适的分子靶标用于防控抗性基因转移导致的细菌耐药奠定基础.     

小鼠白细胞介素4基因的化学合成、克隆与表达

利用381A型DNA合成仪,分29个寡聚核苷酸片段化学合成了小鼠IL-4全基因,共442bp。以pUC12质粒作为载体,将所有合成片段分前后两组进行磷酸化、退火、连接和克隆,经过菌落原位杂交、酶切鉴定和质粒DNA序列分析,分别得到了含有小鼠IL-4前后两半基因片段的两种重组质粒,回收前半基因片段,插入到含有后半基因重组质粒的EcoRI和PstI酶切位点之间,成功地得到了含有小鼠IL-4全基因的重组质粒pFR101。将全合成基因插入到质粒pSM53中,得表达质粒pFR105,转化大肠杆菌TAP106,根据IL-4对CTLL细胞的作用,肯定了TAP106(pFR105)细菌中有小鼠IL-4活性蛋白的表达。     

转化条件对质粒DNA转化大肠杆菌的影响

研究了质粒DNA大小、质粒DNA浓度、CaCl2 浓度、热休克时间及感受态细胞保藏时间等因素对大肠杆菌HB1 0 1和JM1 0 5转化频率的影响 ,并对转化子中质粒DNA进行了分离、酶切、琼脂糖凝胶电泳检测。结果表明 ,CaCl2 浓度、质粒大小和浓度 ,以及感受态细胞的活力对转化频率有重要影响 ,42℃热休克处理可以提高转化频率。     

卡那霉素抗性基因在三孢布拉霉菌种中的表达

利用一个带有自主复制子,但缺失自身启动子的源于转座子Tn-5的卡那霉素抗性基因的质粒PVB32,在大肠杆菌中克隆丝状真菌三孢布拉霉DNA中有启动子功能的DNA片段;通过原生质体转化,获得了三孢布拉霉对卡那霉素抗性的表达,且抗性表现稳定,可通过孢子无性繁殖稳定遗传下去。     

大肠杆菌中整合的F′质粒带动细菌染色体复制需要recA基因

大肠杆菌的dnaA46突变能被F′质粒整合抑制。整合抑制的菌林(Sin菌株)在通过转导引入了recA56突变后又变得不能在40℃中生长。标记转移、吖啶橙敏感性,F′质粒消除和mini-染色体质粒转化等实验说明,Sin菌株中F′质粒始终处于整合状态,并且在40℃中细菌染色体的复制由整合状态的F′质粒所带动。比较了Sin recA~ 和Sin recA~-菌株在不同温度中的DNA、蛋白质的生物合成情况。实验结果说明recA基因在DNA复制过程中起作用。前人的工作证明了recA基因在DNA重组和DHA损伤应急修复(SOS)过程中是一个关键的基因。本文的工作为recA基因的功能提供了新的认识。     

病毒ki-ras表达质粒的构建

用重组DNA技术构建了病毒ki-ras表达质粒,用Eco R Ⅰ和Bam H Ⅰ酶解pUC-9DNA,小牛肠碱性磷酸酶脱磷酸,病毒ki-ras编码基因来源于Hi-Hi-3质粒DNA的Sat Ⅱ和Eco R Ⅰ酶切的0.6kb片段,用连接酶和DNA聚合酶Klenow片段将两个片段连接,转化大肠杆菌JM103,免疫筛选表达质粒,从156个转化克隆中得到两株表达质粒,其中一株pKras83的p21蛋白表达量为细菌总蛋白量的10％。     

不同培养时期的大肠杆菌和枯草芽孢杆菌间发生的跨属自然遗传转化

携带穿梭质粒的大肠杆菌与作为受体的枯草芽孢杆菌分别培养至不同生长阶段混合均匀后静置40min,涂布选择性平板,37℃培养30h后得到一定数目的转化子,DNaseⅠ敏感实验证实质粒是通过自然遗传转化而非其它形式发生转移。实验发现大肠杆菌可以在特定生长时期向胞外分泌DNA,并且在对数期具有最高的提供质粒的能力,而生长后期的细胞因为体系中DNase量的增加转化频率下降。进一步的研究发现枯草芽孢杆菌在营养丰富的LB培养基中也具有与基本培养基中相当的转化能力,并且在对数生长前期具有较高的转化频率。     

Intergeneric natural plasmid transformation between E. coli and a marine Vibrio species

Natural transformation is the mechanism of procaryotic gene transfer that involves the uptake and expression of genetic information encoded in extracellular DNA. This process has been regarded as a mechanism to transfer genes (primarily chromosomal markers) between closely related strains or species. Here we demonstrate the cell-contact-dependent transfer of a non-conjugative plasmid from a laboratory E. coli strain to a marine Vibrio species, the first report of intergeneric natural plasmid transformation involving a marine bacterium. The nucleic acid synthesis inhibitors nalidixic acid and rifampicin inhibited the ability of the E. coli to function as a donor. However, dead cells also served as efficient donors. There was an obligate requirement for cell contact. No transfer occurred in the presence of DNase I, when donors and recipients were separated by a 0.2-micron filter, or when spent medium alone was used as a source of transforming DNA. These results indicate that contact-mediated intergeneric plasmid exchange can occur in the absence of detectable viable donor cells and that small non-conjugative plasmids can be spread through heterogeneous microbial communities by a process previously not recognized, natural plasmid transformation. These findings are important in the assessment of genetic risk to the environment, particularly from wastewater treatment systems and the use of genetically engineered organisms in the environment.     

Putative mechanism of natural transformation as deduced from genome data.

Genetic transformation is widely utilized in molecular biology as a tool for gene cloning in Escherichia coli and for gene mapping in Bacillus subtilis. Several strains of eubacteria can naturally take up exogenous DNA and integrate the DNA into their own genomes. Molecular details of natural transformation, however, remained to be elucidated. The complete genome of a cyanobacterium, Synechocystis sp. PCC6803, has been sequenced. This bacterium has been used to examine functions of a particular gene. The genome is considered to carry information on natural transformable characteristics of Synechocystis. The first step in genetic transformation is the uptake of exogenous DNA. Proteins with non-specific DNA binding features are required, because specificity in the exogenous DNA has not been demonstrated. Such proteins have modules interacting with the phosphate backbone of DNA, including helix-turn-helix modules. Using a consensus pattern of the phosphate-binding helix-turn-helix module, we searched through the genome data of Synechocystis for genes or open reading frame (ORF) products with the pattern in primary structures. We found that an ORF, slr0197, has the pattern in duplicate at the C-terminal region. We also found that the ORF product has a hydrophobic segment at the N-terminal region, which is followed by two internal repeats of the endonuclease domain. Based on these observations, we propose a model for the initial stage of genetic transformation. This is apparently the first report on molecular mechanisms of natural transformation.     

Natural plasmid transformation in<Emphasis Type="Italic">Escherichia coli</Emphasis>

Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli.     

Role of membrane potential on artificial transformation of E. coli with plasmid DNA

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.     

Plasmid uptake by bacteria: a comparison of methods and efficiencies

The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation, (2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method, was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique. We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by which to transform bacteria.     

Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis

Natural competence is a process by which bacteria construct a membrane-associated machine for the uptake and integration of exogenous DNA. Many bacteria harbor genes for the DNA uptake machinery and yet are recalcitrant to DNA uptake for unknown reasons. For example, domesticated laboratory strains of Bacillus subtilis are renowned for high-frequency natural transformation, but the ancestral B. subtilis strain NCIB3610 is poorly competent. Here we find that endogenous plasmid pBS32 encodes a small protein, ComI, that inhibits transformation in the 3610 strain. ComI is a single-pass trans-membrane protein that appears to functionally inhibit the competence DNA uptake machinery. Functional inhibition of transformation may be common, and abolishing such inhibitors could be the key to permitting convenient genetic manipulation of a variety of industrially and medically relevant bacteria.     

Transformation of plasmid DNA into E. coli using the heat shock method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42 degrees C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37 degrees C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.     

Inducible cell lysis system for the study of natural transformation and environmental fate of DNA released by cell death.

Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKL02 consists of lysis genes S, R, and Rz from bacteriophage lambda under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKL02 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. calcoaceticus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. calcoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage phi X174 and causes lysis of E. coli and P. stutzeri bacteria by activating cellular autolysins. Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKL01-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made.