盐生盐杆菌RM07 DNA片段在大肠杆菌中的定点诱变和启动子功能分析

将来源于嗜盐古生菌——盐生盐杆菌(Halobacterium halobium)基因组的RM07 DNA片段以正反两个方向分别插入大肠杆菌启动子探针载体pKK232-8携带的报告基因——氯霉素抗性基因(cat)的上游,得到RM07-cat融合的质粒pRM07-1( )和pRM07-1(-),将其分别转入大肠杆菌HB101,进而检测了不同转化子菌株的氯霉素抗性水平和细胞内氯霉素乙酰转移酶蛋白质浓度。结果表明：正向的RM07片段在真细菌(大肠杆菌)中具有启动子活性,能够驱动cat报告基因的表达;而反向的RM07片段在大肠杆菌中不具有启动子活性。对RM07片段进行了定点诱变分析,检测了特定核苷酸突变对启动子活性的影响,结果进一步精确定位了RM07片段中对在大肠杆菌中的启动子功能有重要作用的关键碱基,并且通过改造RM07片段的碱基组成成分大幅提高了其在大肠杆菌中的启动子活性。     

盐生盐杆菌DNA 片段RM07的-35、-10区缺失分析

RM07DNA片段分离自嗜盐古菌——盐生盐杆菌R1,该片段不仅在嗜盐古菌内具有启动子功能,而且在大肠杆菌中也具有启动子活性。序列分析表明其上确实含有细菌启动子的-35、-10保守区。进一步通过缺失分析证实该片段就是在大肠杆菌中具启动功能的DNA片段：仅含-10区而缺失-35区的RM07a片段基本上无启动活性;而含-35和-10区的0．1kb片段RM07b具有高于RM07的启动活性。研究结果还表明,RM07片段在大肠杆菌中的启动活性是受环境因素调节的,尤其是对氯化钠浓度的提高具有明显的相关性。因此RM07片段有可能成为构建双功能表达载体的新启动子资源,同时对进一步揭示古菌的融合特征及水平基因转移具有特殊意义。     

盐生盐杆菌DNA片段RM07的-35、-10区缺失分析*

RM0 7DNA片段分离自嗜盐古菌———盐生盐杆菌R1 ,该片段不仅在嗜盐古菌内具有启动子功能 ,而且在大肠杆菌中也具有启动子活性。序列分析表明其上确实含有细菌启动子的 35、 1 0保守区。进一步通过缺失分析证实该片段就是在大肠杆菌中具启动功能的DNA片段 :仅含 1 0区而缺失 35区的RM0 7a片段基本上无启动活性 ;而含 35和 1 0区的0 1kb片段RM0 7b具有高于RM0 7的启动活性。研究结果还表明 ,RM0 7片段在大肠杆菌中的启动活性是受环境因素调节的 ,尤其是对氯化钠浓度的提高具     

棒杆菌启动子片段的酶谱分析及定位

用大肠杆菌启动子探测质粒pSDS I (Ap^r,Tc^s)从钝齿棒杆菌(Corynebacterium crenatum)6282染色体的HindⅢ酶切片段中,克隆到两个具有启动功能的DNA片段,分别将两个重组质粒命名为pSDB5和pSDB21。含有这两个质粒的菌株均可以在含300μg/ml Tc的平板上生长。通过酶切分析,pSDB5的插入片段为1.6kb,pSDB21的插入片段为3.4kb,并分别作出了它们的限制性酶切图谱。对pSDB21利用其EcoR Ⅰ和BglⅡ位点,通过亚克隆删除了与启动功能无关片段,构建成pSDB210和pSDB211,从而使启动子定位于约0.1kb的BglⅡ/HindⅢ外源片段上。分子杂交实验证明所得到的这两个具有启动功能的DNA片段确实来源于钝齿棒杆菌6282的染色体DNA。     

大肠杆菌ptsHIcrr操纵子的快速敲除及敲除菌生长性能测定

敲除大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统(简称PTS系统)ptsHIcrr操纵子,考察敲除菌株生长特性并将其与ptsG敲除菌进行比较。利用I-SceⅠ特异性切割和Red同源重组方法成功构建了大肠杆菌DH5α△ptsHIcrr敲除菌。在LB培养基中,DH5α△ptsHIcrr的生长行为与DH5α和DH5α△ptsG明显不同,其最高菌密度是DH5α和DH5α△ptsG的近2倍,而DH5α△ptsG生长行为与DH5α无明显差异。但在含1%葡萄糖的LB中,DH5α△ptsHIcrr和DH5α△ptsG均表现出生长优势,最高菌密度依次是DH5α的2.8和2倍;培养液中最终乙酸含量分别是DH5α的12.2%、47%。在M9修饰培养基中,DH5α△ptsHIcrr比生长速率(1/h)和比葡萄糖消耗速率[g/(g.h)]明显低于DH5α,并略低于DH5α△ptsG。结果说明,ptsHIcrr操纵子敲除菌改变了葡萄糖的代谢速率,并呈现与ptsG基因敲除菌不同的代谢特点。     

人Leptin的基因克隆及其在大肠杆菌中的表达

用 RT- PCR自脂肪细胞 RNA扩增人瘦素 (leptin)基因的 c DNA片段 (约 50 0 bp)并克隆至克隆载体 p SK(+) ,获得重组质粒 p SK- OB,DNA序列分析显示获得的 Leptin基因和文献报道一致 .用限制酶 Eco R 和 Bam H 从 p SK- OB切下并插入原核表达载体 p BV2 2 0的相应限制酶位点 ,转化大肠杆菌 DH5α.转化菌株经 42℃诱导 ,SDS- PAGE检测和 Western印迹鉴定 ,获得高水平重组人瘦素的表达菌株 ,其表达量达菌体总蛋白的 30 %以上 .     

利用绿色荧光蛋白基因gfp研究芽胞杆菌的启动子活性

利用绿色荧光蛋白基因gfpmut3,分别标记苏云金芽胞杆菌(Bacillus thuringiensis)的cry3A启动子Pcry3A、BtI_BtII启动子PBtI_BtII和来自蜡状芽胞杆菌特异启动子P44-12以研究其表达差异。其中,Pcry3A和PBtI_BtII分别与gfpmut3构成融合基因,以调控gfpmut3在苏云金芽胞杆菌中的表达。将重组质粒pGFP_304(含P44-12)、pGFPExpA(含Pcry3A_ gfpmut3融合基因)和pGFPExpB(含PBtI_BtII_ gfpmut3融合基因)分别导入大肠杆菌(Escherichia coli)和苏云金芽胞杆菌后发现,P44-12和PBtI_BtII在大肠杆菌与苏云金芽胞杆菌中均可表达gfpmut3,其中PBtI_BtII在大肠杆菌中具有极强的启动基因表达的能力。而Pcry3A不能启动gfpmut3在大肠杆菌中表达,在苏云金芽胞杆菌中启动的gfpmut3表达的荧光强度也较弱。进一步通过荧光显微镜和生物活性检测器对含重组质粒pGFP_304、pGFPExpA和pGFPExpB的转化子分别进行荧光检测及微量热检测。结果表明,3种启动子驱动下的gfpmut3基因均可在苏云金芽胞杆菌无晶体突变株BMB171中表达并检测得到不同的发光类型。微量热法检测发现P44_12和PBtI_BtII启动gfpmut3表达的代谢热低于Pcry3A驱动gfpmut3表达的代谢热。     

油桐尺蠖核型多角体病毒DNA聚合酶基因的克隆和序列分析

用PCR方法从油桐尺蠖核型多角体病毒 (BusuNPV)中扩增出DNA聚合酶基因片段 ,经pGEM T载体克隆到大肠杆菌DH5α菌株中。经自动序列分析仪测出DNA聚合酶基因 2 379bp长的核苷酸序列 ,推导出 793的氨基酸序列。氨基酸同源性比较显示 ,BusuNPV与HzSNPV的同源性最高 ,达 57% ;与OpMNPV的同源性最低 ,为 39.6 %。     

含G0S2基因启动子的荧光素酶报告基因载体的构建

目的:克隆人G0S2基因启动子并构建荧光素酶报告基因载体,为进一步研究G0S2基因转录调控提供质粒。方法:利用PCR技术从人胚肾293A细胞基因组DNA中克隆获得G0S2基因启动子的DNA片段,将其克隆至pGL3-basic表达载体中,并转化人大肠杆菌DH5α,经限制性内切酶酶切、PCR及测序鉴定得到确认;将重组载体质粒与半乳糖苷酶表达质粒psV-β-Galactosidase共转染至大鼠血管平滑肌细胞(VSMC),检测细胞中荧光素酶的活性。结果:pGL3-G0S2-Promoter重组质粒插入片段和相邻序列正确,克隆的G0S2基因片段有启动子活性(P0.05)。结论:成功构建了pGL3-G0S2-Promoter报告基因质粒,为进一步研究G0S2基因的表达奠定了基础。     

嗜酸氧化亚铁硫杆菌启动子探针载体的构建

目的：为了研究嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans）启动子结构与功能。方法：以pSV-β-galactosidase质粒为骨架,通过定点突变的方法引入一个新的BstBⅠ单酶切位点,构建能在大肠杆菌（Escherichia coli）中正常复制的启动子探针载体。利用PCR的方法将A.ferrooxidans菌cycA2基因上游5＇段上游DNA片段克隆到探针载体β-半乳糖苷酶基因上游以替代其原有启动子（gpt启动子）,并将重组的质粒转化E.coliDH5α菌株。通过检测宿主细胞的β-半乳糖苷酶活性,来鉴定启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。结果：pSV-β-galactosidase质粒被正确突变,成功构建了启动子探针载体pSVB。来源于A. ferrooxidans菌的启动子片段可驱动β-半乳糖苷酶基因在E.coli细胞中表达,转化子酶活性约为gpt 启动子驱动下活性的70 %。结论：启动子探针载体（pSVB）可用于A. ferrooxidans菌或者其它原核生物启动子的分离及进一步的分析研究。酶活性分析结果表明,来源于A. ferrooxidans菌cycA2基因上游5＇段上游DNA片段具有显著启动子活性。     

Microcalorimetry is a sensitive method for studying the effect of nucleotide mutation on promoter activity

Microcalorimetric method was successfully used to study the effect of nucleotide mutations on promoter activity and identify the important nucleotide necessary for the promoter function in Escherichia coli. The thermokinetic parameters, such as k, I and IC(50), were calculated from the metabolic power-time curves obtained by microcalorimetric measurement using the TAM air Isothermal Microcalorimeter (manufactured by Thermometric AB company of Sweden). Analysis of these data revealed that different nucleotide mutations in -10 box sequence of RM07 fragment had different effect on the promoter activity. Our research also suggest that the microcalorimetric method is a very sensitive and easily performed method for investigation of promoter mutation.     

Improving the growth rate of Escherichia coli DH5alpha at low temperature through engineering of GroEL/S chaperone system

GroEL/S is a molecular chaperone system in Escherichia coli which not only assists the folding of intracellular proteins but also affects the cellular activity against the change of environmental condition. Here we show that the growth rate of E. coli DH5alpha can be improved at low temperature by expressing a GroEL/S variant achieved through irrational protein engineering approach. The GroELS variant (GroELS(var)) accelerating the growth of E. coli DH5alpha was screened through enrichment culture of the mutant libraries obtained by random mutagenesis. E. coli DH5alpha harboring the groELS(var) gene exhibited approximately 1.5-2 times higher growth rate compared to the strain with wild-type GroELS at 15-30 degrees C. At 10 degrees C, a temperature that the growth of E. coli DH5alpha almost stops, the GroELS(var) triggered the growth of E. coli DH5alpha. We identified that seven nucleotides of groELS gene and six amino acids of the GroELS were changed through the mutagenesis and screening. Site directed mutagenic analysis revealed that H360 in GroEL(var) is the most crucial residue determining the activity of GroELS(var) and more than one of the other residues in GroEL(var) may be additionally involved in the activity of GroELS(var). The improvement of growth rate induced by the GroELS(var) was observed only in the strain DH5alpha and not detected in other E. coli strains, such as BL21, BW25113, codon+, JM110, Top10, and XL1-blue.     

Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galactosidase activity after maximal induction/specific beta-galactosidase activity before induction.)The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD(600) is congruent to 1.3) before being transferred to the fermentor; the amount of beta-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD(600) = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD(600) became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (c) 1996 John Wiley & Sons, Inc.     

The 492-bp RM07 DNA Fragment from the Halophilic Archaea Confers Promoter Activity in All Three Domains of Life

A 492-bp DNA fragment, designated RM07, was isolated from the chromosomal DNA of the halophilic Archaea, Halobacterium halobium, and was shown to confer promoter activity in Escherichia coli. Sequence analysis revealed that RM07 contained three consensus sequences of the archaeal distal promoter element as well as the typical –35 and –10 box sequences of bacterial promoters. Promoter probe analysis confirmed that RM07 conferred promoter activity in all three domains of life: Archaea (Haloferax volcanii), Eukarya (Saccharomyces cerevisiae) and Bacteria (Escherichia coli). Deletion analysis and site-directed mutagenesis further identified the functional regions within RM07 required for promoter activity. This is the first report of a DNA fragment from Archaea that confers promoter activity in all three domains of life, suggesting that the promoter structure and activity may be viewed as a bridge narrowing the gaps among the different domains of life.