应用绿色荧光蛋白基因标记细菌进行生物膜结构定量化新方法

绿色荧光蛋白基因pEGFP经CaCl₂转化法标记E.coli JM109菌株,获得的标记菌株作为模式细菌接种含50μg/mL氨苄的LB培养基,在摇瓶中与火山岩颗粒共混培养挂膜(37℃,120r/min,16h).用激光共聚焦扫描显微镜摄取获得火山岩填料生物膜250μm×250μm区域不同层面的图片堆,所获图片堆经COMSTAT程序处理可以获得相关的定量化参数,如16h生物膜平均厚度为0.120844μm,生物膜最大厚度为10.5μm,生物膜体积为0.136986μm³/μm²,生物膜表面积21338.1μm²,生物膜比表面积为3.36854μm²/μm³.该方法也可以扩展至其他绿色荧光蛋白基因标记细菌的生物膜结构定量化.     

绿色荧光蛋白标记的大肠杆菌海水中稳定性研究

用绿色荧光蛋白基因( GFP) 标记大肠杆菌K12(K12GFP),检测其在海水中的存活及稳定性.研究结果表明,K12与K12GFP微生物学特性差异不显著,而且两者在高压灭菌海水中的存活曲线也基本一致,说明K12GFP可为进一步研究K12在自然海水中的生态变化提供可靠依据.在高压灭菌海水中,K12GFP存活28 d之久,该菌密度随时间缓慢下降;在过滤海水中,K12GFP存活7 ,该菌密度从107CFU/mL降到102CFU/mL.表明海水的营养成分会对其存活产生较大影响.     

绿色荧光蛋白标记阿特拉津降解基因工程菌的特性

通过转化绿色荧光蛋白基因质粒,对阿特拉津降解基因工程菌进行标记.转化后,绿色荧光蛋白在细胞内表达情况良好.在含抗生素的LB培养基中,绿色荧光蛋白的表达水平高于LB培养基和基础培养基.在细胞生长的稳定期,绿色荧光蛋白表达水平高于停滞期和对数生长期.绿色荧光蛋白基因质粒转化和表达,不会对基因工程菌原有的降解能力产生影响,而且绿色荧光蛋白的表达水平和降解活性存在接近线形的正相关关系.荧光蛋白标记细胞投加到反应器活性污泥中,会以2种状态存在:游离态和附着态,而且初期游离态细胞的数量大于附着态细胞的数量.     

绿色荧光蛋白基因在根际优势菌株中的高效表达

从人工模拟污染土壤中的黄麻根际分离得到两株优势菌株。通过PCR技术对增强绿色荧光蛋白基因与质粒载体pTnMod Ocm进行体外重组构建新的质粒 ,并转化分离得到的优势菌株。结果表明增强绿色荧光蛋白基因在黄麻根际优势菌株中高效表达     

腺病毒气溶胶的实时荧光定量PCR检测和绿色荧光蛋白活细胞检测

将带有绿色荧光蛋白的复制缺陷型重组腺病毒,作为模拟病毒建立一种检测病毒侵袭力的方法.在钢化玻璃箱中通过TK-3型微生物气溶胶发生器将腺病毒形成气溶胶,用FA-1型多级撞击式空气微生物采样器进行气溶胶采样,对采样样品分别进行实时荧光定量PCR检测和表达了绿色荧光蛋白的PK15活细胞定时检测.实时荧光定量PCR检测可测定病毒在大气中存在的相对基因拷贝数,通过在荧光显微镜下计数带绿色荧光的PK15细胞数可直观检测病毒的感染力及活力.结果表明,重组腺病毒气溶胶主要分布在采样器第五级,腺病毒气溶胶与病毒粒子相比较大.     

用于废水处理系统中目标酵母动态解析的绿色荧光蛋白基因质粒构建

绿色荧光蛋白(green fluorescent protein,GFP)可用于研究复合微生物体系中特定目标功能菌的特性及动态变化,本研究为确立用于解析酵母细胞在混合酵母废水处理系统中的动态特性的GFP技术体系奠定了基础.将gfp基因克隆到酵母载体pACT-URA3中,再用构建的重组质粒转化宿主菌大肠杆菌(Escherichia coli JMl09),扩增得到含gfp的重组质粒,荧光显微镜照片显示gfp基因在大肠杆菌内得到了表达,但表达程度不高;电泳图谱及聚合酶链反应结果表明,含gfp基因的质粒不是以游离的形式存在,而可能是以某种特殊的形式和细胞染色体发生了相互作用.     

绿色荧光蛋白标记在生物修复中的应用

通过对铜绿假单胞菌进行增强型绿色荧光蛋白(EGFP)标记,获得带有EGFP遗传标记的铜绿假单胞菌.该菌株绿色荧光标记性能稳定,通过转化子基因组DNAPCR和斑点杂交检测,证明外源EGFP基因存在于转化子染色体上.转化子30h内将溶液的表面张力由70mN/m降至35mN/m,产生的生物表面活性剂性能良好.     

镉对松散和紧密胞外聚合物类蛋白的荧光滴定

采用分子荧光技术和荧光滴定的方法解析了重金属cd与2种不同类型胞外聚合物类蛋白的结合机理;并通过2种胞外聚合物类蛋白与cd结合后荧光光谱的特征,进一步分析松散附着(Loosely Bound,LB)和紧密黏附(Tightly Bound.TB)2种胞外聚合物之间的结构差异.结果表明,在滴定的cd浓度低于1×10-mol·L-1、pH值为4的条件下,cd对LB具有显著的猝灭效应;结合Stern-Volmer分析,这种猝灭现象不仅与生色团的质子化效应有关.而且也和类蛋白与cd结合位点的变化有关.同时.滴定过程中LB均出现不同程度的红移现象,而TB却出现不同程度的蓝移.这说明,cd与2种类蛋白物质的结合机理方面具有显著差异.进一步三维光谱分析结果显示,在滴定前后LB中类蛋白荧光峰的结构与形态并未产生明显分化,由此说明,LB内荧光生色团性质和结构相对单一;而滴定后TB中肩峰的凸现说明.其类蛋白性质、结构与LB相比更具有多样性的特点.pH值为10的条件下,Stem-Volmer分析结果显示,LB和TB类蛋白荧光团的猝灭与2方面因素有关,一是与生成不产生荧光的EPS-Cd络合物有关,二是与分子之间的碰撞所导致的荧光猝灭有关.     

1株2,4-D降解菌的gfp标记及其在废水生物处理系统中的检测方法

通过mini-Tn7转座子系统将绿色荧光蛋白基因(gfp)插入到2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)降解菌Achromobacter sp.的染色体上,考察了标记前后该菌株的生长、发光及降解污染物特性,并探讨了将其投加到不同废水生物处理系统(活性污泥和颗粒污泥系统)后的定量检测方法.结果表明,Achromobacter sp.标记前后生长和降解2,4-D特性基本不变,在103～112 h内可将初始浓度约为100 mg/L的2,4-D完全降解.标记后菌株在生长和降解2,4-D过程中都能够稳定表达绿色荧光,降解过程荧光强度/D600稳定在4 500左右.向活性污泥系统投加该标记菌,可通过直接测定混合液荧光强度对该标记菌进行定量检测,在标记菌质量分数为0～75%的范围内,绿色荧光蛋白的表达水平与该标记菌的质量分数线性相关(R2=0.995 2).向颗粒污泥系统投加该标记菌,需要对混合液破碎均质化处理后测定荧光强度,在标记菌质量分数为0～42%的范围内,绿色荧光蛋白的表达水平与该标记菌的质量分数线性相关(R2=0.980 1).基于Tn7插入gfp的标记方法可以用来跟踪检测生物处理系统中的特异微生物.     

固定化荧光假单胞菌降解阴离子表面活性剂的研究

用海藻酸钠法包埋适冷性微生物荧光假单胞菌株AIS-11,并用于处理阴离子表面活性剂废水,所有实验均在批式反应器中进行。以L_9(3~4)正交试验确定了其对阴离子表面活性剂十二烷基硫酸钠(SDS)和十二烷基苯磺酸钠(SDBS)的最佳包埋条件,并且对游离细胞和固定化菌体的降解过程进行了动力学分析和比较。结果表明,固定化珠体能将吸附和生物催化过程有机地耦联起来,有效地提高了阴离子表面活性剂的降解速率,将底物最大利用速度(mg/L·h)从游离细胞的5.75(SDS)和5.12(SDBS)分别提高至22.89和25.14。     

荧光原位杂交技术解析发酵产氢细菌群落结构

应用荧光原位杂交(FISH)技术,研究了连续流搅拌槽式(CSTR)发酵制氢反应器中2种产氢发酵类型的菌群组成和发酵类型转化过程中的菌群种类和数量变化及其对产氢速率、生物量和发酵产物的影响.结果表明,梭菌属和肠杆菌科在决定产氢发酵类型方面有重要作用.以梭菌为优势种群的乙醇型发酵比以肠杆菌为优势种群的丁酸型发酵具有更佳的产氢能力.实验结果能够与生物发酵系统常规监测指标形成很好的印证.     

Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization(FISH)

A molecular biology method, fluorescent in situ hybridization（FISH）, in which the pre-treatment was improved in allusion to the media of the constructed wetlands（CW）, e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria（AOB） quantity and the relation with oxidation-reduction potential（ORP） in the Typha latifolia constructed wetlands under three different Ioadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.     

Determination of paraquat in water samples using a sensitive fluorescent probe titration method

Paraquat (PQ), a nonselective herbicide, is non-fluorescent in aqueous solutions. Thus, its determination through direct fluorescent methods is not feasible. The supramolecular inclusion interaction of PQ with cucurbit[7]uril was studied by a fluorescent probe titration method. Significant quenching of the fluorescence intensity of the cucurbit[7]uril-coptisine fluorescent probe was observed with the addition of PQ. A new fluorescent probe titration method with high selectivity and sensitivity at the ng/mL level was developed to determine PQ in aqueous solutions with good precision and accuracy based on the significant quenching of the supramolecular complex fluorescence intensity. The proposed method was successfully used in the determination of PQ in lake water, tap water, well water, and ditch water in an agricultural area, with recoveries of 96.73% to 105.77%. The fluorescence quenching values (ΔF) showed a good linear relationship with PQ concentrations from 1.0×10^-8 to 1.2× 10^-5 mol/L with a detection limit of 3.35×10^-9 mol/L. In addition, the interaction models of the supramolecular complexes formed between the host and the guest were established using theoretical calculations. The interaction mechanism between the cucurbit[7]uril and PQ was confirmed by ¹H NMR spectroscopy.     

环境因素对大肠杆菌REP-PCR指纹图谱稳定性影响研究

大肠杆菌作为粪便污染指示生物,其DNA指纹图谱稳定性是制约微生物污染源识别技术发展的瓶颈之一.本研究采用绿色荧光蛋白基因(GFP)标记后的Escherichia coli K12(K12GFP)为受试菌株,利用REP-PCR方法,对大肠杆菌在不同温度、光照、盐度条件下其DNA指纹图谱的稳定性进行了研究.结果表明,绿色荧...     

Preimplantation genetic diagnostic protocols for α- and β-thalassaemias using multiplex fluorescent PCR

Haemoglobinopathies including α- and β-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for β-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting β-thalassaemia mutations (codon 41-42 and IVSI-110) and one for α-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of β-globin and δ-globin was also found to be an important issue that necessitated careful design of primers for the β-globin gene. Copyright © 2001 John Wiley & Sons, Ltd.     

Nutrient depletion is the main limiting factor in the crude oil bioaugmentation process

The biodegradation was considered as the prime mechanism of crude oil degradation. To validate the efficacy and survival of the crude oil-degrading strain in a bioremediation process, the enhanced green fluorescent protein gene (egfp) was introduced into Acinetobacter sp. HC8–3S. In this study, an oil-contaminated sediment microcosm was conducted to investigate the temporal dynamics of the physicochemical characterization and microbial community in response to bacterium amendment. The introduced strains were able to survive, flourish and degrade crude oil quickly in the early stage of the bioremediation. However, the high abundance cannot be maintained due to the ammonium (NH₄⁺-N) and phosphorus (PO₄^3?-P) contents decreased rapidly after 15 days of remediation. The sediment microbial community changed considerably and reached relatively stable after nutrient depletion. Therefore, the addition of crude oil and degrading cells did not show a long-time impact on the original microbial communities, and sufficient nitrogen and phosphorus nutrients ensures the survive and activity of degrader. Our studies expand the understanding of the crude oil degradative processes, which will help to develop more rational bioremediation strategies.     

荧光溶解性有机质EEMs的新旧自组织映射图解析方法比较研究

目前,对地表水体荧光溶解性有机质(Fluorescent Dissolved Organic Matter,FDOM)三维激发发射矩阵(Excitation-Emission Matrices,EEMs)数据的解析仍旧存在挑战.本文结合原始EEMs和从中分离的荧光平行因子分析(Parallel Factor Analysis,PARAFAC)组分,利用自组织映射图(Self-Organizing Maps,SOM),开展了基于传统EEMs-SOM和新型PARAFAC-SOM神经网络模型的复杂荧光数据解析能力对比研究,以此探索和改进EEMs多元解析方法技术体系.模型以已发表论文原始数据为基础构建,研究对象为南太湖重要入湖河流东苕溪水系.结果显示:EEMsSOM模型需依赖常规"摘峰法"对各荧光峰做出主观判断,不能有效识别重叠荧光峰,且人为割裂多激发共发射荧光物质峰之间的联系,从而弱化分析结果的实际环境意义,但神经元的模式荧光光谱可视化效果较佳,且操作简便,耗时较少;PARAFAC-SOM模型克服了上述缺陷,可清除EEMs中的噪声或无用信息,大幅降低输入变量数(从1742个降至4个),缩减运算时间,较大地改善了输出结果的准确性,获得无损可靠且实际环境意义较强的结果,但该法的PARAFAC预处理要求较高,工作量较大;两类模型解析结论大体一致,对前期研究进行了全新的阐述和补充,且生动揭示了FDOM在多级河流水体中的空间赋存变化规律及河流的动态污染过程.综上,推荐在今后研究中综合使用两类优势互补的模型以实现对EEMs的深度解析.