Construction of WCB-11: A novel phiYFP arsenic-resistant whole-cell biosensor

The prediction and assessment of environmental pollution by arsenic are important preconditions of advocating environmental protection and human health risk assessment.A yellow fluorescent protein-based whole-cell biosensor for the detection of arsenite and arsenate was constructed and tested.An arsenic-resistant promoter and the regulatory gene arsR were obtained by PCR from the genome of Escherichia coli DH5α,and phiYFP was introduced into E.coli DH5α as a reporter gene to construct an arsenic-resistant whole-cell biosensor(WCB-11) in which phiYFP was expressed well for the first time.Experimental results demonstrated that the biosensor has a good response to arsenic and the expression of phiYFP.When strain WCB-11 was exposed to As 3+ and As 5+,the expression of yellow fluorescence was time-dependent and dose-dependent.This engineered construct is expected to become established as an inexpensive and convenient method for the detection of arsenic in the field.     

A GFP-based bacterial biosensor with chromosomally integrated sensing cassette for quantitative detection of Hg(II) in environment

A mercury biosensor was constructed by integrating biosensor genetic elements into E. coli JM109 chromosome in a single copy number, using the attP/attB recombination mechanism of λphage. The genetic elements used include a regulatory protein gene (merR) along with operator/promoter (O/P) derived from the mercury resistance operon from pDU1358 plasmid of Serratia marcescens. The expression of reporter gene gfp is also controlled by merR/O/P. Integration of the construct into the chromosome was done to increase the stability and precision of the biosensor. This biosensor could detect Hg(II) ions in the concentration range of 100-1700 nmol/L, and manifest the result as the expression of GFP. The GFP expression was significantly different (P ≤ 0.05) for each concentration of inducing Hg(II) ions in the detection range, which reduces the chances of misinterpretation of results. A model using regression method was also derived for the quantification of the concentration of Hg(II) in water samples.     

微生物全细胞传感器在重金属生物可利用度监测中的研究进展

传统的物理化学方法主要用于测定环境重金属的总量,微生物全细胞传感器可以对土壤及水体环境的重金属生物可利用度进行监测.此外,微生物全细胞传感器还具有操作简单、快速、经济的特点,适用于污染事件的应急监测.微生物全细胞传感器的生物学元件主要由MerR、ArsR、RS等家族的金属调控蛋白和gfp、lux、luc等报告基因组成.调控蛋白、报告基因与微生物全细胞传感器的灵敏度、特异性和监测特点有关.受pH、金属螯合物及检测条件等因素的影响,不同的环境条件下的重金属生物可利用度是不同的.增加重金属在微生物细胞内的累积,进行调控蛋白的分子生物学改造,优化检测条件是提高传感器灵敏度、特异性和准确性的可行方案.实现污染物的原位和在线监测是微生物全细胞传感器的主要发展方向.     

二氯乙腈对大肠杆菌基因表达的影响

为探究二氯乙腈（DCAN）对大肠杆菌（E.coli）基因表达的影响及相应的毒性作用,采用自组织映射（SOM）聚类及剂量效应关系分析方法考察了E.coli在不同剂量DCAN暴露120min过程中其基因表达状况.结果表明：随时间及浓度改变E.coli基因表达具有动态性;在DCAN浓度为1.429×10^-3mg/L时,多个参与应急反应（SOS response）调节、氧化还原应激及普通应激的基因启动子活性发生改变,导致DNA损伤、氧化应激加剧,细胞生物化学和物理稳态可能受到干扰;此外,毒性终点结果表明DNA损伤是DCAN主要的毒性作用模式.     

Hg^2＋诱导发光报告基因系统的构建及其检测红壤中残汞的研究

将汞诱导型启动子PmerT和其调控基因merR与egfp基因融合构建发光报告基因系统.通过mini-Tn5系统把merR-egfp插入到Pseudomonas putida染色体DNA上,构建成为在Hg2＋诱导下能特异性表达绿色荧光蛋白的工程菌,并将其应用于检测江西红壤中汞污染.当土壤中汞含量在0.04～50 mg.k...     

基因重组细胞在环境样品多氯联苯检测中的应用

为发展快速、简便和廉价的检测环境和生物样品中的多氯联苯技术,本研究利用重组有绿色荧光蛋白(GFP)和荧光素酶(Luc)报告基因的2个细胞系,检测从野外环境中所采集的水、底泥和生物样品中的多氯联苯的含量.研究结果表明,GFP和Luc荧光强度与多氯联苯标样浓度的相关性很好,相关系数分别达到0.99188和0.98239;具有很好的剂量-效应关系.与气相色谱-电子捕获器法(GC-ECD)的仪器分析比较,GFP和Luc的荧光强度与环境样品中的多氯联苯化合物含量也具有很好的相关性.因此可用于受多氯联苯污染的环境样品筛选和半定量快速、简便、廉价检测.     

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.     

含荧光素酶四膜虫B2086-LUC全细胞生物传感器的构建及其对重金属离子的响应

为了获得可用于快速检测环境中重金属污染的全细胞生物传感器,本研究将含有HA标签的萤火虫荧光素酶(LUC)基因重组到含有四膜虫金属硫蛋白MTT1启动子和微管蛋白终止子序列的载体pBX中,获得重组质粒pBX-LUC,用基因枪粒子轰击法将pBX-LUC转化入四膜虫细胞中,通过同源重组和巴龙霉素抗性筛选, LUC基因整合到四膜虫大核基因组MTT1 位点,获得含有LUC基因的细胞株B2086-LUC. B2086-LUC对重金属镉和汞的响应敏感,可检测的最低浓度为5~10ng/mL;对铜和锌的响应较弱,可检测的最低浓度为0.5~ 1mg/mL.因此,四膜虫B2086-LUC可作为环境中镉和汞污染快速检测的全细胞生物传感器.     

H_2O_2诱导拟南芥NIT2基因甲基化特征与转录水平改变

用外源H2O2处理拟南芥植株,亚硫酸氢盐修饰后测序法分析胁迫生理中NIT2基因启动子区甲基化特征变化,RT-PCR检测该基因的转录水平.结果显示:用100#mol·L-1的H2O2处理3 h后,NIT2启动子区胞嘧啶总甲基化水平与对照差异不大,但对照组CHG(H为C、A或T)和CG位点甲基化水平分别为35.0%和93.3%,H2O2处理组CHG和CG位点甲基化水平分别为50.0%和96.9%;H2O2处理组CHH位点则表现为甲基化水平升高、降低或去甲基化;H2O2胁迫组拟南芥地上组织中NIT2基因转录水平提高.研究结果表明:NIT2基因的转录应答和DNA甲基化修饰参与了植株的逆境生理过程;氧化胁迫与DNA甲基化改变、基因转录上调同时发生,说明胁迫诱发的活性氧增高可能参与胞嘧啶甲基化修饰和基因转录的调节.     

生物传感细胞ADP1_pWHlux在水环境急性毒性检测中的应用

针对天然发光菌和以模式微生物为宿主构建的生物传感细胞在急性毒性检测应用中对测试条件要求苛刻等适用性问题,本研究将1株基因工程构建的生物传感细胞不动杆菌ADP1_p WHlux应用于急性毒性检测,建立检测方法,考察其灵敏度及检测范围.结果表明,ADP1_p WHlux发光受急性毒物的抑制,毒物剂量与发光抑制存在剂量效应关系.在4 mg·L-1Hg Cl2诱导下仅5 min可作出响应,暴露30~60 min后可以给出较为准确的结果.对Hg Cl2的检出限可达0.04 mg·L-1.对我国生活饮用水卫生标准的指标中Be2+、Ba2+、Cu2+、Ni2+检出效果明显,对Be2+、Ba2+、Cu2+的检测范围均在0.025~250 mg·L-1,对Ni2+的检测范围在0.002 5~250 mg·L-1,对Pb2+、Br O-3、Cl O-2的检出限均在0.002 5 mg·L-1,对Cl O-3检出限为0.025mg·L-1.采用ADP1_p WHlux生物传感细胞检测方法对北京市清河水环境急性毒性进行评价,表明ADP1_p WHlux生物传感细胞检测方法可用于污染水样检测.     

Overexpression of NADH oxidase gene from Deinococcus geothermalis in Escherichia coli

When using stable enzyme genes from a thermophile to create a biosensor in Escherichia coli, it is vital that these genes be overexpressed in order to provide a sufficient supply of enzymes. In this study, overexpression of the NADH oxidase (Nox) gene from the thermophile Deinococcus geothermalis was successfully achieved with the aim of creating a stable biosensor active at room temperatures. To do so, modification of 10 nucleotides, GAAATTAACT, upstream of the start codon of the Nox gene was necessary.     

基于脱氧核酶的水中铀酰离子光纤倏逝波生物传感检测技术研究

为了满足铀的高通量快速筛查及环境突发事件快速检测的需要,本研究利用脱氧核酶的特异性催化反应,结合本课题组自主研制开发的倏逝波光纤生物传感器,通过设计两步法的反应策略,即"均相反应,固相杂交"的检测方案,建立了光纤倏逝波生物传感检测铀酰离子的新型技术方法.结果发现,该方法对于5~100 nmol·L~(-1)内的铀酰离子具有良好的线性检测区间,检测限低至0.5 nmol·L~(-1),整个检测过程仅需16 min即可完成,铅和汞等10种金属离子对于本检测方法无明显干扰,表现出了对铀酰离子的良好选择性.传感光纤可重复使用100次以上,不会有信号的明显衰减,可有效降低检测成本.对丹江口水库水进行的实际水样加标回收实验结果显示,丹江口水库实际水样的加标回收率为91.6%~94.6%.本研究可为利用生物传感器法检测环境水体中的铀酰离子提供技术支撑.     

固定化条件对苯系物细胞传感器检测效果的影响

利用海藻酸钠作为包埋剂对苯系物细胞传感器Pseudomonas fluorescens A506(pTS)进行固定化处理,并对细胞浓度、固定化时间、投加量等检测因素进行了优化.固定化后的细胞与游离细胞对苯系污染物的检测效果进行对比.经过2 h固定化处理的细胞传感器,在检测时间1.5～6.0 h内,信号上升速度为游离细胞的2.26倍,信号最大值为游离细胞的2.23倍.固定化处理后的细胞生长缓慢,且浓度远低于游离细胞,说明了固定化处理后的单位细胞信号强度增加.同时,细胞浓度随着固定化时间的延长而降低.在影响因素方面,细胞浓度和固定化时间对于检测信号的影响较大.固定化后,对高浓度的苯系污染物有着更快的信号响应.     

Immigrant Pantoea agglomerans embedded within indigenous microbial aggregates：A novel spatial distribution of epiphytic bacteria

Immigrant bacteria located on leaf surfaces are important to the health of plants as well as to people who consume fresh fruits and vegetables. However, the spatial distribution and organization of these immigrant bacteria on leaf surfaces are still poorly understood. To examine the spatial organization of these strains, two bacterial strains on tobacco leaves： （1） an indigenous strain, Pseudomonas stutzeri Nov. Y2011 labeled with green fluorescent protein, and （2） an immigrant strain Pantoea agglomerans labeled with cyan fluorescent protein isolated from pear, were studied. Under moist conditions, P. agglomerans cells quickly disappeared from direct observation by laser- scanning confocal microscopy, although elution results indicated that large amounts of live cells were still present on the leaves. Following exposure to desiccation stress, particles of cyan fluorescent protein-labeled P. agglomerans were visible within cracked aggregates of P. stutzeri Nov. Y2011. Detailed observation of sectioned aggregates showed that colonies of immigrant P. agglomerans were embedded within aggregates of P. stutzeri Nov. Y2011. Furthermore, carbon-resource partitioning studies suggested that these two species could coexist without significant nutritional competition. This is the first observation of an immigrant bacterium embedding within aggregates of indigenous bacteria on leaves to evade harsh conditions in the phyllosphere.     

Applications of biosensors for bacteria and virus detection in food and water–A systematic review

Biosensors for sensitive and specific detection of foodborne and waterborne pathogens are particularly valued for their portability, usability, relatively low cost, and real-time or near real-time response. Their application is widespread in several domains, including environmental monitoring. The main limitation of currently developed biosensors is a lack of sensitivity and specificity in complex matrices. Due to increased interest in biosensor development, we conducted a systematic review, com...     

Climate change and the geography of weed damage: Analysis of U.S. maize systems suggests the potential for significant range transformations

By the end of the century, climate change projections under a “business-as-usual” emissions scenario suggest a globally averaged warming of 2.4–6.4 °C. If these forecasts are realized, cropping systems are likely to experience significant geographic range transformations among damaging endemic weed species and new vulnerabilities to exotic weed invasions. To anticipate these changes and to devise management strategies for proactively addressing them, it is necessary to characterize the environmental conditions that make specific weed species abundant, competitive, and therefore damaging the production of particular crops (i.e. defining the damage niche). In this study, U.S. maize is used as a model system to explore the implications of climate change on the distribution of damaging agricultural weeds. To accomplish this, we couple ensemble climate change projections of annual temperature and precipitation with survey data of troublesome weed species in maize. At the state scale, space-for-time substitution techniques are used to suggest the potential magnitude of change among damaging weed communities. To explore how the geography of damage for specific species may evolve over the next century, bioclimatic range rules were derived for two weed species that are pervasive in the Northern (Abutilon theophrasti Medicus, ABUTH) and Southern (Sorghum halepense (L.) Pers., SORHA) U.S. Results from both analyses suggest that the composition of damaging weed communities may be fundamentally altered by climate change. In some states, potential changes in the coming decades are commensurate to those possible by the end of the century. Regions such as the Northeastern U.S. may prove particularly vulnerable with emerging climate conditions favoring few weed species of present-day significance. In contrast, regions like the mid-South are likely to experience fewer shifts even with a similar magnitude in climate change. By the end of the century in the U.S. Corn Belt, cold-tolerant species like A. theophrasti may be of minor importance whereas S. halepense, a predominantly Southern U.S. weed species at present, may become common and damaging to maize production with its damage niche advancing 200–600 km north of its present-day distribution.     

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.     

DEP对蚯蚓抗氧化酶系的影响及DNA损伤

邻苯二甲酸二乙酯(DEP)是一种常见的塑料添加剂,并因塑料制品大量广泛使用而进入土壤环境,但其对土壤动物的毒性及其机制并未完全阐明.本文以赤子爱胜蚓(Eisenia foetida)为研究对象,使其暴露于不同含量DEP的模拟污染土壤,以蚯蚓体内的抗氧化酶活性、ROS含量、GST活性、MDA含量和DNA损伤程度为评估参数,研究含DEP污染土壤对蚯蚓的毒性作用并分析其机制.结果表明,在DEP胁迫作用下,蚯蚓体内的抗氧化酶和谷胱甘肽-S-转移酶(GST)活性、活性氧自由基(ROS)均发生变化并导致基因损伤产生.在28d的实验周期内0.1~50 mg·kg-1DEP的胁迫下,ROS含量水平呈现增加状态,存在"剂量-效应"关系,并且过量的ROS引起脂质过氧化反应造成机体内MDA含量增加.在ROS和MDA共同作用下,蚯蚓体腔内的DNA受到损伤并且损伤程度与DEP含量存在"剂量-效应"关系.从实验结果可以看出,DEP可以对蚯蚓机体和DNA造成一定程度的损伤,表现出较强的生态毒理效应.     

紫外线和次氯酸钠对Escherichia coli和Poliovirus的消毒作用

本研究选用大肠杆菌(Escherichia coli)和脊髓灰质炎病毒(poliovirus)分别作为典型的细菌和病毒,利用培养和定量PCR的检测技术,对比研究紫外线消毒和次氯酸钠消毒对细菌和病毒的作用特点.结果表明:脊髓灰质炎病毒比大肠杆菌更难被灭活,达到1-log所需的氯剂量分别为19.2 mg·L~(-1)·min和10.14 mg·L~(-1)·min;所需的紫外线剂量分别为6.37 m J·cm~(-2)和1.81 m J·cm~(-2).定量PCR方法检测大肠杆菌和脊髓灰质炎病毒达到1-log的核酸损伤所需的紫外线剂量和氯剂量要比培养法高出1~2数量级,紫外线消毒对脊髓灰质炎病毒的RNA损伤量明显大于对大肠杆菌的DNA损伤,病毒的单链RNA对紫外线的敏感性更强,该结果与培养法正好相反.达到1-log核酸损伤脊髓灰质炎病毒所需的紫外线剂量为135 m J·cm~(-2),大肠杆菌所需的剂量为270.3 m J·cm~(-2),核酸损伤需要更多的消毒剂量,可能由于消毒过程微生物进入活性但处于非可培养状态(VBNC),以及灭活对微生物其他分子的损伤和微生物死后核酸的持续性.