恶臭假单胞菌KT2440高效电转化方法

恶臭假单胞菌(Pseudomonas putida)KT2440是模式环境微生物菌株,也是异源表达的良好宿主菌.高效率的电转化方法对于基因的引入及后续实验非常关键.通过条件优化,建立了高效的电转化方法.EM培养基培养菌体至D600nm=0.6～0.75,冰冷的3mmol/L4-羟乙基哌嗪乙磺酸(HEPES,pH=7.0)的缓冲液洗涤菌体3次,菌体浓缩300倍,50μL分装并用于一次电转化.电转化条件:1.2kV,200Ω,25μF.1mL SOC培养基悬浮后,转至15mL聚丙烯离心管培养2h,转化效率可高达3.0×108转化子/μg,比目前为止文献报道的该菌株电转化效率高出30倍左右.实验结果还表明,转化DNA浓度与转化子数目呈线性关系.     

荧光假单胞菌高效广谱载体的构建及分析

为促进高GC含量基因在荧光假单胞菌(Pseudomonas fluorescens)中表达效果更加理想、操作更加简便,本研究首先采用不依赖基因序列和连接反应的克隆(Sequence and ligation independent cloning,SLIC)方法将载体pCIBhis上与复制相关的序列和标记基因片段构建成克隆载体pCIBS1.然后优化荧光假单胞菌转化方法,用电转化法将pCIBS1导入荧光假单胞菌BL915中,随后又将T7和tac基因启动子分别插入pCIBS1中,成功构建了表达载体pCIBS3和pCIBS2.研究发现载体pCIBS1在大肠杆菌和荧光假单胞菌中均较为稳定,并且将绿色荧光蛋白基因插入表达载体中,在大肠杆菌BL21(DE3)中获得表达,验证了表达载体功能.本研究构建的表达载体和建立的荧光假单胞菌BL915电转化方法,为高GC含量基因在荧光假单胞菌中的表达奠定了基础.     

萘降解质粒pND6的分离和鉴定

从工业废水中分离的假单胞菌 (Pseudomonassp .)ND6菌株 ,能以萘为唯一碳源生长 ,使无机盐培养基(MM)中 2g/L的萘在 48h内降解 98% .该菌株含有一个 115kb的大质粒pND6 .DNA杂交实验表明 ,pND6质粒含有与恶臭假单胞菌 (P .putida)G7菌株的NAH7质粒同源的萘降解基因 .图 3表 1参 14     

假单胞菌表达载体pYMB03的构建与性能分析

恶臭假单胞菌(Pseudomonas putida)是具有强抗逆性能的环境优势菌,构建其质粒表达载体有着明显的应用潜力.将恶臭假单胞菌AB92019菌株中肽聚糖相关脂蛋白编码基因的启动子PoprL和质粒载体pTrcHis-B的多克隆位点片段插入到质粒载体pUCPl8的EcoRL/HInIII位点,获得了重组载体pYMB03.用绿色荧光基因gfp作为标记进行外源蛋白表达的结果表明,该载体能分别在恶臭假单胞菌AB92019菌株和大肠杆菌DH5a菌株中,由启动子PoprL启动组成型表达GFP蛋白并使细胞产生可见荧光.经SDS-PAGE验证,所产生的GFP蛋白分别占细胞总蛋白的12.5%和5.O%.重组菌株YMB001中GFP表达量与菌体培养时间有关,在稳定期后期其相对荧光强度达到最大值(D600nm=l.0),但与培养温度未见相父性.对携带该载体的2株重组恶臭假单胞菌7次168 h继代培养测定,载体pYMB03的稳定性均为100%.     

荧光假单胞菌(Pseudomonas fluorescens)工程菌株田间残留和扩散的追踪检测

分别在北京和江苏省连云港市对携带Btcry基因的荧光假单胞菌工程菌进行了田间残留与扩散的追踪检测 .对越冬前后的试验地和保护地土壤样品进行抗生素抗性平板分离 ,能检测到极少量的具有与出发菌株相同抗性的荧光假单胞菌菌落 ,但没有发现工程菌株的残留与扩散 .对江苏试验地样品还进行了工程菌株质粒卡那霉素和壮观霉素抗性标记基因的抗性菌落分离 ,绝大多数样品中都能分离到包括荧光假单胞菌在内的抗性菌落 ,土样中菌密度n(cfu) =10 4 ～ 10 5g-1,但进行Btcry基因PCR -RFLP检测时没有从样品中得到特异扩增产物 .研究结果表明 :工程菌株抗性标记基因在自然界广泛存在 ,工程菌在株环境中没有残留和扩散 ,具有良好的生物安全性 .表 4参 8     

松材线虫PCR标准化阳性对照构建及检测体系的建立

建立了松材线虫PCR检测标准化阳性对照及特异性强的PCR检测体系.根据松材线虫rDNA-ITS区和BxPe12基因的特异基因序列设计引物,并从中筛选出一对特异引物cqubs01/cquba01,该引物能从松材线虫特异性扩增出196 bp片段,而不能对其他种类线虫进行扩增.基于优化PCR反应体系和反应程序,稳定的检测体系得以建立,检测灵敏度为100 ps/μL.以松材线虫基因组DNA为模板,以上述特异引物进行PCR扩增,将纯化后的PCR产物与PMD18-T载体连接之后转入大肠杆菌中,筛选出阳性克隆进行测序验证,最终获得了松材线虫的无害化阳性对照,建立了松材线虫标准化阳性对照的PCR检测体系.对来自于不同地区的12批次近100个样品进行了实际检测验证,其结果与实际发生情况一致,说明本检测体系稳定可靠.图3表2参8     

萘降解质粒pND6-1中萘趋化基因nahY的克隆和核苷酸序列分析

假单胞菌（Pseudomonas）ND6菌株的萘降解基因位于102kb的质粒pND6-1上。以pUC18质粒为载体，制备了含有1.5-3.0kbpND6-1DNA随机片段的基因文库。通过DNA测序和DNA序列的同源性分析，从基因文库中筛选出含有萘趋化基因nahY的克隆。NahY基因的大小为1617bp，编码的萘趋化蛋白由538个氨基酸组成，与假单胞菌G7菌株的nahY基因相比，核苷酸序列的同源性是96.7％，编码的氨基酸序列的同源性是95％。图4参7     

Tet抗性铜绿假单胞菌接合转移pHN102方法研究

二亲本杂交实验通常要求受体菌不能携带有与质粒上相同的抗性.本次实验中受体菌铜绿假单胞菌(Pseudo-monas aeruginosa)和重组质粒pHN102都带有四环素(Tc)抗性.考虑到质粒pHN102导入受体菌后至少带有两个Tc抗性基因片段,转移接合子的抗性会增强,所以提高四环素浓度至20μg/mL,并利用pHN102上携带的luxAB发光酶基因标记受体菌筛选转移接合子,并设立对照菌株.通过抽提质粒、琼脂糖凝胶电泳检测,确定pHN102成功转入P.aeruginosa中.转移接合子经4次转接后,质粒仍可以稳定存在且能够稳定表达.P.aeruginosa(pHN102)的发光动力学曲线表明,加入底物20min后,发光强度趋于稳定.经液体培养稀释后进行发光度和活菌数测定,发现二者呈显著的线性关系(r=0.994).图4表2参6     

铜绿假单胞菌增强型绿色荧光蛋白标记的研究

根据增强型绿色荧光蛋白(EGFP)基因设计了上下游引物P1和P2,通过对铜绿假单胞菌菌株X3绿色荧光蛋白标记研究,构建了铜绿假单胞菌标记系统,获得带有EGFP标记的基因工程菌X9,为进行相关的跟踪研究创造了条件.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,外源EGFP基因存在于转化子染色体上. 图 6参 14     

基于高灵敏度全细胞传感器的汞转化基因元件评价体系

汞是最具毒性的重金属之一;不同形态的汞具有的毒性差异大,发掘高效的不同汞形态转化的基因元件,通过合成生物学手段构建汞的微生物转化减毒体系是未来汞污染治理的重要方向;因此,发展和建立一套基因元件功能和效率的评价体系,是筛选高效汞形态转化基因的关键.基于MerR这一汞离子特异性响应蛋白和绿色荧光蛋白(GFP)报告基因所构建的汞离子大肠杆菌细胞传感器,首先通过引入汞转运蛋白MerT提高汞离子的响应程度,构建含pUC57-MerT-MerR-GFP的细胞传感器.该传感器4h对氯化汞(HgCl₂)的响应浓度为5-200μg/L,响应能力与汞离子浓度线性相关.在此基础上,将含有待测试汞转化基因的pACYC184质粒转入这一传感器,构建含相容性双质粒的大肠杆菌体系,通过GFP荧光的强度(胞内汞离子浓度)来评价待测基因是否具有将汞离子转化成其他形态的功能及其效率.抗生素处理下,相容性双质粒在大肠杆菌中拷贝数保持稳定,可用于基因元件评价.通过生物信息学分析,分别在原核生物蓝藻和单细胞真核生物四膜虫中鉴定和筛选了潜在的汞离子转化相关基因,包括半胱氨酸合成酶、胱硫醚-β-合成酶和半胱氨...     

Conservation by Design

Abstract: Conservation researchers are increasingly aware of the need to conduct interdisciplinary research and to engage nonscientists in practical applications of conservation biology. But so far, industrial designers have been left out of such collaboration and outreach efforts. Conservation of wildlife often depends on products such as nest boxes, feeders, barriers, and corridors, all of which have a designed component that is frequently overlooked. Furthermore, many products are adopted without testing on short or long time scales. We argue that the design of products for conservation, and hence their functionality, effectiveness, and value, can be improved through collaboration with industrial designers. We see four key benefits that can arise from interactions with industrial designers: improvement of product quality and value, innovation and improvement in functionality of products, harmonization of conservation products with local values, and development of a psychological biomimesis approach to design. The role of industrial designers in conservation projects would be to improve factors such as product durability, affordability, functionality, and aesthetic appeal to local people. Designers can also help to create multiple product options whose success can be tested in the field. We propose that collaborations with industrial designers can contribute to the development of improvements to existing products and innovations in the practice of animal conservation.     

Shipboard bilge water treatment by electrocoagulation powered by microbial fuel cells

Reveals the synergy between microbial fuel cells and electrocoagulation. Demonstrates MFC-ECC shipboard wastewater treatment is advantageous. MFC-ECC integration enables energy neutral bilge water treatment. Ships generate large amounts of wastewater including oily bilge water, blackwater and greywater. Traditionally they are treated separately with high energy consumption. In this study we demonstrate the feasibility that these waste streams can be treated using an integrated electrocoagulation cell (ECC) and microbial fuel cell (MFC) process, which not only synergized the contaminants removal but also accomplished energy neutrality by directly powering EC with MFC electricity. Results showed that MFC stack powered ECC removed 93% of oily organics, which is comparable to the performance of an external DC voltage powered ECC. In the meantime, more than 80% of COD was removed from MFCs when fed with either acetate or municipal wastewater. Moreover, the ECC electrode area and distance showed notable effects on current generation and contaminants removal, and further studies should focus on operation optimization to enhance treatment efficiency.     

Products of denitrification by a marine bacterium as revealed by gas chromatography

Gas chromatography was used to analyse the gases released by growing cultures of the denitrifying marine bacterium Pseudomonas perjectomarinus. When nitrate was provided as final oxidant, CO₂ was the first detectable gas released. Following the peak rate of release of CO₂ at approximately 16 h, N₂ liheration began and peaked at 30 h. When N₂O was provided as terminal oxidant, CO₂ and N₂ release began within a few hours and the rate of liberation of both increased for 45 to 50 h before leveling. Cell-free extracts of bacteria from denitrifying cultures of P. perfectomarinus were incubated anaerobically with nitrate, malic acid and electron transfer cofactors provided, and the gases in the atmosphere above the reaction mixtures were analysed. NO was found to be present transiently, and N₂O was discerned after prolonged incubation. The applicability of gas chromatography to analyses of marine ecosystems and the need for better means of sampling the atmosphere above enzymatic reaction mixtures were disoussed.     

Biodeposition by salt-marsh invertebrates

The importance of salt-marsh invertebrate biodeposits in biogeochemical cycles is discussed. Yearly biodeposition rates of nitrogen, phosphorus, potassium, magnesium, maganese, copper, zinc, and molybdenum are reported for 5 invertebrate species. The 5 species (Littorina irrorata, Arcatula demissa, Polymesoda caroliniana, Uca pugnax and U. pugilator) deposit 1709 g dry weight m² year^-1, of which 455 g m² year^-1 is organic material. Calculations suggest that the invertebrates are capable of processing 53% of the marsh's yearly production, but the actual amount utilized is probably substantially less than this.Contribution No. 716 from the Virginia Institute of Marine Science, and Contribution No. 295 from the University of Georgia Marine Institute.     

Echolocation by cave swiftlets

Summary The ability of cave dwelling swiftlets (Collocalia spodiopygius) to detect small cylindrical obstacles by echolocation was tested in a 3x9 m flight chamber. Although there was great individual variability, 6.3 mm obstacles were avoided much more often in total darkness than 1.5 wires (P<0.001). Flash photographs showed that the latter were avoided only by chance (about 50% misses). Obstacles 3.0 mm in diameter were avoided only slightly more often than 1.5 mm wires.Even in the light these swiftlets avoided the obstacles in only about 75% of the trials, probably because gentle touches with the primary feathers caused little discomfort.The 3.0 and 6.3 mm obstacles tended to be avoided more often on first encounters after they had been shifted horizontally by 10–30 cm, probably because the birds became more attentive.     

Human carcinogenesis by arsenic

Arsenic is one of the few human carcinogens for which there is not yet a reliable animal cancer model. As such, the classification of arsenic as a carcinogen is based upon data derived from human epidemiologic studies. Although the mechanisms of action of arsenic as a toxic agent have been known for many years, the inability to produce cancer with arsenic in laboratory animals has confounded the operational characterisation of arsenic as initiator, promoter, complete carcinogen, or cocarcinogen for humans. Arsenic is clearly a genotoxic agent that induces chromosomal aberrations, micronuclei and sister chromatid exchange in mammalian cells as well as neoplastically transforms Syrian hamster embryo cells; however, it is not a classical point mutagen. This paper reviews some of the scientifically based issues relating to arsenic and risk assessment.     

Direct measurement of dissolved organic carbon release by phytoplankton and incorporation by microheterotrophs

It is now possible to divide particulate primary production into algal and heterotrophic components without physical separation. This depends on two innovations, the introduction of isotope in the form of labelled dissolved product(s) of primary production and the employment of a data analysis specifically designed for tracer kinetic incorporation experiments. The ¹⁴C technique described by Steemann Nielsen (1952) is inapplicable in the analyses of certain classes of systems and kinetic tracer incorporation experiments must be employed instead. We show that measurement of PDOC production rate requires such kinetic tracer analyses. Measurements made in the laboratory on water taken from 2 m depth in South West Arm of the Port Hacking estuary showed that: (1) the steady-state rate of PDOC production was 0.10 to 0.13 mg C.m^-3.h^-1; (2) the rate of PDOC incorporation into microheterotroph particulate organic carbon was 0.10 to 0.12 mg C.m^-3.h^-1; (3) the rate at which PDOC was respired to CO₂ was 0.001 to 0.003 mg C.m^-3.h^-1. (4) the PDOC makes up only about 0.1% of the total dissolved organic carbon. The size class of particles associated with PDOC production differed from the size class responsible for uptake of PDOC. More than 50% of the PDOC production was associated with particles having a nominal diameter range of 20 to 63 m, while this fraction was responsible for <10% of the incorporation.     

Rapid degradation of carbon tetrachloride by commercial micro-scale zinc powder assisted by citric acid

We developed an effective method for degradation of carbon tetrachloride (CT) in contaminated water. Zinc metal as a reducing agent for CT in aqueous solutions has been previously studied in some detail, but the rapid corrosion of zinc surface usually reduces its efficiency in removing CT. We assumed that citric acid could enhance the degradation of CT by zinc powder due to the elimination of a passivation layer of Zn(II) (hydr)oxides on the surface of zinc powder through chelating of organic ligands with Zn(II) produced from the reaction and keeping the exposure of active sites to targets. Here the influence of citric acid on the decomposing of CT by commercial micro-scale zinc powder was investigated in a pH range of 3.5–7.5 at 25°C in batch experiments. Reaction mixtures were analysed by gas chromatography/headspace analysis, and Cl⁻ concentration was determined by turbidimetry. The results demonstrate that the degradation of CT by zinc metal alone is very weak, but the addition of citric acid can assist zinc powder to decompose CT more completely and rapidly at all pHs. Degradation of CT took place mainly in the first 10 min of reaction, coupled with 75–95% of CT removal. Maximum dechlorination percentage (82.4%) of CT was obtained at pH 5.5. In that case, chloroform and dichloromethane, as main intermediates, were found at low levels during the whole reaction, suggesting that CT may be sequentially and multiply degraded so quickly that methane is yielded before the intermediates can be desorbed and released to aqueous solution. When compared with the current methods of nano-scale zinc and bimetallic systems, the application of commercial micro-scale zinc particles assisted by organic ligands is of environmental significance since it allows decontamination of aqueous chlorinated organic compounds at low cost and with high efficiency.