Construction of Broad Host Range Cloning Vectors for Gram-negative Bacteria

A new cloning vector, pMFY31, has been constructed based on the high-copy-number, broad-host-range plasmid RSF1010. The plasmid has a size of 13.2 kb and carries the Ap^r, Cm^r, and Tc^r genes. It contains unique PstI, EcoRI, HindIII, BamHI, and SalI sites, all of which are located within the antibiotic resistance genes, therefore all sites are applicable to insertional inactivation. We also constructed pMFY40, a 11.6 kb derivative of pMFY31, by the elimination of the Cm^r gene. Plasmid pMFY31 has been efficiently introduced into a Pseudomonas putida strain not only by plasmid-DNA transformation but also by conjugal co-transfer with the helper plasmid, and was maintained stably in the strain.     

聚合酶链式反应快速鉴定啤酒有害菌研究

建立了PCR快速鉴定啤酒有害菌的新方法。用基于抗酒花基因horA部分序列的特异引物对啤酒污染乳酸菌进行PCR检测,灵敏度可达到3个细胞(CFU),样品的预培养需12~16h。啤酒有害菌检测所需时间从传统的5d减少到24h。     

PCR-RFLP检测LDL受体基因TaqⅠ多态性位点的研究

应用聚合酶链反应(PCR)扩增人类LDL受体基因外显子4-内含子4-外显子5片段,PCR产物为1.55kb,DNA片段经序列鉴定后,进行TaqI酶切位点的RFLP分析。结果显示:中国汉族人群LDL受体基因中存在着TaqⅠ酶切位点多态性; 200个LDL受体等位基因中TaqⅠ酶切位点出现的频率为0.515,该点频率较为适中, 可作为中国汉族人群LDL受体基 因的遗传标志来进行家族性高胆固醇血症(FH)的基因诊断。所建立起的LDL受体基因TaqⅠ位点的PCR -RFLP方法具有快速、简便的特点,在FH的基因诊断上有应用价值。 Abstract:To develop rapid and sensitive technique for detectin the TaqI polymorphism at the human LDL receptor gene in Chinese,the exon4-intron4-exon5 of the human LDL receptor gene was amplified by polymerase chain reaction(PCR).The PCR products were directly analysed by restriction fragment length polymorphisms(RFLP).The results showed that the TaqI polymorphism is associated with the LDL receptor gene in Chinese of Han nationality;The frequency of T= allele (presence of TaqI cutting site)is 0.515 in 200 LDL receptor alleles.This technique may be used for rapid and sensitive screening of the LDL receptor gene for the TaqI polymorphism.     

Taq DNA聚合酶功能区域的定位

通过参Ｕ法定点突变产生了ＴａｑＤＮＡ聚合酶Ｎ端分别缺失３个,２３５个,２８７个和４４３个氨基酸的４个缺失体,利用Ｂａｌ－３１连续缺失法产生了ＴａｑＤＮＡ聚合酶的Ｃ端分别缺失了２个、１６个、２９个、３２个、３４个氨基酸的５个缺失体．经ＤＮＡ聚合酶活性测定表明Ｎ端缺失３个,２３５个,２８７个氨基酸后活力和完整的Ｔａｑ相近,而缺失４４３个氨基酸后则失去了ＤＮＡ聚合酶活力;Ｃ端的５个缺失体都失去了ＤＮＡ聚合酶活性．据此ＴａｑＤＮＡ聚合酶的功能区域被定位在２８７～８３２氨基酸之间．     

Biosynthetic Enhancement of the Detection of Bacteria by the Polymerase Chain Reaction

Molecular viability testing (MVT) was previously reported to specifically detect viable bacterial cells in complex samples. In MVT, brief nutritional stimulation induces viable cells, but not non-viable cells, to produce abundant amounts of species-specific ribosomal RNA precursors (pre-rRNA). Quantitative polymerase chain reaction (qPCR) is used to quantify specific pre-rRNAs in a stimulated aliquot relative to a non-stimulated control. In addition to excluding background signal from non-viable cells and from free DNA, we report here that MVT increases the analytical sensitivity of qPCR when detecting viable cells. Side-by-side limit-of-detection comparisons showed that MVT is 5-fold to >10-fold more sensitive than standard (static) DNA-targeted qPCR when detecting diverse bacterial pathogens (Aeromonas hydrophila, Acinetobacter baumannii, Listeria monocytogenes, Mycobacterium avium, and Staphylococcus aureus) in serum, milk, and tap water. Sensitivity enhancement may come from the elevated copy number of pre-rRNA relative to genomic DNA, and also from the ratiometric measurement which reduces ambiguity associated with weak or borderline signals. We also report that MVT eliminates false positive signals from bacteria that have been inactivated by moderately elevated temperatures (pasteurization), a condition that can confound widely-used cellular integrity tests that utilize membrane-impermeant compounds such as propidium iodide (PI) or propidium monoazide (PMA) to differentiate viable from inactivated bacteria. MVT enables the sensitive and specific detection of very small numbers of viable bacteria in complex matrices.     

Improved Solid Medium for the Detection and Enumeration of Pectolytic Bacteria

An improved solid agar medium (MP medium) has been developed which allows detection of pectolytic activity in bacteria. Organisms tested exhibited a variety of regulatory controls governing pectate lyase synthesis. The medium contains mineral salts, pectin, and yeast extract. After growth of the organisms, the agar plate is flooded with a polysaccharide precipitant, and pectolytic activity is shown by clear zones around active colonies. High concentrations of phosphate are shown to be necessary for pectic enzyme formation on solid media. The medium has successfully been used to detect pectolytic organisms in soil, forest litter, and rotting vegetable samples.     

Taq DNA耐热聚合酶在大肠杆菌中的克隆和高表达

从水生栖热菌(Thermus aquaticus)YT-1中分离得到的Taq DNA 聚合酶是一种广泛应用于PCR的耐热DNA聚合酶。由于天然菌株酶产量较低,培养条件要求严格,酶的纯化过程极为繁琐而使产品成本较高,因而促使人们构建适合于大规模生产的基因工程菌株。已有人分别通过不同的途径,使用不同的载体,成功地在大肠杆菌菌株中表达了Taq 耐热DNA聚合酶基因。我们通过与前人不同的途径,把这一基因克隆到大肠杆菌的载体质粒pJLA503上并使其得以高表达,为降低生产成本和进一步研究该酶的各种特性提供了有利条件。     

Improved Method for Detection of Methanotrophic Bacteria in Forest Soils by PCR

A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils. Received: 1 September 2000 / Accepted: 2 October 2000     

Combining Precursor and Fragment Information for Improved Detection of Differential Abundance in Data Independent Acquisition

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Highlights

• •Modern DIA methods contain high quality MS1 and MS2.
• •We developed a statistical procedure incorporating MS1 and MS2.
• •Benchmarking, the combined method outperformed the individual use of MS1 or MS2.

     

Taq DNA聚合酶结构改造与功能改进及其在医药领域的应用前景

TaqDNA聚合酶（ThermusaquaticusDNApolymerase）是重要的生物技术工具酶,广泛应用于疾病诊断与治疗、传染病检测、药物作用机理等医药领域。TaqDNA聚合酶在结构上由三部分构成：DNA聚合酶区、3’核酸外切酶区和5’核酸外切酶区。由于3’核酸外切酶区的氨基酸序列的变化而使其失去了3’-5＇的外切校读功能,因此TaqDNA聚合酶有错误复制倾向。利用特定位点的突变、与其他酶互换功能区和删除N末端的5’核酸外切酶区等方式可以改进TaqDNA聚合酶的性能,使它的忠实性、热稳定性和聚合性能得到提高。本文介绍了TaqDNA聚合酶的结构与功能改造的研究现状,并展望了TaqDNA聚合酶在医药领域的应用前景。     

Improved Method of Enumeration of Attached Bacteria for Study of Fluctuation in the Abundance of Attached and Free-Living Bacteria in Response to Diel Variation in Seawater Turbidity

Sample preparation for enumerating attached bacteria in turbid seawater by epifluorescence microscopy was improved by treating samples with a surfactant (Tween 80) followed by sonication. With optimal treatment with Tween 80 (final concentration, 10 ppm [10 μg/ml]) and sonication, as many as 10 times more attached bacteria were enumerated from turbid seawater relative to the number enumerated from an untreated control. Dispersion of bacteria by sonication alone resulted in the enumeration of only 42 to 72% of the attached bacteria. By this technique, fluctuations in the number of attached and free-living bacteria were determined in water from Aransas Pass, Tex., where surface sediments are resuspended on a regular basis by tidal currents. The abundance of attached bacteria increased in proportion to the seawater turbidity that resulted from sediment resuspension. The variation in abundance of free-living bacteria was not directly related to seawater turbidity. However, the magnitude of fluctuation in the abundance of free-living bacteria was related to the extent of turbidity variation during diurnal tides.     

Elevated Abundance of Bacteriophage Infecting Bacteria in Soil

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 10⁷ g⁻¹), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 10⁸ g⁻¹, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.     

Development of a Rapid Assay for Determining the Relative Abundance of Bacteria

A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 10³ to 10⁴ and 10⁴ to 10⁵ bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.