Inhibition of Real-Time PCR in DNA Extracts from Aquifer Sediment

Variation in inhibition of real-time PCR was investigated with DNA extracts from 50 aquifer sediment core samples of 5 cm length collected through a 2.5 meter vertical profile across a landfill leachate plume. The inhibition was quantified using an internal control of the green fluorescent protein ( gfp ) gene, which was spiked into the real-time PCR reactions. The inhibition was investigated at two gfp gene concentrations: at 1.7 · 10 ⁷ gfp gene copies/g sediment (5.1 · 10 ⁴ gfp gene copies/PCR reaction) and at 1.7 · 10 ⁵ gfp gene copies/g sediment (5.1 · 10 ² gfp gene copies/PCR reaction). Despite the low TOC content of the sediment (average 0.4 mg C/g dw) the average real-time PCR response was partially inhibited, compared to a reference (pure water), at both high and low gfp concentrations. The relative amplification (reference = 1) was 0.85 ± 0.20 (high) and 0.66 ± 0.23 (low), showing significantly (P < 0.05) stronger inhibition at the lower target gene concentration. The inhibition of the real-time PCR did not show a systematic variation in the vertical profile related to plume position but variations were significant on a small scale of 5–15 cm depth intervals. One of the 50 samples failed to produce a signal with either concentration of the gfp internal control and three other samples inhibited real-time PCR at both high and low gfp concentration. These 4 samples, which were the samples with the highest inhibition, were the only DNA extracts with a visible brown colouration, indicating contents of humic-like substances. Elevated absorbance at 400 nm of these samples also indicated that humic-like substances were responsible for inhibition. However, other factors not associated with either absorbance or TOC may have contributed to the inhibition in less inhibited samples since the variation in real-time PCR response could not be sufficiently explained by absorbance or TOC. The results of this study suggest that an internal control is needed in real-time PCR reactions with DNA from environmental samples due to variation in inhibition to correctly quantify the number of target genes, especially at low target gene concentrations, when dilution of DNA extracts is not practical.     

Versatile biosensor vectors for detection and quantification of mercury

Three different whole cell biosensor constructs were made by fusing the mercury inducible promoter, P(mer), and its regulatory gene, merR, from transposon Tn21 with reporter genes luxCDABE, lacZYA, or gfp. In Escherichia coli these biosensor constructs responded to low levels of mercury by producing light, beta-galactosidase or green fluorescent protein, respectively. Since the responses were quantitative, the constructs were used to quantify bioavailable mercury in different environments. The constructs were cloned into mini-Tn5 delivery vectors, thus enabling the transfer of the mer-lux, mer-lac or mer-gfp cassettes to a variety of Gram-negative bacteria. The mer-lux cassette was transferred to a Pseudomonas putida strain, which was used to quantify water-extractable mercury in contaminated soil.     

Development of methods for detection and Agrobacterium-mediated transformation of the sterile,endophytic fungus Muscodor albus

Symbiotic endophytes, unlike plant pathogens, do not usually induce visible host response. This may constraint the researcher's decision whether a plant has been successfully infected by the endophyte. In order to properly study the establishment, development and progress of an endophyte in the host plant and host-endophyte interactions, methods for the identification and localization of endophytic microorganisms are needed. Towards this aim, we focused at two levels: (A) We constructed M. albus-specific primers for polymerase chain reaction (PCR). In vitro, these primers specifically detected only M. albus strains and not isolates of related fungi (such as Daldinia sp. and a Xylariaceae sp.). (B) For direct visualization of the fungi, we inserted a reporter gene (gfp) into M. albus hyphae using Agrobacterium-mediated transformation. Since M. albus is a sterile fungus (i.e., without spores or fungal fruiting bodies), we used chopped fungal mycelium for the transformation procedure. We transformed three different isolates of M. albus using Agrobacterium-mediated transformation. Fifty-nine different transformants were collected with a transformation efficacy of 0.0004–0.0026%. Although PCR-based detection and direct visualization of the transformants in planta were unsuccessful, all tested transformants (with one exception) exhibited similar biological activity to their cognate wild type. This work provides a significant step forward in molecular research of the relationships between this endophytic genus and their hosts.     

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。     

REEVALUATION OF THE NITROGEN FIXATION BEHAVIOR IN THE MARINE NON‐HETEROCYSTOUS CYANOBACTERIUM LYNGBYA MAJUSCULA1

Lyngbya majuscula Harvey ex Gomont is a common marine cyanobacterium in tropical and subtropical near‐shore waters. A few reports have indicated that L. majuscula fixes nitrogen only in the light. Because this feature is uncommon among non‐heterocystous cyanobacteria, we attempted a reevaluation. Nitrogenase activity, regulation, and localization were examined over diel cycles on natural populations of L. majuscula growing in subtidal zones off Zanzibar in the western Indian Ocean. The data show that L. majuscula fixed nitrogen and synthesized nitrogenase in all cells during the dark phase of a diel cycle. During the light phase, nitrogenase was degraded to undetectable levels.     

DNA重组酶Cre介导载体间基因的重组转移

DNA重组酶Cre可以识别LoxP位点,使含有LoxP位点的DNA分子发生重组:2个同向LoxP之间的DNA片段被删除,2个环状DNA分子被整合为一个大分子.基于Cre酶的这些作用特性,构建了一套载体间基因的重组转移体系,在Cre酶的作用下,gfp基因被从基因供体pTLG上切除下来,然后转移到基因受体pET-LoxP上,从而快速、简便地完成了gfp基因高效表达载体pET-gfp的构建.gfp基因在大肠杆菌BL21(DE3)中被诱导表达,使菌落产生了可视的绿色荧光.通过对荧光菌落的计数分析,比较了环状基因供体pTLG和线性基因供体pTLG对有效重组率的影响.使繁琐的传统载体构建变为简单的酶促反应,极大地简化了载体构建步骤,为Cre酶在基因克隆和亚克隆中的应用提供了很好的研究基础.