甘油脱水酶再激活酶的克隆表达及活性鉴定

运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a( )上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。     

一种大肠杆菌快速连续CRISPR基因组编辑的新策略

【背景】通过CRISPR结合λ-Red同源重组技术进行染色体编辑是大肠杆菌遗传改造的重要手段。虽然目前已经有多个以CRISPR为基础的大肠杆菌基因组编辑策略,但这些方法往往涉及单独质粒消除、多片段组装等过程,存在效率低、操作繁琐、耗时长等问题。【目的】建立一种基于不同温度敏感程度的多种质粒协同使用的大肠杆菌快速、连续、高效的CRISPR基因组编辑方法,提高CRISPR在大肠杆菌基因编辑过程中的效率。【方法】将传统CRISPR方法中使用的pTarget质粒改造为RK2ts型温敏型质粒,并采用pTW-A/S两种抗性标记质粒交替使用的方法消除假阳性,实现质粒消除与下一步基因组编辑同步进行。【结果】在相同温度下RK2ts型质粒先于pSC101ts型质粒发生丢失,从而能够选择性缺失RK2ts型质粒pTW-A/S。同时实现pTW-A/S质粒的消除和下一轮基因整合过程中质粒与打靶片段的转入。利用该方法基因敲除/整合效率高达100%。通过对菌株BP01基因Bspan D与asp A进行高效连续整合,构建得到菌株BP03,成功提升产物β-丙氨酸的产量。【结论】建立起一种新的高效、方便、灵活的CRISPR/Cas9介导的大肠杆菌基因组连续编辑策略。通过这种不同温度敏感程度的多种质粒协同使用方法,一方面解决了传统方法中质粒消除繁琐等问题,另一方面也避免了大质粒多片段连接等步骤,并极大地缩短了实验周期,为代谢工程菌株改造提供有力工具。     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

微囊藻毒素生物治理技术研究进展*

近年来,随着全球气候变暖和水体富营养化程度加深,蓝藻水华频繁暴发。微囊藻毒素是有害蓝藻产生及释放的危害最大的一类蓝藻毒素,对生态环境和公众健康造成了严重的威胁。因此,寻求有效的微囊藻毒素降解方法已成为全球科学领域的研究热点。针对微囊藻毒素生物治理技术展开综述,阐述了微囊藻毒素的产生、理化性质及生物毒性,总结了微生物、水生植物、浮游动物等自然生物降解微囊藻毒素的能力。在此基础上概述了生物滤池、人工湿地、生态浮床、膜生物膜反应器等生物治理技术对微囊藻毒素的去除效果,分析了现有微囊藻毒素生物处理方法的优势和局限性,并对今后的研究方向提出展望,为解决水环境中微囊藻毒素的污染问题提供思路。     

利用两种不相容质粒在大肠杆菌中共表达DFF45和DFF40

DNA断裂因子（DNA fragmentation factor,DFF）是细胞凋亡过程中起重要作用的蛋白质之一,它由分子量为45kD和40kD的两个亚基构成,分别称为DFF45和DFF40。利用RT－PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了DFF45和DFF40的cDNA,分别克隆到到卡那霉素抗性表达载体pET-28a( )中,构建了pET28a-DFF45和pET28a-DFF40。用它们单独转化大肠杆菌BL21（DE3）后,经IPTG诱导都可获得高效表达。表达的重组蛋白质各自约占菌体总蛋白质的56％和22％。再将DFF45的cDNA克隆到氨苄青霉素抗性表达载体pET-21a( )中,得到了pET21a-DFF45。利用二者的不同抗性,将pET28a-DFF40和pET21a-DFF45共同转化大肠杆菌BL21（DE3）,工程菌经IPTG诱导后实现了DFF45和DFF40的共表达,表达产物各约占菌体总蛋白质的30％和17％。为了研究这两种不相容质粒在细菌中共存的稳定性,我们将共转化子在同时含有卡那霉素和氨苄青霉素的液体培养基中连续培养14h,发现此时仍有75％以上的细菌可同时耐受两种抗生素,即同时含有pET28a-DFF45和pET28a-DFF40,说明利用两个具有不同抗性的不相容载体进行蛋白质共表达的方法是可行的。     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Reverse genetics system for human rotaviruses

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.     

Use of cholinesterase activity as an ecotoxicological marker to assess anatoxin-a(s) exposure: Responses of two cladoceran species belonging to contrasting geographical regions

The specificity of cholinesterase (ChE) activity to detect the presence of anatoxin-a(s) and sublethal effects of a 7-day exposure to Anabaena spiroides extract containing anatoxin-a(s) were assessed in two freshwater cladoceran species. Activities of ChE of both Pseudosida ramosa and Daphnia magna can be used to indicate the presence of the neurotoxin anatoxin-a(s), but not for the hepatotoxic microcystin. Activity of ChE of P. ramosa, however, performed better as a biomarker of exposure to A. spiroides than that of D. magna. Furthermore, sublethal exposure to A. spiroides extract significantly inhibited the ChE activity in P. ramosa and negatively affected both individual and population endpoints. For D. magna, the inhibition of ChE activity was not related to effects at higher levels of biological organization, since no direct effect was recorded on the individual and population endpoints. The activity of ChE in P. ramosa also proved to be a good predictor of chronic effects of the A. spiroides extract at higher levels of biological organization, since 48-h ChE inhibition was linked to the sublethal effects on the individual and population. These relationships could not be established for D. magna. Since relationships between the effects of A. spiroides extract at different levels of biological organization were species-specific, it can be concluded that the choice of test organism interferes with the accuracy of the environment risk assessment of this neurotoxin and, hence, the use of native species is recommended for its assessment.     

Phosphorus removal coupled to bioenergy production by three cyanobacterial isolates in a biofilm dynamic growth system

In the present study a closed incubator, designed for biofilm growth on artificial substrata, was used to grow three isolates of biofilm-forming heterocytous cyanobacteria using an artificial wastewater secondary effluent as the culture medium. We evaluated biofilm efficiency in removing phosphorus, by simulating biofilm-based tertiary wastewater treatment and coupled this process with biodiesel production from the developed biomass. The three strains were able to grow in the synthetic medium and remove phosphorus in percentages, between 6 and 43%, which varied between strains and also among each strain according to the biofilm growth phase. Calothrix sp. biofilm turned out to be a good candidate for tertiary treatment, showing phosphorus reducing capacity (during the exponential biofilm growth) at the regulatory level for the treated effluent water being discharged into natural water systems.

Besides phosphorus removal, the three cyanobacterial biofilms produced high quality lipids, whose profile showed promising chemical stability and combustion behavior. Further integration of the proposed processes could include the integration of oil extracted from these cyanobacterial biofilms with microalgal oil known for high monounsaturated fatty acids content, in order to enhance biodiesel cold flow characteristics.     

Effective aerial monitoring of cyanobacterial harmful algal blooms is dependent on understanding cellular migration

Cyanobacterial harmful algal blooms (CHABs) degrade water quality and may produce toxins. The distribution of CHABs can change rapidly due to variations in population dynamics and environmental conditions. Biological and ecological aspects of CHABs were studied in order to better understand CHABs dynamics. Field experiments were conducted near Hartington, Ontario, Canada in ponds dominated by Microcystis aeruginosa and CHABs floating experiments were conducted at Lake Taihu during the summers of 2015 and 2016. Single colonies composed of hundreds to thousands of cells with an average median of 0.2–0.5 mm in diameter were the basic form assumed by the Microcystis, and this remained the same throughout the growing season. Thorough mixing of the water column followed by calm conditions resulted in over 90% of the cyanobacteria floating after 1 h. Multiple colonies floated on the water surface in four types of assemblages: aggregates, ribbons, patches, and mats. It is the mats that are conventionally considered the blooming stage of cyanobacteria.Presence of CHABs on open water surfaces also depends on environmental influences such as direct and indirect wind effects. For example, field tests revealed that the surface coverage of CHABs can be reduced to half within an hour at wind speeds of 0.5 m/s.Because our findings indicated that blooming involves surface display of cyanobacteria essentially presenting as a two-dimensional plane under defined conditions, the use of surface imagery to quantify CHABs was justified. This is particularly important in light of the fact that traditional detection methods do not provide accurate distribution information. Nor do they portray CHABs events in a real-time manner due to limitations in on-demand surveillance and delays between sample time and analyzed results. Therefore, a new CHAB detection method using small unmanned aerial systems with consumer-grade cameras was developed at a maximum detection altitude of 80 m. When cyanobacteria were floating on the surface, CHABs detection through RGB band cameras and spectral enhancement techniques was efficient and accurate. Small unmanned aerial systems were capable of providing coverage up to 1 km² per mission and the short intervals between sampling and results (approx. 2 h) allowed for the rapid analysis of data and for implementing follow-up monitoring or treatments. This method is very cost-effective at an estimate of as low as $100 CAD per mission with an average cyanobacterial detection accuracy of 86%. Thus, it is a good candidate method to fill the urgent need for CHABs detection, providing cost effective, rapid, and accurate information to improve risk management at a local level as well as to help quickly allocate resources for purposes of mitigation.