Tn5是一种细菌转座子。经改造的Tn5能够高效地切割DNA,同时连接上特定的接头序列,因而广泛应用于高通量二代测序文库构建中。CUT&Tag(Cleavage Under Target&Tagmentation)是一种改进的研究蛋白质与DNA互作的技术,具有重复性好...Tn5是一种细菌转座子。经改造的Tn5能够高效地切割DNA,同时连接上特定的接头序列,因而广泛应用于高通量二代测序文库构建中。CUT&Tag(Cleavage Under Target&Tagmentation)是一种改进的研究蛋白质与DNA互作的技术,具有重复性好、信噪比高及操作简便等优点。该技术采用pA(Protein A)或pG(Protein G)与Tn5形成融合蛋白,定位于特定抗体(用于识别目标蛋白),利用Tn5的特性,在目标位点附近打断DNA的同时引入测序接头,随后提取DNA,再进行PCR扩增即可获得测序文库。但不同类型的抗体与pA或pG的亲和力不同,因此限制了部分抗体在CUT&Tag技术中的应用。为克服这一局限,该文构建了pG与Tn5的融合蛋白表达载体,通过原核表达及亲和纯化的方式获得pG-Tn5重组蛋白;并以RNA聚合酶Ⅱ(PolⅡ)特异性抗体PolⅡSer5P(小鼠IgG1型抗体和兔IgG型抗体)为例,在模式植物拟南芥(Arabidopsis thaliana)中评估pA-Tn5与pG-Tn5在不同类型抗体的CUT&Tag测序文库构建中的效果。结果表明,IgG1型抗体与p G-Tn5的亲和力更高,构建的文库质量更好,而IgG型抗体与2种酶的亲和力相当;同时,较低起始量的植物材料也能获得较好的效果,证明了CUT&Tag的应用优势。该研究优化了CUT&Tag技术,可为后续CUT&Tag实验中针对不同抗体时Tn5融合蛋白的选择提供参考。展开更多
Strain S2 is a lecithin (or phosphatidylcholine)- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, p...Strain S2 is a lecithin (or phosphatidylcholine)- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, physiological and biochemical tests, 16S rDNA sequence analysis, G+C content determination and DNA-DNA hybridizations analy-sis, strain S2 was identified as Pseudomonas alcaligenes. P. alcaligenes S2 was mutagenized with Tn5 and four mutants showing decreased or increased solubilizing ability of lecithin were isolated based on the halo size around colonies on the solid plate supplemented with egg yolk. To characterize the genes of P. alcaligenes S2 involved in solubilization of lecithin, the EcoR I fragments of the chromosomes from the four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. The Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decreased solubilizing ability of lecithin, were found to be located in the xcpS, xcpX and xcpW , respectively, whose products XcpS, XcpX and XcpW were the components of type II secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize lecithin. The data suggested that mutation in one of these xcp genes would lead to the absence of mature lecithinase secretion into the extracellular medium. The data also indicated that the secretion of leci-thin-hydrolyzing enzyme of P. alcaligenes was via type II secretion pathway. In the mutant M20 showing increasing lecithin-hydrolyzing activity, the interrupted gene showed 86% identity with chpA of Pseudomonas aeruginosa PAO1, whose product plays an important role in controlling twitch-ing motility of the bacterial cells.展开更多
The moblizable plashed pBF101 carrying Tn5-nifA of E. cloacae E26 was introduced into K. oxytoca NG13 with high frequency from donor E. coli S17-1 by mating. Tn5-nifA was transposed spontaneously from PBF101 into the ...The moblizable plashed pBF101 carrying Tn5-nifA of E. cloacae E26 was introduced into K. oxytoca NG13 with high frequency from donor E. coli S17-1 by mating. Tn5-nifA was transposed spontaneously from PBF101 into the genome of NG13 with a frequency of 6 ×10-5. Strain NG1394 was obtamed by selecting KmrCms colonies,which had lost plashed PBF101 and into which transposition of Tns-nifA had occurred. The integrahon of nifA gene into the genome in NG1394 was verified by agarose gel electrophoresis (Fig. 1) and curing of plasdrid with acridine orange. The synthesis of nitrogrnase in NG1394 was not repressed by (Table 1 ). Constitutive nifA could be stably maintained in NG1394 without antibiotic selection stress. borne nitrogenase activity was detected in NG1394 at 39℃ while NG13 (wild type) competely lost the activity at 39℃ (table 2).展开更多
Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture.Effective exploitation of this wealth of chemicals requires the functional expres...Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture.Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts.We have previously established the TREX system which facilitates the transfer,integration and expression of biosynthetic gene clusters in various bacterial hosts.Here,we describe the yTREX system,a new tool adapted for one-step yeast recombinational cloning of gene clusters.We show that with yTREX,Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition.Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning,transfer and expression of the respective biosynthesis genes from Serratia marcescens.Furthermore,the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum,producing pathway metabolites violacein,deoxyviolacein,prodeoxyviolacein,and deoxychromoviridans.Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype.Finally,the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa.All compounds accumulated to substantial titers in the mg range.We thus corroborate here the suitability of P.putida for the biosynthesis of diverse natural products,and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters,applicable for natural compound discovery and combinatorial biosynthesis.展开更多
转座酶因其能够插入目的片段的能力正受到越来越多的关注,经过改造的高活性转座酶可用于标记DNA,具有较大的应用价值,如ATAC-seq(Assay for transposase-accessible chromatin using sequencing)以及TrAC-looping(Transposase-mediated ...转座酶因其能够插入目的片段的能力正受到越来越多的关注,经过改造的高活性转座酶可用于标记DNA,具有较大的应用价值,如ATAC-seq(Assay for transposase-accessible chromatin using sequencing)以及TrAC-looping(Transposase-mediated analysis of chromatin looping)等染色质高级结构捕获实验,使用高活性转座酶为染色质开放区DNA或存在染色质相互作用的片段加上标记,从而分离出目的片段。天然转座酶普遍活性较低,缺乏实际应用价值,而经过改造的高活性转座酶因其可能导致的致死突变,对宿主具有较大毒性,难以制备。本研究优化了高活性转座酶Tn5的原核表达以及纯化方法,将已转化pet15b His6Tn5质粒的改良型工程菌BL21-Gold(DE3)以及毒性蛋白诱导条件进行原核表达,借助高活性转座酶Tn5带有的His tag标签进行纯化,通过Western blotting鉴定了纯化所得蛋白,并使用考马斯亮蓝染色测定了目的蛋白浓度,采取在标准反应条件下与斑马鱼ZF4细胞基因组DNA反应的方法,测试了纯化所得Tn5的活性。本研究为进一步探测ZF4细胞基因组的三维结构提供了研究基础。展开更多
With the help of Tn5 transposon technique, gene yfj B encoding NAD kinase in Escherichia coli(E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate(PHB) containing PHB synthesis operon integ...With the help of Tn5 transposon technique, gene yfj B encoding NAD kinase in Escherichia coli(E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate(PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfj B gene copy into genome, the selected recombinant named E. coli PHBTY4 showed stronger NAD kinase activity than that of E. coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E. coli PHBTY4 compared with that of the control. E. coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E. coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions.展开更多
细菌性果斑病(Bacterial Fruit Blotch,BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌株MH21,经鉴定为燕麦食酸菌西瓜亚种(Acidovorax avenaesubsp.citrulli)。利用转座子Mini-Tn5构建MH21菌株的突变体库,Souther...细菌性果斑病(Bacterial Fruit Blotch,BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌株MH21,经鉴定为燕麦食酸菌西瓜亚种(Acidovorax avenaesubsp.citrulli)。利用转座子Mini-Tn5构建MH21菌株的突变体库,Southern印迹杂交结果显示Mini-Tn5在菌株MH21染色体上可随机、单拷贝插入。通过浸种处理甜瓜种子进行突变体的致病性检查,筛选得到1株致病性完全丧失的突变体M543。对M543中转座子插入基因的克隆和测序表明其突变基因为燕麦食酸菌Ⅲ型分泌系统(TTSS)中的保守基因hrcR。推测菌株MH21中hrcR基因编码的蛋白定位于细菌内膜,是分泌通道主要成分之一。hrcR基因互补菌株的致病性检查结果显示其致病力恢复到野生型的84%。研究同时发现hrcR基因突变后MH21菌株丧失了在烟草上诱导过敏性坏死反应的能力。本研究从遗传学角度说明TTSS是西甜瓜果斑病菌致病性的重要因子。展开更多
文摘Strain S2 is a lecithin (or phosphatidylcholine)- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, physiological and biochemical tests, 16S rDNA sequence analysis, G+C content determination and DNA-DNA hybridizations analy-sis, strain S2 was identified as Pseudomonas alcaligenes. P. alcaligenes S2 was mutagenized with Tn5 and four mutants showing decreased or increased solubilizing ability of lecithin were isolated based on the halo size around colonies on the solid plate supplemented with egg yolk. To characterize the genes of P. alcaligenes S2 involved in solubilization of lecithin, the EcoR I fragments of the chromosomes from the four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. The Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decreased solubilizing ability of lecithin, were found to be located in the xcpS, xcpX and xcpW , respectively, whose products XcpS, XcpX and XcpW were the components of type II secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize lecithin. The data suggested that mutation in one of these xcp genes would lead to the absence of mature lecithinase secretion into the extracellular medium. The data also indicated that the secretion of leci-thin-hydrolyzing enzyme of P. alcaligenes was via type II secretion pathway. In the mutant M20 showing increasing lecithin-hydrolyzing activity, the interrupted gene showed 86% identity with chpA of Pseudomonas aeruginosa PAO1, whose product plays an important role in controlling twitch-ing motility of the bacterial cells.
文摘The moblizable plashed pBF101 carrying Tn5-nifA of E. cloacae E26 was introduced into K. oxytoca NG13 with high frequency from donor E. coli S17-1 by mating. Tn5-nifA was transposed spontaneously from PBF101 into the genome of NG13 with a frequency of 6 ×10-5. Strain NG1394 was obtamed by selecting KmrCms colonies,which had lost plashed PBF101 and into which transposition of Tns-nifA had occurred. The integrahon of nifA gene into the genome in NG1394 was verified by agarose gel electrophoresis (Fig. 1) and curing of plasdrid with acridine orange. The synthesis of nitrogrnase in NG1394 was not repressed by (Table 1 ). Constitutive nifA could be stably maintained in NG1394 without antibiotic selection stress. borne nitrogenase activity was detected in NG1394 at 39℃ while NG13 (wild type) competely lost the activity at 39℃ (table 2).
基金The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation,Science and Research of the German federal state of North Rhine-Westphalia MIWF within the framework of the NRW Strategieprojekt BioSC(No.313/323-400-00213).
文摘Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture.Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts.We have previously established the TREX system which facilitates the transfer,integration and expression of biosynthetic gene clusters in various bacterial hosts.Here,we describe the yTREX system,a new tool adapted for one-step yeast recombinational cloning of gene clusters.We show that with yTREX,Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition.Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning,transfer and expression of the respective biosynthesis genes from Serratia marcescens.Furthermore,the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum,producing pathway metabolites violacein,deoxyviolacein,prodeoxyviolacein,and deoxychromoviridans.Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype.Finally,the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa.All compounds accumulated to substantial titers in the mg range.We thus corroborate here the suitability of P.putida for the biosynthesis of diverse natural products,and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters,applicable for natural compound discovery and combinatorial biosynthesis.
文摘转座酶因其能够插入目的片段的能力正受到越来越多的关注,经过改造的高活性转座酶可用于标记DNA,具有较大的应用价值,如ATAC-seq(Assay for transposase-accessible chromatin using sequencing)以及TrAC-looping(Transposase-mediated analysis of chromatin looping)等染色质高级结构捕获实验,使用高活性转座酶为染色质开放区DNA或存在染色质相互作用的片段加上标记,从而分离出目的片段。天然转座酶普遍活性较低,缺乏实际应用价值,而经过改造的高活性转座酶因其可能导致的致死突变,对宿主具有较大毒性,难以制备。本研究优化了高活性转座酶Tn5的原核表达以及纯化方法,将已转化pet15b His6Tn5质粒的改良型工程菌BL21-Gold(DE3)以及毒性蛋白诱导条件进行原核表达,借助高活性转座酶Tn5带有的His tag标签进行纯化,通过Western blotting鉴定了纯化所得蛋白,并使用考马斯亮蓝染色测定了目的蛋白浓度,采取在标准反应条件下与斑马鱼ZF4细胞基因组DNA反应的方法,测试了纯化所得Tn5的活性。本研究为进一步探测ZF4细胞基因组的三维结构提供了研究基础。
基金supported by the National Basic Research Program of China (2012CB725201)the National Natural Science Foundation of China (31430003, 31270146)
文摘With the help of Tn5 transposon technique, gene yfj B encoding NAD kinase in Escherichia coli(E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate(PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfj B gene copy into genome, the selected recombinant named E. coli PHBTY4 showed stronger NAD kinase activity than that of E. coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E. coli PHBTY4 compared with that of the control. E. coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E. coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions.
文摘细菌性果斑病(Bacterial Fruit Blotch,BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌株MH21,经鉴定为燕麦食酸菌西瓜亚种(Acidovorax avenaesubsp.citrulli)。利用转座子Mini-Tn5构建MH21菌株的突变体库,Southern印迹杂交结果显示Mini-Tn5在菌株MH21染色体上可随机、单拷贝插入。通过浸种处理甜瓜种子进行突变体的致病性检查,筛选得到1株致病性完全丧失的突变体M543。对M543中转座子插入基因的克隆和测序表明其突变基因为燕麦食酸菌Ⅲ型分泌系统(TTSS)中的保守基因hrcR。推测菌株MH21中hrcR基因编码的蛋白定位于细菌内膜,是分泌通道主要成分之一。hrcR基因互补菌株的致病性检查结果显示其致病力恢复到野生型的84%。研究同时发现hrcR基因突变后MH21菌株丧失了在烟草上诱导过敏性坏死反应的能力。本研究从遗传学角度说明TTSS是西甜瓜果斑病菌致病性的重要因子。
