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    • 33. 发明申请
    • IDENTIFYING GENES INVOLVED IN ANTIBIOTIC RESISTANCE AND SENSITIVITY IN BACTERIA USING MICROCULTURES
    • 微生物鉴定参与细菌耐药性和敏感性的基因
    • WO2016102927A3
    • 2016-08-11
    • PCT/GB2015053770
    • 2015-12-09
    • DISCUVA LTD
    • WILLIAMS DAVID HUGHWAIN JOHN RICHARDWOOD STUART ROBERT
    • C12N15/10C40B40/08
    • C12N15/1082C12M25/01C12N15/102C12N15/1058C12N15/1075C12Q1/18C12Q1/6876C12Q2600/156C40B40/08
    • Described is a method for identifying a gene which mediates antibiotic sensitivity or resistance in a target bacterium, the method comprising the steps of: (a) generating a pool of mutant target bacteria by transposon mutagenesis with an activating transposon (TnA), wherein the TnA comprises an outward-facing promoter (TnAP) capable of increasing transcription of a gene at or near its insertion site in the DNA of said target cells; (b) generating a control microdroplet library by encapsulating individual members of the pool of step (a) in microdroplets, the microdroplets comprising a volume of aqueous growth media suspended in an immiscible carrier liquid, each microdroplet comprising a single mutant target cell; (c)generating a test microdroplet library by encapsulating individual members of the pool of step (a) in microdroplets, the microdroplets comprising a volume of aqueous growth media containing the antibiotic and suspended in an immiscible carrier liquid, each microdroplet comprising a single mutant target cell; (d) incubating the control and test microdroplet libraries to produce control and test microcultures; and (e) comparing the distribution of TnA insertions between control and test microcultures to identify a gene which mediates antibiotic sensitivity or resistance in said target bacterium.
    • 描述了用于鉴定在靶细菌中介导抗生素敏感性或抗性的基因的方法,所述方法包括以下步骤:(a)通过用活化转座子(TnA)转座子诱变产生突变靶细菌库,其中所述TnA 包含能够增加基因在所述靶细胞的DNA的插入位点处或其附近的基因转录的面向外启动子(TnAP); (b)通过将步骤(a)的池的各个成员包封在微滴中产生对照微滴文库,所述微滴包含悬浮在不混溶的载体液体中的一定体积的水性生长培养基,每个微滴包含单一突变靶细胞; (c)通过将步骤(a)的池的各个成员包封在微滴中产生测试微滴文库,所述微滴包含一定体积的含有所述抗生素并悬浮在不混溶的载体液体中的水性生长培养基,每个微滴包含单一突变目标 细胞; (d)孵育对照和测试微滴库以产生对照和测试微量培养物; 和(e)比较对照和测试微培养物之间TnA插入的分布,以鉴定介导所述靶细菌中抗生素敏感性或抗性的基因。
    • 34. 发明申请
    • IDENTIFYING GENES INVOLVED IN ANTIBIOTIC RESISTANCE AND SENSITIVITY IN BACTERIA USING MICROCULTURES
    • 使用微生物鉴定参与细菌抗生素和敏感性的基因
    • WO2016102927A2
    • 2016-06-30
    • PCT/GB2015/053770
    • 2015-12-09
    • DISCUVA LIMITED
    • WILLIAMS, David HughWAIN, John RichardWOOD, Stuart Robert
    • C12N15/10C12M1/12
    • C12N15/1082C12M25/01C12N15/102C12N15/1058C12N15/1075C12Q1/18C12Q1/6876C12Q2600/156C40B40/08
    • Described is a method for identifying a gene which mediates antibiotic sensitivity or resistance in a target bacterium, the method comprising the steps of: (a) generating a pool of mutant target bacteria by transposon mutagenesis with an activating transposon (TnA), wherein the TnA comprises an outward-facing promoter (TnAP) capable of increasing transcription of a gene at or near its insertion site in the DNA of said target cells; (b) generating a control microdroplet library by encapsulating individual members of the pool of step (a) in microdroplets, the microdroplets comprising a volume of aqueous growth media suspended in an immiscible carrier liquid, each microdroplet comprising a single mutant target cell; (c)generating a test microdroplet library by encapsulating individual members of the pool of step (a) in microdroplets, the microdroplets comprising a volume of aqueous growth media containing the antibiotic and suspended in an immiscible carrier liquid, each microdroplet comprising a single mutant target cell; (d) incubating the control and test microdroplet libraries to produce control and test microcultures; and (e) comparing the distribution of TnA insertions between control and test microcultures to identify a gene which mediates antibiotic sensitivity or resistance in said target bacterium.
    • 描述了一种用于鉴定介导目标细菌中的抗生素敏感性或抗性的基因的方法,所述方法包括以下步骤:(a)通过用活化转座子(TnA)通过转座子诱变产生突变靶细菌池,其中所述TnA 包括能够在所述靶细胞的DNA中的其插入位点处或其附近增加基因转录的向外启动子(TnAP); (b)通过将步骤(a)的池的各个成员封装在微滴中来产生对照微滴文库,所述微滴包含悬浮在不混溶载体液体中的一定体积的水性生长培养基,每个微滴包含单个突变靶细胞; (c)通过将步骤(a)的池的各个成员包封在微滴中来产生测试微滴文库,所述微滴包含一定体积的含有抗生素的水性生长培养基并悬浮在不混溶的载体液体中,每个微滴包含单个突变体靶 细胞; (d)孵育控制和测试微滴文库以产生对照和测试微量培养物; 和(e)比较对照和测试微培养之间的TnA插入的分布,以鉴定介导所述目标细菌中的抗生素敏感性或抗性的基因。
    • 35. 发明申请
    • METHOD FOR SCREENING FOR BIOACTIVE NATURAL PRODUCTS
    • 用于生物活性天然产物筛选的方法
    • WO2016092304A1
    • 2016-06-16
    • PCT/GB2015/053774
    • 2015-12-09
    • BACTEVO LIMITED
    • WILLIAMS, David HughWAIN, John RichardWOODS, Stuart Robert
    • C12N15/10C12M1/12
    • C12Q1/18C12M25/01C12N1/20C12N15/102C12N15/1058C12N15/1082C12Q2600/136
    • Describe is a method for screening mutant prokaryotic cells to identify producers of a cytotoxic agent active against a target cell, the method comprising the steps of: (a) providing cells of a producer prokaryotic species; (b) generating a pool of mutant producer cells by transposon mutagenesis of the cells of step (a) with an activating transposon (TnA), wherein the TnA comprises an outward-facing promoter (TnAP) capable of increasing transcription of a gene at or near its insertion site in the DNA of said producer cells; (c) co-encapsulating individual members of the pool of step (b) with one or more target cells in microdroplets, the microdroplets comprising a volume of aqueous growth media suspended in an immiscible carrier liquid, thereby generating a library of microdroplets each comprising a single mutant producer cell and one or more target cell(s); (d) incubating the microdroplet library of step (c) under conditions suitable for co-culture of the single mutant producer cell and target cell(s) to produce a library of microcultures, whereby mutant producer cells producing a cytotoxic agent active against the target cell(s) outgrow target cells in each microculture; and (e) screening the library of microcultures of step (d) for microcultures in which target cells have been outgrown or overgrown to extinction by mutant producer cells.
    • 描述是筛选突变体原核细胞以鉴定对靶细胞有活性的细胞毒性剂的生产者的方法,所述方法包括以下步骤:(a)提供生产者原核物种的细胞; (b)通过用活化转座子(TnA)通过对步骤(a)的细胞进行转座子诱变来产生突变体生成细胞池,其中所述TnA包含能够增加基因的转录的向外启动子(TnAP) 靠近其在所述生产细胞的DNA中的插入位点; (c)将步骤(b)的池的单个成员与微滴中的一个或多个靶细胞共同包封,微滴包含悬浮在不混溶的载体液体中的体积的水性生长培养基,从而产生微滴文库,每个微滴包含 单个突变体生产细胞和一个或多个靶细胞; (d)在适合于单个突变体生产细胞和靶细胞共培养的条件下培养步骤(c)的微滴文库以产生微培养物文库,由此产生对靶标有活性的细胞毒性剂的突变体生产细胞 细胞在每个微量培养物中扩大靶细胞; 和(e)筛选步骤(d)的微培养物文库用于微培养物,其中靶细胞已经长出或过度生长以被突变体生产细胞灭活。