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公开(公告)号：US09493770B2
公开(公告)日：2016-11-15
申请号：US14071618
申请日：2013-11-04
Applicant: DISCUVA LIMITED
Inventor： David Hugh Williams , Arthur Keith Turner
CPC classification number: C12N15/1082 , C12N15/102 , C40B30/06 , G06F19/18
Abstract: Provided herein are methods, and related compositions for identifying a putative essential gene which serves as an antibiotic target in a bacterium, the method, in some embodiments, comprising (a) generating an antibiotic resistant mutant of the bacterium by a method comprising the step of selecting for growth in the presence of an antibiotic to produce an antibiotic resistant mutant clone (AbR mutant); (b) transforming the AbR mutant with one or more essential genes of the bacterium and a transposon which insertionally inactivates bacterial DNA to produce a pool of transposon mutants which are merodiploid for the one or more essential genes; (c) growing bacteria from the merodiploid pool in the presence of different amounts of the antibiotic to produce two or more test cultures; and (d) comparing the distribution of transposon insertions between the test cultures to identify a putative essential gene serving as a target of the antibiotic in the bacterium.
Abstract translation: 本文提供了用于鉴定在细菌中用作抗生素靶标的推定必需基因的方法和相关组合物，在一些实施方案中，所述方法包括（a）通过包括以下步骤的方法产生所述细菌的抗生素抗性突变体： 在抗生素存在下选择生长以产生抗生素抗性突变体克隆（AbR突变体）; （b）用所述细菌的一种或多种必需基因转化所述AbR突变体，以及转座子，其插入地灭活细菌DNA以产生用于所述一种或多种必需基因的二聚体的转座子突变体库; （c）在不同量的抗生素存在下从单体二聚体培养细菌产生两种或更多种测试培养物; 和（d）比较测试培养物之间的转座子插入的分布，以鉴定用作细菌中抗生素靶标的推定的必需基因。
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公开(公告)号：US11359195B2
公开(公告)日：2022-06-14
申请号：US15655784
申请日：2017-07-20
Applicant: DISCUVA LIMITED
Inventor： David Hugh Williams , Arthur Keith Turner
Abstract: Disclosed are methods related to identifying an essential gene which serves as an antibiotic target in a bacterium.
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公开(公告)号：US20180010122A1
公开(公告)日：2018-01-11
申请号：US15655784
申请日：2017-07-20
Applicant: David Hugh Williams , DISCUVA LIMITED
Inventor： David Hugh Williams , Arthur Keith Turner
CPC classification number: C12N15/1082 , C12N15/102 , C40B30/06 , G16B20/00
Abstract: Disclosed are methods related to identifying an essential gene which serves as an antibiotic target in a bacterium.
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4.发明申请公开(公告)号：US20180179518A9
公开(公告)日：2018-06-28
申请号：US15617797
申请日：2017-06-08
Applicant: DISCUVA LIMITED
Inventor： David Hugh Williams , John Richard Wain , Stuart Robert Wood
CPC classification number: C12N15/1082 , C12M25/01 , C12N15/102 , C12N15/1058 , C12N15/1075 , C12Q1/18 , C12Q1/6876 , C12Q2600/156 , C40B40/08
Abstract: 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.
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Patent Agency Ranking
5.发明申请公开(公告)号：US20170342407A1
公开(公告)日：2017-11-30
申请号：US15617797
申请日：2017-06-08
Applicant: DISCUVA LIMITED
Inventor： David Hugh Williams , John Richard Wain , Stuart Robert Wood
CPC classification number: C12N15/1082 , C12M25/01 , C12N15/102 , C12N15/1058 , C12N15/1075 , C12Q1/18 , C12Q1/6876 , C12Q2600/156 , C40B40/08
Abstract: 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.
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公开(公告)号：US20170204449A1
公开(公告)日：2017-07-20
申请号：US15415361
申请日：2017-01-25
Applicant: DISCUVA LIMITED
Inventor： David Hugh Williams , John Richard Wain , Stuart Robert Woods
CPC classification number: C12Q1/18 , C12N15/102 , C12N15/1082 , C12Q1/689 , C12Q2600/158 , G01N2333/195 , G01N2500/10
Abstract: Disclosed is a method for characterizing the effect of an antibiotic on a bacterium, the method comprising the steps of: (a) generating a pool of mutant bacteria by transposon mutagenesis of a culture of said bacterium with an activating transposon (TnA) which comprises an outward-facing promoter (TnAP) capable of increasing transcription of a gene at or near its insertion site in the DNA of said bacterium; (b) growing bacteria from the mutant pool in the presence of different amounts of said antibiotic to produce two or more test cultures; (c) sequencing mRNA transcripts produced by TnAP in each of said test cultures to produce an mRNA transcript profile for each of the test cultures; and (d) comparing the mRNA transcript profiles of the test cultures.
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公开(公告)号：US20220186211A9
公开(公告)日：2022-06-16
申请号：US15655784
申请日：2017-07-20
Applicant: David Hugh Williams , DISCUVA LIMITED
Inventor： David Hugh Williams , Arthur Keith Turner
Abstract: Disclosed are methods related to identifying an essential gene which serves as an antibiotic target in a bacterium.
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公开(公告)号：US09745572B2
公开(公告)日：2017-08-29
申请号：US14071615
申请日：2013-11-04
Applicant: DISCUVA LIMITED
Inventor： David Hugh Williams , Arthur Keith Turner
CPC classification number: C12N15/1082 , C12N15/102 , C40B30/06 , G06F19/18
Abstract: Provided are methods for identifying differentially represented genes, the method comprising the steps of generating a pool of mutant bacteria by transposon mutagenesis with an activating transposon (TnA), wherein the TnA comprises a promoter such that transposon insertion into bacterial DNA increases the transcription of a gene at or near the insertion site; growing bacteria from the mutant pool in the presence of different amounts of said antibiotic to produce two or more test cultures; and comparing the distribution of TnA insertions between test cultures.
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公开(公告)号：US20170204403A1
公开(公告)日：2017-07-20
申请号：US15415357
申请日：2017-01-25
Applicant: DISCUVA LIMITED
Inventor： David Hugh Williams , John Richard Wain , Stuart Robert Woods
CPC classification number: C12N15/1058 , C12N15/102 , C12N15/1082 , C12Q1/689 , C12Q2600/158
Abstract: Disclosed is a process for producing a mutant bacterium which exhibits improved survival and/or growth under a selected growth condition, the process comprising the steps of: (a) generating a pool of mutant 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 bacterium; (b) growing bacteria from the mutant pool under the selected growth condition and under one or more reference conditions to produce two or more test cultures; and (c) sequencing mRNA transcripts produced by TnAP in each of said test cultures to produce an mRNA transcript profile for each of the test cultures; and (d) comparing the mRNA transcript profiles of the test cultures to identify a first class of genes which are disadvantageous for growth and/or survival under the selected growth condition and a second class of genes which are advantageous for growth and/or survival under the selected growth condition.
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