Abstract: Disclosed are methods related to identifying an essential gene which serves as an antibiotic target in a bacterium.

Abstract: Disclosed are methods related to identifying an essential gene which serves as an antibiotic target in a bacterium.

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 t

Abstract: Disclosed are methods related to identifying an essential gene which serves as an antibiotic target in a bacterium.

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 t

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.

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.

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 surviva

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: Disclosed is a method for identifying an essential gene which serves as an antibiotic target in a bacterium, the method comprising the steps of: (a) 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; (b) growing bacteria from the mutant pool in the presence of different amounts of said antibiotic to produce two or more test cultures; and (c) comparing the distribution of TnA insertions between test cultures to identify a putative essential gene serving as a target of said antibiotic in said bacterium.

Abstract: Disclosed is a method for identifying an essential gene which serves as an antibiotic target in a bacterium, the method comprising the steps of: (a) generating an antibiotic resistant mutant of said bacterium by a method comprising the step of selecting for growth in the presence of said antibiotic to produce an antibiotic resistant mutant clone (AbR mutant); (b) transforming the AbR mutant with: (i) one or more essential genes of said bacterium; and (ii) a transposon which insertionally inactivates bacterial DNA, to produce a pool of transposon mutants which are merodiploid for said one or more essential genes; (c) growing bacteria from the merodiploid pool in the presence of different amounts of said antibiotic to produce two or more test cultures; and (d) comparing the distribution of transposon insertions between test cultures to identify a putative essential gene serving as a target of said antibiotic in said bacterium.