The present invention relates to methods and means for implementing evolution of a gene of interest inside bacterial cells.

The invention relates to the field of genome editing and provides a method of generating DNA recombinases or provides DNA recombinases, which efficiently and specifically recombine genomic target sequences as obligate DNA recombinase enzymes. The invention provides a genetically engineered DNA recombining enzyme comprising a complex of a first and a second recombinase enzyme, wherein said first recombinase enzyme and said second recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and/or a downstream target site of a DNA recombinase, wherein said first recombinase enzyme and said second recombinase enzyme each comprises at least one mutation in its catalytic site, wherein said first recombinase enzyme and said second recombinase enzyme carrying said at last one mutation in their catalytic site, when expressed in isolation, do not show the catalytic activity of a DNA recombinase, and wherein said first DNA recombinase enzyme and said second DNA recombinase enzyme carrying said at least one mutation in their catalytic site when co-expressed and forming a complex show the catalytic activity of a DNA recombinase. The invention further relates to nucleic acid molecules encoding said genetically engineered DNA recombinases and complexes, as well as to pharmaceutical compositions comprising the same. The invention further provides the use of said genetically engineered DNA recombinases, complexes, nucleic acid molecules and pharmaceutical compositions in medicine and specifically for treating genetic disorders such as hemophilia A.

The invention relates to the field of genome editing and provides a method of generating DNA recombinases or provides DNA recombinases, which efficiently and specifically recombine genomic target sequences as obligate DNA recombinase enzymes. The invention provides a genetically engineered DNA recombining enzyme comprising a complex of a first and a second recombinase enzyme, wherein said first recombinase enzyme and said second recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and/or a downstream target site of a DNA recombinase, wherein said first recombinase enzyme and said second recombinase enzyme each comprises at least one mutation in its catalytic site, wherein said first recombinase enzyme and said second recombinase enzyme carrying said at last one mutation in their catalytic site, when expressed in isolation, do not show the catalytic activity of a DNA recombinase, and wherein said first DNA recombinase enzyme and said second DNA recombinase enzyme carrying said at least one mutation in their catalytic site when co-expressed and forming a complex show the catalytic activity of a DNA recombinase. The invention further relates to nucleic acid molecules encoding said genetically engineered DNA recombinases and complexes, as well as to pharmaceutical compositions comprising the same. The invention further provides the use of said genetically engineered DNA recombinases, complexes, nucleic acid molecules and pharmaceutical compositions in medicine and specifically for treating genetic disorders such as hemophilia A.

The present disclosure provides methods of identifying source organisms for antibiotic agents and methods of producing novel antibiotic agents. In particular, the disclosure provides methods of identifying novel source organisms for antibiotic agents by sing functionally significant structural motifs to select probes, and mining genome sequences using the selected probes to identify suitable source organisms for production and isolation of novel antibiotic agents.

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.

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.

The present application discloses methods useful for the multiplex measurement of enzymatic activities. Also disclosed are polynucleotide constructs, constructs libraries and compartments related suitable for use in the methods.

A method of making a polypeptide including at least one covalent bond between a pair of reactive side chains of corresponding amino acids, wherein the covalent bond is insensitive to reduction is provided including genetically modifying a genomically recoded organism to express a corresponding synthetase, tRNA or synthetase/tRNA pair for translating mRNA encoding the corresponding amino acids having the reactive side chains into the polypeptide and to express the polypeptide including the at least one pair of the reactive side chains wherein the reactive side chains are oriented near one another when the expressed polypeptide is in a folded configuration, wherein the reactive side chains react to form the covalent bond that is insensitive to reduction.

Provided herein are systems and methods for screening desirable biological variants using a high-throughput integrated system. The integrated system may be configured to input a plurality of parameters from functional studies of biological variants under applied conditions, in conjunction with integrated libraries of biological variants, and filter the inputs to produce desirable biological variants based on an input performance requirement. The system may output optimized strains, molecules, or novel molecules expected to have a desirable functional characteristic. Accordingly, the methods and systems disclosed herein enable multi-parametric studies of biological diversity and conditional diversity in systems biology.

The disclosure relates to methods for the screening, identification, and/or application of one or more microorganisms of use in imparting one or more beneficial properties to one or more plants.