Engineered light-emitting reporter genes
AssigneeCobalt Technologies, Inc.
InventorsSTACY M. BURNS-GUYDISH, PAMELA REILLY CONTAG
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Abstract
Compositions and methods are provided for enhanced expression of light emitting reporters. Such reporters are used in methods for monitoring cultures for production of target compounds.
Claims70
An isolated non-natural nucleic acid molecule comprising a nucleotide sequence encoding a light-emitting reporter, wherein the nucleotide sequence has an A/T content between about 62% and about 75%.
A recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%.
A recombinant cell comprising a recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%.
An isolated polypeptide comprising a light-emitting reporter, wherein the polypeptide is encoded by a nucleotide sequence having an A/T content from between about 62% and about 75%.
A method comprising:
- a) culturing a recombinant cell comprising a recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%;
- and
- b) measuring the light emitted from the reporter in the culture.
A method for regulating fermentation in a bacterial cell culture comprising;
- monitoring expression of a light emitting reporter in bacteria in said culture, wherein said light emitting reporter is encoded by a nucleic acid sequence comprising total A/T content of about 62% to about 75%;
- wherein said bacteria is low-GC bacteria;
- and regulating conditions in said culture based on said monitoring.
A method for identifying and/or optimizing fermentation culture conditions comprising:
- culturing a plurality of cultures, wherein the bacteria are the same, wherein the culture conditions are different, and wherein one culture condition serves as a control condition;
- monitoring the expression of a light emitting reporter in said bacteria in said cultures, wherein said light emitting reporter is encoded by a nucleic acid sequence comprising total A/T content of about 62% to about 75%;
- and wherein said bacteria is low-GC bacteria;
- and identifying said cultures that have a higher expression of the light emitting reporter compared to a control culture.
A method for identifying mutants with higher productivity comprising:
- mutagenizing a plurality of bacteria that express a recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%;
- isolating pure cultures derived from individual mutants;
- culturing the pure cultures of mutants;
- measuring the light emitted from the reporter in the cultures;
- and selecting mutants that have a higher emission of light than an unmutagenized parent strain.
A kit comprising:
- a) a first container containing a first nucleic acid molecule comprising an expression control sequence and
- b) a second container containing a second nucleic acid molecule comprising a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 65% and about 75%;
- c) wherein the first and second nucleic acid molecules comprise compatible restriction sequences which, when the first and second nucleic acid molecules are ligated together, put the expression control sequence in operative linkage with the coding nucleotide sequence and create a restriction sequence.
A kit containing:
- a) a Clostridium cell;
- and
- b) recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a light-emitting reporter, wherein the coding nucleotide sequence has an A/T content between about 62% and about 75%.
Dependencies are inferred from the claim text.