Johns Hopkins University

New York University

The General Hospital Corporation

Albert Einstein College of Medicine

University of Pennsylvania

Opentrons Labworks Inc.

Neochromosome Inc.

Cdi Laboratories, Inc.

Jef D Boeke

Neta Agmon

Leslie A Mitchell

Ed Harlow

Marc Vidal

Rainer K Brachmann

Yizhi Cai

Lelita T Braiterman

Scott E Devine

Vern L Schramm

Anthony Sauvé

Ivana Celic

David M Truong

Andrew H Martin

Michael Y Shen

Philip A Cole

Min Li

Heng Zhu

Bradley P Barnett

Haig H Kazazian, Jr

Georges Natsoulis

Shigehito Ikushima

Yi Wu

Joe De Angelo

Eric C Bolton

Joel S Bader

Jeffrey S Han

Jon Laurent

Paolo Mita

John V Moran

Shunyou Long

Beth A Dombroski

Seth Blackshaw

Yousang Hwang

Daniel J Eichinger

Ignacio Pino

Provided are compositions and methods for making a diploid yeast having heterozygosity for at least one chromosome. The method includes mating haploid yeast having at least a first modified chromosome comprising a synthetic chromosome suitable for recombination-site-mediated evolution (as a SCRaMbLE-ready modification). The SCRaMbLE-ready modification includes introduced site-specific recombinase recognition sites that can be recognized by a recombinase. Yeast that have at least one SCRaMbLE-ready modification of a chromosome are mated with a haploid yeast devoid of the SCRaMbLE-ready modification to obtain diploid SCRaMbLE-ready yeast. Subsequent to mating the haploid SCRaMbLE-ready yeast to yeast devoid of the SCRaMbLE-ready modification the method includes using the recombinase to recombine the first modified chromosome to obtain heterozygous diploid yeast comprising at least one recombined (SCRaMbLEd) chromosome and a homologous non-SCRaMbLEd chromosome. The method further includes identifying heterozygous diploid yeast that comprise the at least one SCRaMbLEd chromosome that have a changed phenotype that is different from the phenotype of the diploid SCRaMbLE-ready yeast. Yeast made by the methods, compositions of matter made by the yeast, spores made by the yeast, and kits containing the yeast and/or their spores are included.

SCRaMbLE of heterozygous diploid yeast

Yeast having modified chromosomes are provided. The chromosomes are modified such that at least one of yeast histones H3, H4, H2A or H2B are fully or partially replaced by their human histone counterparts H3, H4, H2A or H2B, respectively. Histone amino acid substitutions are included. Cell fusions with the yeast having the modified chromosomes and non-yeast cells are provided. Methods for screening test agents using the yeast are also provided. Yeast with a mutated yeast DAD1 gene, the mutated DAD1 gene encoding an E50D mutation in yeast DAD1 protein, are provided, and provide a useful genetic background for making the yeast with partially or fully replaced histone(s).

Budding yeast with human chromatin

Compositions, methods and kits are provided. The compositions, methods and kits are for assembly of series of DNA segments in yeast using homologous recombination. The assembled DNA segments are maintained episomally. Yeast made using the methods are included, as are methods of using the yeast to express proteins, and for screening test agents that can affect yeast that are modified to include the assembled DNA segments.

Extrachromosomal switching auxotrophies progressively by integration (eSwAP-In) for assembly of DNA sequences in yeast

Provided herein are methods and compositions for diagnosing a disease or an infection in a high throughput manner. Also provided herein are methods and compositions for diagnosing a disease or an infection with high specificity and high sensitivity.

Methods and compositions for rapid and high throughput diagnosis

Provided are modified microorganisms which are modified such that their growth can be controlled using exogenously provided compounds. The microorganisms can be modified by genetic alterations that include a promoter inducible by a first exogenously supplied compound. The promoter can be configured to drive expression of an RNA coding sequence that may be essential to growth of the microorganism. The microorganisms may also be modified to include site specific recombinase recognition sites flanking or within the RNA coding sequence so that expression of the corresponding site specific recombinase will disrupt transcription of the RNA. The site specific recombinase can be configured such that it expression and/or activity is suppressed by a second exogenously supplied compound. Methods of making the modified microorganisms and kits that contain reagents for making and using the modified microorganisms are also provided.

Compositions and methods for controlling microbial growth

Provided is an in vitro method for making a recombinant DNA molecule. The method includes combining in vitro a recombination-site-mediated evolution (a SCRaMbLE) ready DNA polynucleotide that contains at least one transcription unit (TU) and an introduced site-specific recombinase recognition sites that can be recognized by a recombinase, with a recombinase that recognizes the site-specific recombinase recognition sites. The method results in a polynucleotide that is recombined to provide a recombined polynucleotide. The method may further include determining the sequence, or determining the expression of the recombined polynucleotide. Polynucleotides made by this process, and modified yeast that contain the modified polynucleotides, are also provided.

In vitro DNA SCRaMbLE

Provided are compositions and methods for compound discovery. Modified yeast that have their endogenous yeast shikimate pathway disrupted or deleted, and replaced with homologous pathway genes from one or more distinct organisms, are provided and used in assays of test agents. The homologous pathway genes are designed to supplement the disrupted or deleted shikimate pathway genes. The assays are designed to identify whether or not the test agents can interfere with the function of enzymes in the shikimate pathway from organisms that are distinct from the yeast avatar hosts. In embodiments, the disruption/deletion of the yeast endogenous shikimate pathway results in the yeast being incapable of producing chorismic acid.

Yeast synthetic biology platform for identifying shikimate pathway enzyme inhibitors

A system for controlling gene expression in yeast comprises a repressible gene expression plasmid that has a regulator binding sequence for camR and a target gene sequence. The system also includes a transcription enhancer expression plasmid; wherein said transcriptional activator protein binds to the regulator binding sequence in the absence of a transcriptional inhibitor. The system is used in a method for controlling expression of the target gene through the use of camphor. The target gene is expressed in the absence of camphor but unexpressed if camphor is added to a solution of cells containing the plasmids.

Regulated switch for gene expression

The present invention relates to the field of chromosome engineering. More specifically, the present invention provides methods and compositions useful for inducibly linearizing circular DNA molecules in vivo in yeast. In one embodiment, a comprises a nucleic acid encoding a selectable marker, wherein the nucleic acid encoding a selectable marker comprises an intron comprising an endonuclease recognition site flanked by telomere seed sequences.

Telomerator-a tool for chromosome engineering

Provided are compositions and methods for use in assembling and expressing a plurality of transcription units using, in one aspect, homologous recombination in yeast. Yeast containing the plurality of transcription units, and isolated transcription units, are also provided. Kits for use in making the yeast and the transcript units are further included.

Versatile genetic assembly system (VEGAS) to assemble pathways for expression

The invention relates to synthetic transposon and retrotransposon genes that exhibit higher levels of expression relative to natural transposon and retrotransposon genes. The invention further relates to transposons and retrotransposons comprising such synthetic genes.

Synthetic mammalian retrotransposon gene

The invention provides inhibitors of ghrelin O-acyltransferase, and methods of making and using them. In some embodiments, the invention provides bisubstrate analog inhibitors of ghrelin O-acyltransferase, which can be effective in treating, for example, obesity and diabetes mellitus.

Methods for synthesis and uses of inhibitors of ghrelin O-acyltransferase as potential therapeutic agents for obesity and diabetes

Growing community of inventors

Baltimore, MD, United States of America

Jef D Boeke