公开(公告)号：US11693017B2

公开(公告)日：2023-07-04

申请号：US15760352

申请日：2016-01-12

Applicant: President and Fellows of Harvard College

Inventor： Daniel J. Mandell ,  Justin Feng ,  Xavier Rios Villanueva ,  Rajagopal Chari

IPC: G01N33/74 ,  C07K14/00 ,  C12N15/62 ,  C12N15/63 ,  C12N9/22 ,  C12N15/11 ,  C12N15/90

CPC classification number: G01N33/743 ,  C07K14/00 ,  C12N9/22 ,  C12N15/11 ,  C12N15/62 ,  C12N15/63 ,  C12N15/902 ,  C07K2319/00 ,  C12N2310/20 ,  C12N2320/50 ,  C12N2800/22 ,  C12N2800/80

Abstract: Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Biosensors are produced based on a ligand-binding domain (LBD) using a method that, in principle, can be applied for any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. The power of this method is illustrated by developing biosensors for digoxin and progesterone. Addition of ligand to cells expressing a biosensor activates transcription in yeast, mammalian cells and plants, with a dynamic range of up to about 100-fold or more. The biosensors are used to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules.

公开(公告)号：US20170363648A1

公开(公告)日：2017-12-21

申请号：US15692211

申请日：2017-08-31

Applicant: President and Fellows of Harvard College ,  University of Washington

Inventor： George M. Church ,  Justin Feng ,  Daniel J. Mandell ,  David Baker ,  Stanley Fields ,  Benjamin Ward Jester ,  Christine Elaine Tinberg

IPC: G01N33/74

CPC classification number: G01N33/743 ,  G01N2500/10

Abstract: Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

公开(公告)号：US09766255B2

公开(公告)日：2017-09-19

申请号：US14993509

申请日：2016-01-12

Applicant: President and Fellows of Harvard College ,  University of Washington

Inventor： George M. Church ,  Justin Feng ,  Daniel J. Mandell ,  David Baker ,  Stanley Fields ,  Benjamin Ward Jester ,  Christine Elaine Tinberg

IPC: C12N7/00 ,  G01N33/74

CPC classification number: G01N33/743 ,  G01N2500/10

Abstract: Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

公开(公告)号：US20200292544A1

公开(公告)日：2020-09-17

申请号：US16083935

申请日：2017-03-09

Applicant: President and Fellows of Harvard College

Inventor： George M. Church ,  Daniel J. Mandell ,  Justin Feng

IPC: G01N33/566 ,  C07K14/435

Abstract: The disclosure provides a biosensor having a ligand binding domain (LBD) or its variant, wherein the stability of the LBD or its variant is conditioned on the presence of specific small molecule ligands, and wherein the LBD or its variant is fused to a reporter protein. The disclosure also provides a method of screening for small molecules that modulate protein stability.

公开(公告)号：US20180259541A1

公开(公告)日：2018-09-13

申请号：US15760352

申请日：2016-01-12

Applicant: President and Fellows of Harvard College

Inventor： Daniel J. Mandell ,  Justin Feng ,  Xavier Rios Villanueva ,  Rajagopal Chari

IPC: G01N33/74 ,  C12N15/62 ,  C12N15/63 ,  C12N9/22 ,  C12N15/90 ,  C12N15/11 ,  C07K14/00

CPC classification number: G01N33/743 ,  C07K14/00 ,  C07K2319/00 ,  C12N9/22 ,  C12N15/11 ,  C12N15/62 ,  C12N15/63 ,  C12N15/902 ,  C12N2310/20 ,  C12N2320/50 ,  C12N2800/22 ,  C12N2800/80

Abstract: Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Biosensors are produced based on a ligand-binding domain (LBD) using a method that, in principle, can be applied for any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. The power of this method is illustrated by developing biosensors for digoxin and progesterone. Addition of ligand to cells expressing a biosensor activates transcription in yeast, mammalian cells and plants, with a dynamic range of up to about 100-fold or more. The biosensors are used to improve the biotrans-formation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules.

公开(公告)号：US20160202256A1

公开(公告)日：2016-07-14

申请号：US14993509

申请日：2016-01-12

Applicant: President and Fellows of Harvard College ,  University of Washington

Inventor： George M. Church ,  Justin Feng ,  Daniel J. Mandell ,  David Baker ,  Stanley Fields ,  Benjamin Ward Jester ,  Christine Elaine Tinberg

IPC: G01N33/566 ,  G01N33/74

CPC classification number: G01N33/743 ,  G01N2500/10

Abstract: Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

Abstract translation: 公开了一种工程化以有条件地响应特定小分子的存在的生物传感器，生物传感器包括响应特定小分子存在的条件稳定的配体结合结构域（LBD），其中结合的读出由报告基因或转录因子 （TF）与LBD融合。