Project acronym BIOSILICA
Project From gene to biomineral: Biosynthesis and application of sponge biosilica
Researcher (PI) Werner Ernst Ludwig Georg Müller
Host Institution (HI) UNIVERSITAETSMEDIZIN DER JOHANNES GUTENBERG-UNIVERSITAET MAINZ
Call Details Advanced Grant (AdG), LS9, ERC-2010-AdG_20100317
Summary During the last decade, the principles of biomineralization have increasingly attracted multidisciplinary scientific attention, not only because they touch the interface between the organic/inorganic world but also because they offer fascinating bioinspired solutions to notorious problems in the fields of biotechnology and medicine. However, only one group of animals has the necessary genetic/enzymatic toolkit to control biomineralization: siliceous sponges (Porifera). Based on his pioneering discoveries in poriferan molecular biology and physiological chemistry, the PI has brought biosilicification into the focus of basic and applied research. Through multiple trendsetting approaches the molecular key components for the enzymatic synthesis of polymorphic siliceous skeletal elements in sponges have been elucidated and characterized. Subsequently, they have been employed to synthesize innovative composite materials in vitro. Nonetheless, knowledge of the functional mechanisms involved remains sketchy and harnessing biosilicification, beyond the in vitro synthesis of amorphous nanocomposites, is still impossible. Using a unique blend of cutting-edge techniques in molecular/structural biology, biochemistry, bioengineering, and material sciences, the PI approaches for the first time a comprehensive analysis of natural biomineralization, from gene to biomineral to hierarchically ordered structures of increasing complexity. The groundbreaking discoveries expected will be of extreme importance for understanding poriferan biosilicification. Concurrently, they will contribute to the development of innovative nano-biotechnological and -medical approaches that aim to elicit novel (biogenous) optical waveguide fibers and self-repairing inorganic-organic bone substitution materials.
Summary
During the last decade, the principles of biomineralization have increasingly attracted multidisciplinary scientific attention, not only because they touch the interface between the organic/inorganic world but also because they offer fascinating bioinspired solutions to notorious problems in the fields of biotechnology and medicine. However, only one group of animals has the necessary genetic/enzymatic toolkit to control biomineralization: siliceous sponges (Porifera). Based on his pioneering discoveries in poriferan molecular biology and physiological chemistry, the PI has brought biosilicification into the focus of basic and applied research. Through multiple trendsetting approaches the molecular key components for the enzymatic synthesis of polymorphic siliceous skeletal elements in sponges have been elucidated and characterized. Subsequently, they have been employed to synthesize innovative composite materials in vitro. Nonetheless, knowledge of the functional mechanisms involved remains sketchy and harnessing biosilicification, beyond the in vitro synthesis of amorphous nanocomposites, is still impossible. Using a unique blend of cutting-edge techniques in molecular/structural biology, biochemistry, bioengineering, and material sciences, the PI approaches for the first time a comprehensive analysis of natural biomineralization, from gene to biomineral to hierarchically ordered structures of increasing complexity. The groundbreaking discoveries expected will be of extreme importance for understanding poriferan biosilicification. Concurrently, they will contribute to the development of innovative nano-biotechnological and -medical approaches that aim to elicit novel (biogenous) optical waveguide fibers and self-repairing inorganic-organic bone substitution materials.
Max ERC Funding
2 183 600 €
Duration
Start date: 2011-06-01, End date: 2017-05-31
Project acronym COMREC
Project Designed Plant Breeding by Control of Meiotic Recombination
Researcher (PI) Holger Alexander Puchta
Host Institution (HI) KARLSRUHER INSTITUT FUER TECHNOLOGIE
Call Details Advanced Grant (AdG), LS9, ERC-2010-AdG_20100317
Summary Agriculture depends on breeding. Breeders try to combining desirable and eliminating unfavourable traits of crop plants. Changes in genetic linkage are based on meiotic recombination. Although techniques for the transfer of single traits have been developed, resulting in genetically modified organisms (GMOs), hardly any effort has been undertaken to control the exchange between parental genomes as such. By applying new molecular tools to control recombination the current project aims to establish a new kind of ¿designed¿ plant breeding. Thus, not only transfer or elimination of specific traits should become possible in a programmable way, but also the access to the complete gene pool of natural species and its widening by crossing in closely related species should become feasible. Suppression of recombination should result in an apomixis-like propagation of elite cultivars. Mainly two different levels of control will be addressed in the project: the induction of recombination at predefined specific sites in the genome and the regulation of the level of genome-wide exchange. For the former approach we will apply specifically tailored sequence-specific zinc-finger and meganucleases. Global changes should be achieved by modulating the expression of factors involved in the resolution of recombination intermediates. As the strategy relies on the exploitation of the natural mechanism of recombination, biotechnologically improved plants without transgenes will be obtained after outcrossing. Thus, public concerns raised by GMOs brought out in the field should be avoided. As recent technical improvements make the elucidation of genomic sequences possible at moderate cost and time requirements, the setup of ¿designed¿ breeding should become especially useful in the near future.
Summary
Agriculture depends on breeding. Breeders try to combining desirable and eliminating unfavourable traits of crop plants. Changes in genetic linkage are based on meiotic recombination. Although techniques for the transfer of single traits have been developed, resulting in genetically modified organisms (GMOs), hardly any effort has been undertaken to control the exchange between parental genomes as such. By applying new molecular tools to control recombination the current project aims to establish a new kind of ¿designed¿ plant breeding. Thus, not only transfer or elimination of specific traits should become possible in a programmable way, but also the access to the complete gene pool of natural species and its widening by crossing in closely related species should become feasible. Suppression of recombination should result in an apomixis-like propagation of elite cultivars. Mainly two different levels of control will be addressed in the project: the induction of recombination at predefined specific sites in the genome and the regulation of the level of genome-wide exchange. For the former approach we will apply specifically tailored sequence-specific zinc-finger and meganucleases. Global changes should be achieved by modulating the expression of factors involved in the resolution of recombination intermediates. As the strategy relies on the exploitation of the natural mechanism of recombination, biotechnologically improved plants without transgenes will be obtained after outcrossing. Thus, public concerns raised by GMOs brought out in the field should be avoided. As recent technical improvements make the elucidation of genomic sequences possible at moderate cost and time requirements, the setup of ¿designed¿ breeding should become especially useful in the near future.
Max ERC Funding
2 493 000 €
Duration
Start date: 2011-09-01, End date: 2016-08-31
Project acronym ELECTROTALK
Project Starting an electrical conversation between microorganisms and electrodes to achieve bioproduction
Researcher (PI) Korneel Pieter Herman Leo Ann Rabaey
Host Institution (HI) UNIVERSITEIT GENT
Call Details Starting Grant (StG), LS9, ERC-2012-StG_20111109
Summary "Electrochemically active bacteria enable a host of novel processes in bioproduction, bioenergy and bioremediation. Key to the success of these processes is effective adherence of the bacterial cells to an electrode surface and subsequent equally effective electron exchange with the electrode. While the cellular mechanisms for electron transfer are increasingly known, what drives bacterial adsorption and desorption to positively or negatively polarized electrodes is largely unknown. Particularly processes driven by cathodes tend to be slow, and suffer from limited microbial adherence and lack of growth of the microorganisms. ELECTROTALK aims at developing a mechanistic understanding of mobility towards and microbial adherence at surfaces, from single cell level to complete biofilm formation. Based on this knowledge, effectively catalyzed bio-electrodes will be developed for novel bioproduction processes. Such bioproduction processes, termed microbial electrosynthesis, are independent of arable land availability, promise high production densities and enable the capture of CO2 or more efficient resource-usage for a range of products. Understanding the nature of the microorganism-electrode interaction will create a window of opportunity to improve this process and achieve effective bioproduction. Moreover, as the electrical interaction directly relates to microbial activity electrodes may serve as a means to start up a conversation with the cells. To achieve our aims we will: (i) select and characterize biocatalysts both as pure cultures and microbial communities; (ii) investigate cell adherence and electron transfer in function of electrode topography and chemistry as well as under different operational conditions; (iii) develop an electrode-microorganism combination achieving effective electron transfer; and (iv) electrochemically construct biofilms with defined structure or stratification."
Summary
"Electrochemically active bacteria enable a host of novel processes in bioproduction, bioenergy and bioremediation. Key to the success of these processes is effective adherence of the bacterial cells to an electrode surface and subsequent equally effective electron exchange with the electrode. While the cellular mechanisms for electron transfer are increasingly known, what drives bacterial adsorption and desorption to positively or negatively polarized electrodes is largely unknown. Particularly processes driven by cathodes tend to be slow, and suffer from limited microbial adherence and lack of growth of the microorganisms. ELECTROTALK aims at developing a mechanistic understanding of mobility towards and microbial adherence at surfaces, from single cell level to complete biofilm formation. Based on this knowledge, effectively catalyzed bio-electrodes will be developed for novel bioproduction processes. Such bioproduction processes, termed microbial electrosynthesis, are independent of arable land availability, promise high production densities and enable the capture of CO2 or more efficient resource-usage for a range of products. Understanding the nature of the microorganism-electrode interaction will create a window of opportunity to improve this process and achieve effective bioproduction. Moreover, as the electrical interaction directly relates to microbial activity electrodes may serve as a means to start up a conversation with the cells. To achieve our aims we will: (i) select and characterize biocatalysts both as pure cultures and microbial communities; (ii) investigate cell adherence and electron transfer in function of electrode topography and chemistry as well as under different operational conditions; (iii) develop an electrode-microorganism combination achieving effective electron transfer; and (iv) electrochemically construct biofilms with defined structure or stratification."
Max ERC Funding
1 494 126 €
Duration
Start date: 2012-10-01, End date: 2017-09-30
Project acronym HOSTRESPONSE
Project Host molecular and cellular responses to anti-cancer drug treatment as a potential biomarker for treatment outcome
Researcher (PI) Yuval Shaked
Host Institution (HI) TECHNION - ISRAEL INSTITUTE OF TECHNOLOGY
Call Details Starting Grant (StG), LS9, ERC-2010-StG_20091118
Summary Chemotherapy remains one of the most common treatment modalities for cancer. It is typically administered in cycles of bolus injections following 21 days of drug-free break periods. However, tumor regrowth between drug intervals is often observed, due in part, to rebound angiogenesis. Our previous studies demonstrated that bone marrow derived proangiogenic cells are acutely mobilized following certain chemotherapy treatments, accompanied by enhanced tumor angiogenesis, which can be blocked by prior treatment with antiangiogenic drugs. These findings indicate that unknown host-derived mechanisms induced by chemotherapy, can stimulate tumor growth. Since the efficacy of antiangiogenic drugs is dependent on several parameters such as tumor type, stage, and the type of chemotherapy, such a therapy is not beneficial for all patients, and thus, necessitates the identification of surrogate biomarkers to predict clinical outcome. To address this issue, we will integrate basic, translational, and clinical approaches to:
(i) identify molecular and cellular host systemic responses following treatments;
(ii) isolate novel factors by proteomic approaches which are altered during the course of the treatment, and evaluate their feasibility as biomarkers to predict clinical outcome;
(iii) determine the relevance of these factors in clinical specimens;
(iv) screen for therapeutic compounds which can block host responses mediating tumor growth in order to increase treatment efficacy.
We believe that this strategy of combined approach will lead to the development of new tools in clinical oncology. Profiling individual host response to anti-cancer drug treatment may serve as a biomarker for treatment outcome and further promote the concept of personalised medicine in cancer therapy.
Summary
Chemotherapy remains one of the most common treatment modalities for cancer. It is typically administered in cycles of bolus injections following 21 days of drug-free break periods. However, tumor regrowth between drug intervals is often observed, due in part, to rebound angiogenesis. Our previous studies demonstrated that bone marrow derived proangiogenic cells are acutely mobilized following certain chemotherapy treatments, accompanied by enhanced tumor angiogenesis, which can be blocked by prior treatment with antiangiogenic drugs. These findings indicate that unknown host-derived mechanisms induced by chemotherapy, can stimulate tumor growth. Since the efficacy of antiangiogenic drugs is dependent on several parameters such as tumor type, stage, and the type of chemotherapy, such a therapy is not beneficial for all patients, and thus, necessitates the identification of surrogate biomarkers to predict clinical outcome. To address this issue, we will integrate basic, translational, and clinical approaches to:
(i) identify molecular and cellular host systemic responses following treatments;
(ii) isolate novel factors by proteomic approaches which are altered during the course of the treatment, and evaluate their feasibility as biomarkers to predict clinical outcome;
(iii) determine the relevance of these factors in clinical specimens;
(iv) screen for therapeutic compounds which can block host responses mediating tumor growth in order to increase treatment efficacy.
We believe that this strategy of combined approach will lead to the development of new tools in clinical oncology. Profiling individual host response to anti-cancer drug treatment may serve as a biomarker for treatment outcome and further promote the concept of personalised medicine in cancer therapy.
Max ERC Funding
1 499 622 €
Duration
Start date: 2011-03-01, End date: 2017-02-28
Project acronym MAGNETOGENETICS
Project Reverse engineering the vertebrate molecular machinery for magnetic biomineralisation
Researcher (PI) Gil Gregor Westmeyer
Host Institution (HI) KLINIKUM RECHTS DER ISAR DER TECHNISCHEN UNIVERSITAT MUNCHEN
Call Details Starting Grant (StG), LS9, ERC-2012-StG_20111109
Summary In order to understand distributed biological networks and invent effective molecular therapies, we need to be able to image and control molecular processes across entire live organisms.
Although an arsenal of genetically encoded fluorescent sensors and light-activated proteins has recently been developed to visualize and manipulate cellular activity, these methods all suffer from the poor penetration of light through biological tissue, which makes them rather invasive for practical in vivo use in animal models and prevents whole-organ coverage.
To overcome these substantial limitations of optical methods, I thus propose a reverse engineering project to functionally dissect the molecular machinery for magnetic biomineralisation in eukaryotic cells and build a new bioengineering platform for genetically controlled bio-magnetic interfaces. To achieve this goal, we will use a combination of magnetic cell sorting and biophysical characterization, targeted analysis and -omics approaches as well as genetic tools. This scientific program will break the ground for non-invasive sensing and actuation of molecular processes via magnetic fields based on genetic control over the biosynthesis of magnetic biominerals.
Summary
In order to understand distributed biological networks and invent effective molecular therapies, we need to be able to image and control molecular processes across entire live organisms.
Although an arsenal of genetically encoded fluorescent sensors and light-activated proteins has recently been developed to visualize and manipulate cellular activity, these methods all suffer from the poor penetration of light through biological tissue, which makes them rather invasive for practical in vivo use in animal models and prevents whole-organ coverage.
To overcome these substantial limitations of optical methods, I thus propose a reverse engineering project to functionally dissect the molecular machinery for magnetic biomineralisation in eukaryotic cells and build a new bioengineering platform for genetically controlled bio-magnetic interfaces. To achieve this goal, we will use a combination of magnetic cell sorting and biophysical characterization, targeted analysis and -omics approaches as well as genetic tools. This scientific program will break the ground for non-invasive sensing and actuation of molecular processes via magnetic fields based on genetic control over the biosynthesis of magnetic biominerals.
Max ERC Funding
1 469 100 €
Duration
Start date: 2013-01-01, End date: 2017-12-31
Project acronym MuDLOC
Project Multi-Dimensional Lab-On-Chip
Researcher (PI) Doron Gerber
Host Institution (HI) BAR ILAN UNIVERSITY
Call Details Starting Grant (StG), LS9, ERC-2012-StG_20111109
Summary There are several bottlenecks that hinder certain aspects of proteomics, in particular, incompatibility of high throughput technologies with certain protein types or modifications, low sensitivity and lack of quantitative data. I have developed a microfluidics affinity assay compatible with transmembrane proteins and post-translational modifications that is highly sensitive and can provide quantitative data.
The primary objective of this proposal is to bioengineer, using the abovementioned building blocks, a multi-functional microfluidic-based human protein arrays. The platform will enable addressing important scientific questions not otherwise possible. Specifically, the process of DNA demethylation, which is poorly characterised due to technological limitations. The biological aspects of chromatin methylation and their regulators that are crucial for cell differentiation and disease will be studied.
Work in MuDLOC will include the following: i) Bioengineering of a microfluidic-based platform that expresses thousands of human genes; ii) Design new tools for post-translational modifications and chromatin modifications; iii) Search for chromatin modifiers and their regulators; and iv) Exploration of specific inhibitors using a microfluidic inhibitor screen.
Beyond studying chromatin methylation from a new perspective, MuDLOC will greatly benefit a plethora of disciplines, such as proteomics, genomics and cancer research. At the end of the project my vision is to capture under one platform a whole pathway, including protein interactions, post-translational modifications and chromatin modifications.
Summary
There are several bottlenecks that hinder certain aspects of proteomics, in particular, incompatibility of high throughput technologies with certain protein types or modifications, low sensitivity and lack of quantitative data. I have developed a microfluidics affinity assay compatible with transmembrane proteins and post-translational modifications that is highly sensitive and can provide quantitative data.
The primary objective of this proposal is to bioengineer, using the abovementioned building blocks, a multi-functional microfluidic-based human protein arrays. The platform will enable addressing important scientific questions not otherwise possible. Specifically, the process of DNA demethylation, which is poorly characterised due to technological limitations. The biological aspects of chromatin methylation and their regulators that are crucial for cell differentiation and disease will be studied.
Work in MuDLOC will include the following: i) Bioengineering of a microfluidic-based platform that expresses thousands of human genes; ii) Design new tools for post-translational modifications and chromatin modifications; iii) Search for chromatin modifiers and their regulators; and iv) Exploration of specific inhibitors using a microfluidic inhibitor screen.
Beyond studying chromatin methylation from a new perspective, MuDLOC will greatly benefit a plethora of disciplines, such as proteomics, genomics and cancer research. At the end of the project my vision is to capture under one platform a whole pathway, including protein interactions, post-translational modifications and chromatin modifications.
Max ERC Funding
1 497 990 €
Duration
Start date: 2013-02-01, End date: 2018-01-31
Project acronym N-ABLE
Project Nitrogenase and Nitrous Oxide Reductase:
Biomolecular Engineering of Complex Redox Enzymes
Researcher (PI) Oliver Florian Einsle
Host Institution (HI) ALBERT-LUDWIGS-UNIVERSITAET FREIBURG
Call Details Starting Grant (StG), LS9, ERC-2012-StG_20111109
Summary "Nitrogenase and nitrous oxide reductase are complex metalloenzymes that are highly sensitive to dioxygen. They catalyze economically and ecologically important chemistry, but their function is not understood in full detail. We propose to analyze the mechanisms of these enzymes and elucidate the complex biogenesis of their intricate metal cofactors, the FeMo cofactor and the CuZ site, respectively. To this end we will combine protein biochemistry under anoxic conditions with the systematic, stepwise re-assembly of the structural genes and maturation factors of nitrogenase and nitrous oxide reductase into a heterologous expression system in Escherichia coli as a host system with a facultatively anaerobic lifestyle.
This approach combines the principles of protein biochemistry and synthetic biology and aims at conveying the catalytic capacities of nitrogenase and nitrous oxide reductase to a new host. Once recombinant proteins can be produced we will use directed evolution under a selection conditions of dioxygen stress to increase the oxygen-tolerance of the enzymes and extend the usability of the expression plasmids. With nitrogenase, we will then aim at transferring the ability to fix dinitrogen to eukaryotic hosts and will start with the well-established Pichia pastoris. As a long-term goal, the expression of nitrogenase in plant cells will provide the basis for a new era of green biotechnology that does not rely on chemical nitrogen fertilization."
Summary
"Nitrogenase and nitrous oxide reductase are complex metalloenzymes that are highly sensitive to dioxygen. They catalyze economically and ecologically important chemistry, but their function is not understood in full detail. We propose to analyze the mechanisms of these enzymes and elucidate the complex biogenesis of their intricate metal cofactors, the FeMo cofactor and the CuZ site, respectively. To this end we will combine protein biochemistry under anoxic conditions with the systematic, stepwise re-assembly of the structural genes and maturation factors of nitrogenase and nitrous oxide reductase into a heterologous expression system in Escherichia coli as a host system with a facultatively anaerobic lifestyle.
This approach combines the principles of protein biochemistry and synthetic biology and aims at conveying the catalytic capacities of nitrogenase and nitrous oxide reductase to a new host. Once recombinant proteins can be produced we will use directed evolution under a selection conditions of dioxygen stress to increase the oxygen-tolerance of the enzymes and extend the usability of the expression plasmids. With nitrogenase, we will then aim at transferring the ability to fix dinitrogen to eukaryotic hosts and will start with the well-established Pichia pastoris. As a long-term goal, the expression of nitrogenase in plant cells will provide the basis for a new era of green biotechnology that does not rely on chemical nitrogen fertilization."
Max ERC Funding
1 641 000 €
Duration
Start date: 2013-01-01, End date: 2017-12-31
Project acronym PROSECMET
Project Function and production of secondary metabolites
Researcher (PI) Helge Björn Bode
Host Institution (HI) JOHANN WOLFGANG GOETHE-UNIVERSITATFRANKFURT AM MAIN
Call Details Starting Grant (StG), LS9, ERC-2012-StG_20111109
Summary "Secondary metabolites (SMs) of bacterial origin play a pivotal role in modern therapy of various diseases. Whole-genome sequencing projects have revealed that all SM producing organisms have the capacity to produce many more compounds than reported from the respective strains. Despite their importance and their broad use in medicine, we hardly know anything about (i) the natural function of these important compounds, (ii) the underlying regulatory networks, and (iii) how to increase the number and yield of SMs produced by a single organism.
PROSECMET will address all these issues through the activities in four different work packages. As model organisms Photorhabdus and Xenorhabdus bacteria will be used that live in symbiosis with nematodes and together with them form an entomopathogenic complex.
WP1-Identification of SM targets. Derivatives of SMs will be synthesized allowing the identification of their molecular targets in bacteria, nematodes, insects and/or food competitors using methods of chemical biology.
WP2-Regulatory networks. External factors that activate SM production and if and how SMs act as internal signals in the producer will be identified (eg. by flow cytometry) with specific strains carrying SM biosynthesis gene promoters or promoter libraries fused to genes encoding different reporters (fluorescent proteins, resistance genes).
WP3-Increasing chemical diversity. Factors identified in WP2 will be applied to at least 100 different Photorhabdus and Xenorhabdus isolates in order to increase the number and the yield of SMs produced by them. Additionally, the genome of selected isolates will be sequenced in order to exchange the promoters responsible for SM biosynthesis leading to the production of additional compounds
WP4-Optimization of SM production. Using either regulatory elements identified in WP2 or artificially designed regulators, cell growth will be coupled to SM production, thus enabling the systematic construction of SM overproducers."
Summary
"Secondary metabolites (SMs) of bacterial origin play a pivotal role in modern therapy of various diseases. Whole-genome sequencing projects have revealed that all SM producing organisms have the capacity to produce many more compounds than reported from the respective strains. Despite their importance and their broad use in medicine, we hardly know anything about (i) the natural function of these important compounds, (ii) the underlying regulatory networks, and (iii) how to increase the number and yield of SMs produced by a single organism.
PROSECMET will address all these issues through the activities in four different work packages. As model organisms Photorhabdus and Xenorhabdus bacteria will be used that live in symbiosis with nematodes and together with them form an entomopathogenic complex.
WP1-Identification of SM targets. Derivatives of SMs will be synthesized allowing the identification of their molecular targets in bacteria, nematodes, insects and/or food competitors using methods of chemical biology.
WP2-Regulatory networks. External factors that activate SM production and if and how SMs act as internal signals in the producer will be identified (eg. by flow cytometry) with specific strains carrying SM biosynthesis gene promoters or promoter libraries fused to genes encoding different reporters (fluorescent proteins, resistance genes).
WP3-Increasing chemical diversity. Factors identified in WP2 will be applied to at least 100 different Photorhabdus and Xenorhabdus isolates in order to increase the number and the yield of SMs produced by them. Additionally, the genome of selected isolates will be sequenced in order to exchange the promoters responsible for SM biosynthesis leading to the production of additional compounds
WP4-Optimization of SM production. Using either regulatory elements identified in WP2 or artificially designed regulators, cell growth will be coupled to SM production, thus enabling the systematic construction of SM overproducers."
Max ERC Funding
1 754 700 €
Duration
Start date: 2013-03-01, End date: 2018-02-28
Project acronym PROTLEGO
Project Development of an accessible platform for ex vivo site specific post-translational modifications of proteins
Researcher (PI) Lital Yamna Alfonta
Host Institution (HI) BEN-GURION UNIVERSITY OF THE NEGEV
Call Details Starting Grant (StG), LS9, ERC-2010-StG_20091118
Summary The incorporation of unnatural amino acids (more than 50 to date) into proteins in vivo has resulted in the generation of
proteins with novel chemical, biological, and physical properties. However, some unnatural amino acids possess properties,
such as an inability to cross the cell membrane or a level of toxicity dangerous to the organism, that restrict their incorporation
into proteins in vivo. In addition, even when an unnatural amino acid crosses the cell membrane, its transport efficiency
within the cell is very low. We propose to overcome these limitations by exploiting translational components evolved
tRNA-synthetases and their cognate suppressor-tRNA from Archea for the incorporation of an array of unnatural amino acids
into proteins in vitro in a cell-free protein translation system. The expressed recombinant proteins containing the unnatural
amino acids will be purified from the reaction mixture and used for further research. Using the cell free system, first we will
demonstrate our new approach by incorporating novel unnatural amino acids, i.e., thiolysine analogues, into proteins using
the broad substrate specificity of evolved tRNA synthetases. We will then incorporate a thiolysine analogue into PCNA for
the site-specific ubiquitination and SUMOylation of these proteins for in vitro studies of the interactions between PCNA and
interacting proteins and to follow the progress of the replication fork. This unique approach will show for the first time the use
of evolved synthetases in a cell free translation system, with the advantage being that previously un-incorporable unnatural
amino acids can be incorporated using this approach. Our overall aim is to enable the introduction of new functionalities into
proteins.
Summary
The incorporation of unnatural amino acids (more than 50 to date) into proteins in vivo has resulted in the generation of
proteins with novel chemical, biological, and physical properties. However, some unnatural amino acids possess properties,
such as an inability to cross the cell membrane or a level of toxicity dangerous to the organism, that restrict their incorporation
into proteins in vivo. In addition, even when an unnatural amino acid crosses the cell membrane, its transport efficiency
within the cell is very low. We propose to overcome these limitations by exploiting translational components evolved
tRNA-synthetases and their cognate suppressor-tRNA from Archea for the incorporation of an array of unnatural amino acids
into proteins in vitro in a cell-free protein translation system. The expressed recombinant proteins containing the unnatural
amino acids will be purified from the reaction mixture and used for further research. Using the cell free system, first we will
demonstrate our new approach by incorporating novel unnatural amino acids, i.e., thiolysine analogues, into proteins using
the broad substrate specificity of evolved tRNA synthetases. We will then incorporate a thiolysine analogue into PCNA for
the site-specific ubiquitination and SUMOylation of these proteins for in vitro studies of the interactions between PCNA and
interacting proteins and to follow the progress of the replication fork. This unique approach will show for the first time the use
of evolved synthetases in a cell free translation system, with the advantage being that previously un-incorporable unnatural
amino acids can be incorporated using this approach. Our overall aim is to enable the introduction of new functionalities into
proteins.
Max ERC Funding
1 398 000 €
Duration
Start date: 2010-10-01, End date: 2016-03-31
Project acronym SIMBA
Project Single-Molecule BioAssays at Elevated Concentrations
Researcher (PI) Philip Karl-Josef Tinnefeld
Host Institution (HI) TECHNISCHE UNIVERSITAET BRAUNSCHWEIG
Call Details Starting Grant (StG), LS9, ERC-2010-StG_20091118
Summary In order to advance single-molecule fluorescence spectroscopy to the next level, handling and analysis of single molecules has to become broadly available. A further quantum leap is required to proceed to commercially successful applications such as drug screening and medical diagnostics.
In this project, I suggest a strategy to overcome the fundamental gap between the nanomolar concentration regime of current optical single-molecule spectroscopy and the nano- to millimolar dissociation constants of typical biomolecular interactions. I will use nano-apertures, which confine the detection to sub-attoliter volumes and allow single-molecule studies at elevated concentrations. To overcome unspecific binding and deteriorated fluorescence signals in the nano-apertures, I will use tailor-made DNA nanostructures produced by DNA origami. These nanostructures will match the nano-apertures like a plug in a socket. Inserting molecules at programmed positions in the nanostructures will open up a new realm of applications by the ability to immobilize exactly one molecule per nano-aperture and by obtaining comparable signals from every nano-aperture. I will spectroscopically characterize the nano-apertures creating a fluorescence map of their inside. I will exemplarily use the new abilities for previously impossible applications such as several folds improvement of single-molecule DNA sequencing, direct single-molecule RNA sequencing by reverse transcriptase for cancer screening, for paralleled drug screening of HIV protease inhibitors and for studying the chemomechanical coupling of single helicases.
In summary, I envision a broadly applicable platform that has the potential to become a golden standard by enabling both ground breaking fundamental research and commercial applications.
Summary
In order to advance single-molecule fluorescence spectroscopy to the next level, handling and analysis of single molecules has to become broadly available. A further quantum leap is required to proceed to commercially successful applications such as drug screening and medical diagnostics.
In this project, I suggest a strategy to overcome the fundamental gap between the nanomolar concentration regime of current optical single-molecule spectroscopy and the nano- to millimolar dissociation constants of typical biomolecular interactions. I will use nano-apertures, which confine the detection to sub-attoliter volumes and allow single-molecule studies at elevated concentrations. To overcome unspecific binding and deteriorated fluorescence signals in the nano-apertures, I will use tailor-made DNA nanostructures produced by DNA origami. These nanostructures will match the nano-apertures like a plug in a socket. Inserting molecules at programmed positions in the nanostructures will open up a new realm of applications by the ability to immobilize exactly one molecule per nano-aperture and by obtaining comparable signals from every nano-aperture. I will spectroscopically characterize the nano-apertures creating a fluorescence map of their inside. I will exemplarily use the new abilities for previously impossible applications such as several folds improvement of single-molecule DNA sequencing, direct single-molecule RNA sequencing by reverse transcriptase for cancer screening, for paralleled drug screening of HIV protease inhibitors and for studying the chemomechanical coupling of single helicases.
In summary, I envision a broadly applicable platform that has the potential to become a golden standard by enabling both ground breaking fundamental research and commercial applications.
Max ERC Funding
1 456 374 €
Duration
Start date: 2010-11-01, End date: 2016-10-31
