Final Report Summary - ST-FLOW (Standarization and orthogonalization of the gene expression flow for robust engineering of NTN (new-to-nature) biological properties.)
Executive Summary:
The ST-FLOW Project has merged the efforts of 15 leading European research groups for developing material and computational standards that enable the forward-design of prokaryotic systems with a degree of robustness and predictability that is not possible with customary Genetic Engineering. The central issue at stake has been the identification and implementation of rules that allow the conversion of given biological parts assembled with a set of principles for physical composition into predictable functional properties of the resulting devices, modules and entire systems. ST-FLOW has focused on each of the steps that go from assembling a DNA sequence encoding all necessary expression signals in a prokaryotic host (by default, E. coli, but also others e.g. Pseudomonas putida) all the way to the making of the final product or to the behaviour of single cells and populations. To this end, two complementary approaches have been adopted to solve the conundrum of physical composition vs. biological functionality of thereby engineered devices. In one case (bottom up), large combinatorial libraries of gene expression signals were merged with suitable reporter systems and the input/output functions examined and parameterized in detail. The outcome of this effort was a series of experience-based but still reliable rules and criteria for the assembly of new devices and systems -following the same physical composition rules and in some instances adopting a degree of CAD design. Along the way, many outliers (combinations that do not follow the rules) were observed. To make sense of them we took the complementary top-down approach. This involved revisiting some of gaps in our knowledge of the gene expression flow (transcription, mRNA fate, translation) that need to be addressed for engineering functional devices from first principles. Ethical, legal and societal issues were also examined in a context of public dialogue (including a significant transatlantic dialogue) and sound science communication.
To meet all these challenges, ST-FLOW undertook the mission of producing foundational technologies, which have included:
• Coherent vector platforms for physical/automated assembly of DNA pieces, for testing device performance and for delivering constructs to deployment strains as plasmids or as genomic implants
• Standardized methods for connecting the DNA sequence of distinct functional parts following an automated stepwise workflow.
• Definition and experimental substantiation of PoPs (polymerase per second) units and implementation of robust methods for its quantification.
• Identification and formatting of mRNA motifs that influence degradation and translation of given transcripts
• Development of (metabolically) orthogonal heterologous expression systems for bacteria
• Application of standard tools to design and manufacture bacterial strains tailored for biocatalysis and environmental biosensing
• Deployment of a dissemination and training effort targeting a new generation of scientists; sorting out of intellectual property issues re. SynBio access; addressing public debate on ethical and safety issues.
Project Context and Objectives:
The context. Standards are traditionally claimed to be one of the pillars of modern engineering and as such they are also vindicated as one of the core tenets of contemporary synthetic biology – which is basically looking at biological systems through the eyes of an engineer. Standardization of physical assembly of DNA-encoded genetic parts was one of the first issues that the early pioneers of synthetic biology at the MIT pointed to as being critical for the development of the field. This is still today one of the principles of the iGEM students’ competition and its associated repository of biological parts. But soon after the issue was raised more than a decade ago, an avalanche of criticism followed, because regardless of how one standardizes physical composition, the result is not a predictable functional outcome, as biological activities delivered by given DNA segments are context-dependent in practically all cases. This raised the question: should we simply give up robust design of biological systems with new-to-nature properties? A lot has happened since those days. There has been an increased effort to develop orthogonal devices and even complete systems that are intended to work in a fashion minimally dependent and even autonomous of the biological host. These involve not only a suite of genetic patches and expression systems based on phage polymerases, but also recoding and/or expansion of the genetic code. Also, physical assembly of DNA pieces is no longer an issue, due to the ease of chemical synthesis and the onset of many procedures for composing genetic constructs that do not use restriction enzymes. More importantly, the debate on standards has moved beyond technicalities on DNA composition, now focusing on what else can and should be standardized. For example, how do we measure biological activities? And, along the way, the sector has added benchmarks for synbio practices, including risk assessment methods. At the same time, the growing awareness that synbio can ultimately become a transformative technology has prompted a (mostly implicit) footrace for who will succeed in establishing the rules and standards that will shape the field of synbio for the future. There is a general sentiment that the level of knowledge right now is not sufficient to address standards in biological design with the same rigour as electric or civil engineering does. There have indeed been partial advances in metrology and proposition of operating systems in living organisms, but most standards proposed thus far have not made it beyond very limited communities of users. There is still a considerable wander in the wilderness that the synbio community has to go through before reaching the promised land of full-fledged standardized biology.
The objectives. ST-FLOW Project has taken as its scientific and technical mission: [i] what can be standardized now in Biology for the sake of reliable bioengineering, [ii] what could be standardized provided if there were more data on the cognate gaps of fundamental knowledge and [iii] what may never be standardized in view of inherent qualities of living systems. The central issue at stake the ST-FLOW Project is therefore the identification and implementation of rules that allow the conversion of given biological parts assembled with a set of principles for physical composition into predictable functional properties of the resulting devices, modules and entire systems. To this end large combinatorial libraries of gene expression signals are merged with suitable reporter systems and the input/output functions examined and parameterized in a high-throughput fashion. The expected outcome of this effort is to establish experience-based but still reliable rules and criteria for the assembly of new devices and systems -following the same physical composition rules. In this context, ST-FLOW revisits some of gaps in our knowledge of the gene expression flow (transcription, mRNA fate, translation) that need to be addressed for engineering functional devices. Ethical, legal and societal issues have also been examined in a context of public dialogue and sound science communication.
The main emphases have thus taken place on the recurrent 5 pillars of the Project:
• Development of coherent vector platforms for physical/automated assembly of DNA pieces, for testing device performance and for delivering constructs to deployment strains as plasmids or as genomic implants
• Establishment of conventions for connecting the DNA sequence of distinct functional parts following an automated stepwise workflow. Eventual CAD-based erasing of the cloning scars in the optimized devices
• Fact-finding of basic aspects of transcription aimed at defining PoPs (polymerase per second) units and implementation of robust methods for its quantification. This includes identification and formatting of mRNA motifs that influence degradation and translation of given transcripts
• Application of standards to design and manufacture of bacterial strains tailored for biocatalysis, for biosensing of medically-important small molecules and for detection of environmental pollutants
• Deployment of a dissemination and training effort targeting a new generation of scientists; sorting out of intellectual property issues re. SynBio access; addressing public debate on ethical and safety issues
Project Results:
• A complete system for automated DNA assembly has been developed. New vectors and collections of genetic tools have been put in place under the denomination SEVA (Standard European Vector Architecture), the only European initiative thus far to standardize parts and devices for Synthetic Biology and available to the Syn Bio academic and industrial community on an Open Access regime.
• The issue of PoPs (polymerase per second), one of the basic concepts in Synthetic Biology has been visited and substantiated experimentally. Wet procedures have been developed to measure the frequency of passing of RNA polymerase through a given promoter position and its translation in reporter output. The resulting data raises a new paradigm in the way we envision transcription to occur inside the bacterial cells.
• A complete and coherent platform for data exchange on biological parts and their combination has been designed that allows different SynBio stakeholders to work together by adopting a central repository in which new results on standardized parts and can be entered, edited and shared at all times by means of the SBOL (Synthetic Biology Open Language) language, which is emerging as the best choice to this end.
• Computational platforms have been developed for assembly of new pathways with optimized ratios among their enzymatic constituents. The platforms integrate methods for calculating intergenic regions in between genes of interest as well as metabolic and physiological constraints of their expression.
• A whole series of new optical an enzymatic reporters have been designed that expand the choices that the SynBio professional has for monitoring gene expression in vivo and in real time. Their characterization has shed light on a large number of constraints in the gene expression flow and the cognate transfer functions.
• New protocols with a computational and a wet component have been set for engineering protease cleaving sites in the structure of any protein of interest. This tool will be invaluable for designing proteomic switches able to change the whole metabolic regime of the bacteria under examination, mostly when combined with multiplexed genome engineering methods
• A whole collection of bacterial strains with new-to-nature activities (eg refactored central metabolism and biosensing capabilities for eg Arsenic have been put together with the tools and concepts stemming from the standardization effort that is at the basis of ST-FLOW.
• A large set of resources for popularization and divulgation of SynBio in general and of standards in particular has been produced and delivered to the public domain in the format of videos, courses, debates, media releases and participation of ST-FLOW partners in social outreach activities, including political and ethical questions.
• The Project has provided the formal umbrella to host an unprecedented US-EU meeting on standards for Synthetic Biology (Valencia, march 2015) under the aegis of the US-EU Task force in Synthetic Biology which was instrumental to set priorities and start a transatlantic debate on scientific bottlenecks that have to be overcome to promulgate reliable standards.
Potential Impact:
This Project has made a key contribution to set the technical and conceptual platforms and standards that are required to take modern Biotechnology into a new stage since its foundation in the late 70s. We are convinced that our developments will bring a breakthrough of at least 10-20 fold of the current limitations for engineering Biological systems, which are currently constrained to the somehow modest dimension of ~20 kb or ~20-25 genes and just a few chassis. The results and technologies originating from this Project will be instrumental for the materialization of a Knowledge-based Bio-Economy (KBBE), because they endow the chemical and biotechnological sectors with materials, methodologies and references that will provide them with ways to capitalize on the unlimited catalytic capacity of live organisms. The introduction of composable biological parts will enable an engineering discipline similar to the ones that resulted in modern aviation and information technology. The case studies targeted in the ST-FLOW project have addressed a number of highly recurring biotechnological needs. This ensures a high degree of generality of the proposed strategies, rather than a single-issue specific solution. Many aspects of our work will positively influence other biotechnological fields, such as Bioremediation and Bioprocessing. The Project has had a strategic role in promoting the value of Biotechnology-relevant socioeconomic questions. The combination of Synthetic Biology, Engineering and Bioinformatics provide powerful instruments to explore and industrially exploit the immense potential of the biological world. Crucial to the future development of technologies used to engineer biological systems is the explicit acknowledgement by practitioners, policy makers, and consumers that Biology is itself a technology. But this is just the first step in implementing biological manufacturing, and it is important to highlight the contrast with technologies to come. More and more of the total production of economically important compounds is being miniaturized and the same may soon be true when dealing with biological objects. Today, academic programs or departments dedicated to Synthetic Biology are emerging in national laboratories and universities, with quite different intellectual roots and ultimate goals than traditional Bioengineering programs. They will all benefit from the large body of knowledge emerging from ST-FLOW. An added value in this respect has been the considerable expansion of the network of European Synthetic Biologists towards the US counterparts, an interaction which is bound to bear fruit in the next few years.
List of Websites:
http://www.cnb.csic.es/~stflow-project
The ST-FLOW Project has merged the efforts of 15 leading European research groups for developing material and computational standards that enable the forward-design of prokaryotic systems with a degree of robustness and predictability that is not possible with customary Genetic Engineering. The central issue at stake has been the identification and implementation of rules that allow the conversion of given biological parts assembled with a set of principles for physical composition into predictable functional properties of the resulting devices, modules and entire systems. ST-FLOW has focused on each of the steps that go from assembling a DNA sequence encoding all necessary expression signals in a prokaryotic host (by default, E. coli, but also others e.g. Pseudomonas putida) all the way to the making of the final product or to the behaviour of single cells and populations. To this end, two complementary approaches have been adopted to solve the conundrum of physical composition vs. biological functionality of thereby engineered devices. In one case (bottom up), large combinatorial libraries of gene expression signals were merged with suitable reporter systems and the input/output functions examined and parameterized in detail. The outcome of this effort was a series of experience-based but still reliable rules and criteria for the assembly of new devices and systems -following the same physical composition rules and in some instances adopting a degree of CAD design. Along the way, many outliers (combinations that do not follow the rules) were observed. To make sense of them we took the complementary top-down approach. This involved revisiting some of gaps in our knowledge of the gene expression flow (transcription, mRNA fate, translation) that need to be addressed for engineering functional devices from first principles. Ethical, legal and societal issues were also examined in a context of public dialogue (including a significant transatlantic dialogue) and sound science communication.
To meet all these challenges, ST-FLOW undertook the mission of producing foundational technologies, which have included:
• Coherent vector platforms for physical/automated assembly of DNA pieces, for testing device performance and for delivering constructs to deployment strains as plasmids or as genomic implants
• Standardized methods for connecting the DNA sequence of distinct functional parts following an automated stepwise workflow.
• Definition and experimental substantiation of PoPs (polymerase per second) units and implementation of robust methods for its quantification.
• Identification and formatting of mRNA motifs that influence degradation and translation of given transcripts
• Development of (metabolically) orthogonal heterologous expression systems for bacteria
• Application of standard tools to design and manufacture bacterial strains tailored for biocatalysis and environmental biosensing
• Deployment of a dissemination and training effort targeting a new generation of scientists; sorting out of intellectual property issues re. SynBio access; addressing public debate on ethical and safety issues.
Project Context and Objectives:
The context. Standards are traditionally claimed to be one of the pillars of modern engineering and as such they are also vindicated as one of the core tenets of contemporary synthetic biology – which is basically looking at biological systems through the eyes of an engineer. Standardization of physical assembly of DNA-encoded genetic parts was one of the first issues that the early pioneers of synthetic biology at the MIT pointed to as being critical for the development of the field. This is still today one of the principles of the iGEM students’ competition and its associated repository of biological parts. But soon after the issue was raised more than a decade ago, an avalanche of criticism followed, because regardless of how one standardizes physical composition, the result is not a predictable functional outcome, as biological activities delivered by given DNA segments are context-dependent in practically all cases. This raised the question: should we simply give up robust design of biological systems with new-to-nature properties? A lot has happened since those days. There has been an increased effort to develop orthogonal devices and even complete systems that are intended to work in a fashion minimally dependent and even autonomous of the biological host. These involve not only a suite of genetic patches and expression systems based on phage polymerases, but also recoding and/or expansion of the genetic code. Also, physical assembly of DNA pieces is no longer an issue, due to the ease of chemical synthesis and the onset of many procedures for composing genetic constructs that do not use restriction enzymes. More importantly, the debate on standards has moved beyond technicalities on DNA composition, now focusing on what else can and should be standardized. For example, how do we measure biological activities? And, along the way, the sector has added benchmarks for synbio practices, including risk assessment methods. At the same time, the growing awareness that synbio can ultimately become a transformative technology has prompted a (mostly implicit) footrace for who will succeed in establishing the rules and standards that will shape the field of synbio for the future. There is a general sentiment that the level of knowledge right now is not sufficient to address standards in biological design with the same rigour as electric or civil engineering does. There have indeed been partial advances in metrology and proposition of operating systems in living organisms, but most standards proposed thus far have not made it beyond very limited communities of users. There is still a considerable wander in the wilderness that the synbio community has to go through before reaching the promised land of full-fledged standardized biology.
The objectives. ST-FLOW Project has taken as its scientific and technical mission: [i] what can be standardized now in Biology for the sake of reliable bioengineering, [ii] what could be standardized provided if there were more data on the cognate gaps of fundamental knowledge and [iii] what may never be standardized in view of inherent qualities of living systems. The central issue at stake the ST-FLOW Project is therefore the identification and implementation of rules that allow the conversion of given biological parts assembled with a set of principles for physical composition into predictable functional properties of the resulting devices, modules and entire systems. To this end large combinatorial libraries of gene expression signals are merged with suitable reporter systems and the input/output functions examined and parameterized in a high-throughput fashion. The expected outcome of this effort is to establish experience-based but still reliable rules and criteria for the assembly of new devices and systems -following the same physical composition rules. In this context, ST-FLOW revisits some of gaps in our knowledge of the gene expression flow (transcription, mRNA fate, translation) that need to be addressed for engineering functional devices. Ethical, legal and societal issues have also been examined in a context of public dialogue and sound science communication.
The main emphases have thus taken place on the recurrent 5 pillars of the Project:
• Development of coherent vector platforms for physical/automated assembly of DNA pieces, for testing device performance and for delivering constructs to deployment strains as plasmids or as genomic implants
• Establishment of conventions for connecting the DNA sequence of distinct functional parts following an automated stepwise workflow. Eventual CAD-based erasing of the cloning scars in the optimized devices
• Fact-finding of basic aspects of transcription aimed at defining PoPs (polymerase per second) units and implementation of robust methods for its quantification. This includes identification and formatting of mRNA motifs that influence degradation and translation of given transcripts
• Application of standards to design and manufacture of bacterial strains tailored for biocatalysis, for biosensing of medically-important small molecules and for detection of environmental pollutants
• Deployment of a dissemination and training effort targeting a new generation of scientists; sorting out of intellectual property issues re. SynBio access; addressing public debate on ethical and safety issues
Project Results:
• A complete system for automated DNA assembly has been developed. New vectors and collections of genetic tools have been put in place under the denomination SEVA (Standard European Vector Architecture), the only European initiative thus far to standardize parts and devices for Synthetic Biology and available to the Syn Bio academic and industrial community on an Open Access regime.
• The issue of PoPs (polymerase per second), one of the basic concepts in Synthetic Biology has been visited and substantiated experimentally. Wet procedures have been developed to measure the frequency of passing of RNA polymerase through a given promoter position and its translation in reporter output. The resulting data raises a new paradigm in the way we envision transcription to occur inside the bacterial cells.
• A complete and coherent platform for data exchange on biological parts and their combination has been designed that allows different SynBio stakeholders to work together by adopting a central repository in which new results on standardized parts and can be entered, edited and shared at all times by means of the SBOL (Synthetic Biology Open Language) language, which is emerging as the best choice to this end.
• Computational platforms have been developed for assembly of new pathways with optimized ratios among their enzymatic constituents. The platforms integrate methods for calculating intergenic regions in between genes of interest as well as metabolic and physiological constraints of their expression.
• A whole series of new optical an enzymatic reporters have been designed that expand the choices that the SynBio professional has for monitoring gene expression in vivo and in real time. Their characterization has shed light on a large number of constraints in the gene expression flow and the cognate transfer functions.
• New protocols with a computational and a wet component have been set for engineering protease cleaving sites in the structure of any protein of interest. This tool will be invaluable for designing proteomic switches able to change the whole metabolic regime of the bacteria under examination, mostly when combined with multiplexed genome engineering methods
• A whole collection of bacterial strains with new-to-nature activities (eg refactored central metabolism and biosensing capabilities for eg Arsenic have been put together with the tools and concepts stemming from the standardization effort that is at the basis of ST-FLOW.
• A large set of resources for popularization and divulgation of SynBio in general and of standards in particular has been produced and delivered to the public domain in the format of videos, courses, debates, media releases and participation of ST-FLOW partners in social outreach activities, including political and ethical questions.
• The Project has provided the formal umbrella to host an unprecedented US-EU meeting on standards for Synthetic Biology (Valencia, march 2015) under the aegis of the US-EU Task force in Synthetic Biology which was instrumental to set priorities and start a transatlantic debate on scientific bottlenecks that have to be overcome to promulgate reliable standards.
Potential Impact:
This Project has made a key contribution to set the technical and conceptual platforms and standards that are required to take modern Biotechnology into a new stage since its foundation in the late 70s. We are convinced that our developments will bring a breakthrough of at least 10-20 fold of the current limitations for engineering Biological systems, which are currently constrained to the somehow modest dimension of ~20 kb or ~20-25 genes and just a few chassis. The results and technologies originating from this Project will be instrumental for the materialization of a Knowledge-based Bio-Economy (KBBE), because they endow the chemical and biotechnological sectors with materials, methodologies and references that will provide them with ways to capitalize on the unlimited catalytic capacity of live organisms. The introduction of composable biological parts will enable an engineering discipline similar to the ones that resulted in modern aviation and information technology. The case studies targeted in the ST-FLOW project have addressed a number of highly recurring biotechnological needs. This ensures a high degree of generality of the proposed strategies, rather than a single-issue specific solution. Many aspects of our work will positively influence other biotechnological fields, such as Bioremediation and Bioprocessing. The Project has had a strategic role in promoting the value of Biotechnology-relevant socioeconomic questions. The combination of Synthetic Biology, Engineering and Bioinformatics provide powerful instruments to explore and industrially exploit the immense potential of the biological world. Crucial to the future development of technologies used to engineer biological systems is the explicit acknowledgement by practitioners, policy makers, and consumers that Biology is itself a technology. But this is just the first step in implementing biological manufacturing, and it is important to highlight the contrast with technologies to come. More and more of the total production of economically important compounds is being miniaturized and the same may soon be true when dealing with biological objects. Today, academic programs or departments dedicated to Synthetic Biology are emerging in national laboratories and universities, with quite different intellectual roots and ultimate goals than traditional Bioengineering programs. They will all benefit from the large body of knowledge emerging from ST-FLOW. An added value in this respect has been the considerable expansion of the network of European Synthetic Biologists towards the US counterparts, an interaction which is bound to bear fruit in the next few years.
List of Websites:
http://www.cnb.csic.es/~stflow-project