Final Report Summary - TARPOL (Targeting environmental pollution with engineered microbial systems a la carte)
The development of synthetic biology (SB) in Europe faces three major obstacles that have, up to date, hampered the development of an SB-based framework in our continent:
[i] The new field is still missing a comprehensive language and a shared conceptual framework for the description of minimally functional biological.
[ii] Scientists and technicians, particularly in Europe, have so far failed to recognise their latent capacity to shape a brand-new discipline within their scope.
[iii] Promotion of SB might touch upon social sensitivities related to recreating 'life-in-the-test-tube', which threatens a re-enactment of the controversy stirred up by GMOs. TARPOL, led by the Universitat de Valencia and composed of 18 partners (see http://www.sb-tarpol.eu/ for details), has developed a dynamic two-year programme of activities, run by a large collection of European stakeholders.
TARPOL has been successful in recruiting environmental competences from neighbouring disciplines and developing numerous material and computational resources for advanced refactoring of biological systems. TARPOL has progressed in laying the foundations of SB, particularly on genome reduction strategies including a novel view of living organisms as information traps and contributed with dozens of basic, conceptual and applied publications. The consortium has paid attention to education. A range of conferences, dissemination and training activities have been organised, including the support to a successful iGEM team and the organisation of two intensive SB summer schools in Valencia (Spain) and Basel (Switzerland).
Mobilisation, interdisciplinary collaboration and training were the objectives concerning SB converge into a single goal: to set up a brand-new discipline which may have revolutionary effects on the lives of people in Europe. TARPOL has significantly contributed to the objectives as a key step to implement a powerful, sustainable knowledge-based bio-economy in our old continent.
Project context and objectives
The basic premise of SB is that methods commonly used to design and construct non-biological systems, such as those employed in computational sciences and engineering disciplines, could also be used to model and programme novel synthetic biological systems. SB is intrinsically trans-disciplinary and draws expertise from biology, chemistry, physics, computer science, mathematics and engineering. Synthetic biologists are attempting to develop 'artificial life', for both its tremendous applications in biotechnology and as a proxy for shedding light into the question of the origins of life. This is attempted by taking two separate and competing routes: the 'top-down' and 'bottom-up' approaches to minimal cells. In the former, a primordial or minimal cell is generated by systematically reducing a biological cell's genome until it no longer functions. The bottom-up methodology seeks to assemble from scratch components or information units until an aspect of life emerges. The overall intellectual and experimental challenges of implementing artificial life remain relatively long-term goals. However, guiding principles, experimental methodologies and theoretical insights from biomimetic chemistry and SB can be adopted in new ways for practical applications on a realistic, yet not necessarily immediate, time-frame.
Based on SB achievements, there is a possibility of building a living being, or a part of it, which either already exists or is a non-natural entity. This view on SB represents a promising applied field to areas as different as biomedicine, bioenergy, environment, etc. This type of programme requires a serious reflexion on the acquired responsibilities, as a consequence of the new natures that humans may create. In addition, SB demands a philosophical thinking on the panoply of futures that, more than ever, are close to man-made reality.
The main objective is to catalyse the shaping of the European SB community for translating this new conceptual and technical field into relevant environmental applications.
This goal comprises several specific objectives:
- to foster the transfer of SB conceptual fields into technical applications directed to solve environmental problems;
- to brand the conceptual and material interfaces between various disciplines required for surfacing and establishment of a European community on SB focused on tackling, monitoring and preventing environmental problems;
- to empower the SB environmental biotechnology field with material, technical and web based resources;
- to ensure that all research areas of SB are coordinated at European level so there is a maximum exchange of knowledge;
- to enable the development of a conceptual frame in which ethical and safety aspects of novel SB-related technologies, especially those related with environmental applications, can be optimally integrated into an emerging community;
- to promote scientific training and specialisation on SB, especially among scientists in early stages of their careers;
- to identify European research and development (R&D) needs and priorities on SB and to make recommendations for future cooperation areas and innovative research activities to be launched in the EU;
- to create awareness at academic, industry, social, policy and decision-makers level on the economic and scientific potential of SB for environmental applications.
Project results
We believe that the project has led to important advances in the conceptual field of SB. For instance, the state of the art of cell engineering in the context of genome research has been reviewed by UVEG during the first year, paying particular attention to what was learned on naturally reduced genomes from either symbiotic or free living bacteria. The identification of a genomic core for a cyanobacterium (Synechococcus elongatus) was accomplished by UVEG. This analysis will set the basis for designing a minimal photoautotrophic system suitable for a plethora of biotechnological applications. During the whole project, UVEG focused on the general coordination of the project, management, dissemination activities, and conceptual frame and consensual language definitions. The latter task has been broadly covered with a range of peer-reviewed contributions on the definition of the minimal genetic array for life to exist; the evolution of prokaryote-animal symbiosis; and theoretical limitations of synthetic life. Examples of this production are a review paper dealing with the milestones and challenges of synthetic biologists, co-authored by several TARPOL partners and coordinated by UVEG several book chapters on minimal cells and a myriad of publications on the origin of life, ethics of teaching evolution and a range of conceptual aspects of SB.
Dissemination and training activities organised by UVEG were key points of the project, with activities including a summer course on SB and the international meeting on synthetic and streamlined genomes. Thinking of SB as a new field of technology in the intersection of biology and engineering, the importance of attracting and training new researchers in this discipline becomes evident. With this objective, the Cavanilles Institute (UVEG) and the Intertech Group (UPVLC) coorganised the summer course on SB. This activity was developed from two sides: training and motivational. For this reason, the programme offered combined expertise of the organisers to train scientists from an interdisciplinary perspective in order to offer a range of activities for young researchers from a wide range of fields. The workshop on streamlined and synthetic genomes was organised as part of the TARPOL and EMERGENCE programmes to promote SB in Europe.
The workshop joined worldwide-recognised experts. The objectives were to assess the status, identify constraints and discuss the potential of research on minimal/streamlined genomes from different perspectives. This included the minimal and sufficient features of life, the study of naturally evolved reduced genomes, the engineering of minimal cells from bottom-up and top-down approaches, as well as various practical applications derived from research on minimal living systems. The workshop consisted of four sessions:
- Synthetic and digital biology, chaired by V. Martins dos Santos;
- Streamlining genomes, chaired by I. Economidis;
- Building genomes, chaired by G. Posfai; and
- Circuit design and evolution, chaired by V. de Lorenzo.
A satellite meeting on insect symbiosis in the era of systems and SB was held and allowed many of the attendants to join the community of synthetic biologists and discuss topics at the interphase between these closely related disciplines.
UVEG-led initiatives also served as a forum on which partners had the opportunity to discuss and prepare further research projects. We also participated in a course hold at Spetses (Greece) focusing on how Bioscience will generate major advances in our understanding of how molecular systems can support all properties associated with living organisms. The key to progress here was concluded to be to increase the number of significant inputs from the physical sciences, engineering, computation and mathematics, leading to powerful new quantitative and precise methods of analysis and far deeper insight into the fundamental principles of living systems. Introductory parts of the course explored how one can build up a comprehensive picture of a living system starting with principles of macromolecular structure and function, how molecules in living cells self-organise, how macromolecules assemble to form complexes, pathways and subcellular structures, how they function in pathways, how these are all networked together and how they are controlled and regulated. Emphasis was placed on the full range of quantitative techniques required to study bio systems.
Different approaches to capture the kinetics of pathways using mathematical representations such as biochemical systems theory and metabolic control analysis were presented and the analysis of rate control and regulation discussed. Particular consideration was given to the challenges for modelling posed by gene expression systems and macromolecular assembly pathways. Following on from this, it was shown how the design, modelling, construction and testing of man-made bio molecular systems can be developed from a thorough understanding of naturally evolved bio molecular systems. This unique course was of value to talented young PhD students and postdocs who are keen to engage with opportunities provided by these burgeoning interdisciplinary areas of research. More information is available on http://www.mib.ac.uk/spetses2010/index.html
In addition to courses and meetings, the interaction between TARPOL members resulted in the possible germ of new European research projects. Organisational meetings took place in Kolymbari and Vienna in order to set up a promising SB-based engineering of endosymbionts with medical applications. In the intersection among research, collaboration, training and dissemination, a TARPOL-linked iGEM team, the Valencia team, attended the international genetic engineered machine competition and got a gold medal with an innovative project on the implementation of a prion-based control circuit for tuning the Martian climate. The project was entitled 'Mad yeasts on Mars?' and contains the project in which the students presented an intermediate scenario in the pathway towards the terraforming of Mars. The idea is that, after preliminary changes devoted to make Mars conditions suitable for life, it can be colonised by microorganisms that will accelerate changes to make the planet conditions acceptable for plant life, which then will be able to generate enough oxygen to eventually allow the colonisation by animals and humans.
The project also included a human practices report with the largest inquiry on SB ever made, with more than 1000 interviews (see http://2009.igem.org/wiki/images/0/0d/Sins,_Ethics_and_Biology.pdf online). The iGEM 2009 research project, describing the first biological lightning display with aequorin-expressing yeasts as living pixels reported (see http://2009.igem.org/Team:Valencia; http://en.wikipedia.org/wiki/ILCD online) had, besides the recognition of the iGEM organisation committee, an unprecedented impact on international media. The success of European teams in the competition demonstrates that the combined effort of European universities and research centres may be able to change the fate of the geographic location of the excellence core on SB.
iGEM might be called the major educational success in the area of SB. In our estimate, the following elements in iGEM together provide an essentially 'irresistible' attraction to students. Given the fact that iGEM projects are a rather expensive form of education that does not (yet) mix well with more traditional educational programmes in the life sciences, the consortium suggests that the European Commission (EC) takes the funding of iGEM projects under consideration under the auspices of its research or education funding schemes.
UVEG has focused on training and theoretical development. But it is obvious that, in addition to the theoretical framework defining how a basic artificial cell should be, a real chassis is needed. In order to achieve this, BCR-HAS has carried out an applied work by developing core-genome E. coli strains to serve as host cells for various SB applications. Reduced complexity of the cells allows for more precise control/programmeming of cellular processes and for increased stability of engineered, synthetic genetic circuits. A set of bacterial strains is now available for use. In order to construct streamlined genome E. coli strains, up to 75 deletions were combined in a single genome, representing all together a 25 % genome reduction. This reduced genome approaches the core genome size predicted by comparative genomics for intestinal E. coli strains. The strain series was characterised for growth, transformation efficiency, mutation rate and other practical parameters. Milestone strains with best phenotypic characteristics were further modified for specific tasks. These include stable maintenance of normally unstable DNA constructs, and inducible, high efficiency recombinant protein expression. Strains with enhanced mutagenic capability, serving as host for in vivo mutagenesis, are also available.
Specific attention was paid to reduced-evolvability variants. Evolvability is an intrinsic feature of all living cells. Newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches are installed in the cell. BCR-HAS has shown that delayed genetic adaptation of reduced-genome host cells, devoid of all mutation-generating mobile genetic elements, improve maintenance of unstable genetic constructs. To further reduce the mutability of the host, point-mutation rates were significantly reduced by specific gene deletions. It was shown that various stress conditions, including recombinant protein expression, induce mutation-generating mechanisms to a high level in regular E. coli hosts. In contrast, the modified multi-deletional strains display low mutation rates, even under stress conditions. These minimalised, genetically stabilised strains are suggested to be beneficial hosts (SB chassis / engine) in both laboratory and industrial settings. Through re-sequencing and re-annotating Bacillus subtilis, a better comprehension of the limits between the paleome and the cenome has been achieved by IP. A genomic database for B. subtilis has been created by IP partner (BacilluScope), and another one is in process (SubtiliCyc).
Regarding advances in molecular assets, CSIC partner has developed a fully synthetic mini-Tn5 delivery vector in the first year and has also developed an application of one Pseudomonas putida strain for detection of 2,4 DNT in soil. During the second year, CSCIC has focused on synthetic genetic and molecular tools. The functioning of complex regulatory networks, or even a single gene, is revealed only when perturbations are entered in the corresponding dynamic systems and the outcome monitored. These endeavours rely on the availability of genetic tools to successfully modify à la carte the chromosome of target bacteria. Key aspects to this end include the removal of undesired genomic segments, systems for production of directed mutants and allelic replacements, random mutant libraries to discover new functions and means to stably implant larger genetic networks into the genome of specific hosts. The list of Gram-negative species that are appealing for such genetic refactoring operations is growingly expanding. However, the repertoire of available molecular techniques to do so is very limited beyond Escherichia coli.
Utilisation of novel tools is described (exemplified in two plasmids systems: pBAM1 and pEMG) tailored for facilitating chromosomal engineering procedures in a wide variety of Gram-negative microorganisms. The way that goes from genetically engineered microorganisms to synthetic involves the stepwise replacement of growing portions or their naturally occurring genomes by rationally designed and chemically manufactured DNA. Although the current ability to synthesise long genomic segments is in the range of 1 Mb, the contemporary level of knowledge does not allow assembling new activities or genetic circuits involving more than 20-30 kb of engineered DNA. It is thus likely that most SB endeavours of this sort will focus on handling a relatively short range of DNA sizes, whether for deletions from existing chromosomes, for genomic replacements of alleles by designed variants, or by straight implantation of new sequences. Numerous genetic tools exist in E. coli to this end but the state of affairs for other biotechnologically relevant Gram-negative bacteria such as Pseudomonas putida is far less satisfactory. Two strategies are described for implementing a large number of genetic manipulations in the chromosome of a large variety of Gram-negative microorganisms. P. putida was used as the target bacterium and the constructs named pBAM1 and pEMG adopted to give details of the underlying concepts and their practical application. These plasmids are tailored for implantation/insertion of heterologous DNA segments in the genome of the targeted strain as well as for directed mutagenesis or deletion or pre-specified chromosomal regions. Even though the procedures are different for each plasmid system, their utilisation share a good deal of the biological materials listed in the corresponding section below. The organisation and properties of pBAM1 make it suitable to be used either for creating saturated random transposon mutant libraries or for stably introducing gene networks or functional cassettes into the genome of a specific bacterial host. Both of these properties are due to the special characteristics of mini-transposons.
The pBAM1 plasmid is composed of four blocks. The first segment corresponds to the plasmid selectable marker, ampicillin. Next there is an R6K origin of replication that makes its maintenance dependent of the trans-supply of the p protein (pir gene). Thus, pBAM1 must be replicated in specialised E. coli strains, which expresses the p protein from a lysogenic phage, such as E. coli CC118lpir. A Tn5 transposase borne by the same plasmid (tnpA) recognises the end sequences of the mini-Tn5 transposon module (ME-I and ME-O) and catalyses the random motion of the mini-Tn5 cassette the target genome. All features have been individually optimised by CSIC, cured of the most common restriction sites present within its sequence, and then assembled and chemically synthesised de novo.
The pBAM1 frequencies of transposon insertions when applied to P. putida are in the range of 10-3 when the plasmid is delivered to the recipient by mating, or 10-7 when electroporation is used as an alternative method of suicide donation. On the other hand, the pEMG plasmid is used to generate directed scar-less deletions, as well as allelic replacements in the genome (Martinez-Garcia and de Lorenzo in preparation). This genetic system is a recreation of the method developed before for the same purpose in E. coli. The procedure is based on the homologous recombination forced by the appearance of double strand breaks (DSB) in the genome of the target bacterium upon cleavage in vivo by I-SceI, a homing endonuclease from Saccharomyces cerevisiae that recognises an 18-bp DNA sequence.
The I-SceI recognition sequence is not present in any of the microbial genomes sequenced so far, as revealed by blastn search against the 1379 completed (see http://www.ncbi.nlm.nih. gov/sutils/genom_table.cgi for details). In this way, integration of pEMG into the chromosome of choice endows flanking I-SceI target sequences to the extremes of a lacZ-alpha pUC19-based polylinker. Intracellular expression of the I-SceI enzyme in live bacteria is brought about in vivo by the cognate pSW plasmid. Transient expression of the nuclease is tightly controlled in pSW by means of the 3-methylbenzoate-inducible promoter Pm. The steps of the deletion strategy process include:
[i] cloning regions homologous to those flanking the desired deletion / replacement into pEMG;
[ii] cointegrating the resulting plasmid into the genome of the target host;
[iii] introduction of pSW into cells bearing the cointegrate;
[iv] induction of the DSBs;
[v] selection of the deleted/replaced strain and
[vi] pSW curation.
For this deletion/replacement process, the I-SceI expressing plasmid (pSW) can be introduced before or after obtaining the co-integrate. The delivery of the pEMG plasmid could be done either by mating or electroporation.
To summarise the CSIC's work, a synthetic plasmid composed of multiple formatted and optimised functional parts that behave as predicted -both individually and as an integrated system- has been created. Since the 1990s this is the first report that describes a fully edited genetic tool optimised and streamlined for its final applications rather than relying on cutting and pasting naturally occurring sequences. In short, non-functional DNA sequences were trimmed-off, common restriction sites present outside the multiple cloning site inside the mobile element were eliminated and the plasmid was designed following a modular pattern in which each business sequence was flanked by non-frequent restriction sites. In this respect, the key features of pBAM1 include not only the removal of many bottlenecks that flaw utilisation of its predecessors, but also the incorporation of a fixed standard for physical assembly and exchange of new DNA pieces while maintaining its overall layout. The modularity of the design and the origin of the parts enable pBAM1 for two specific applications.
The first is the exploitation of the cargo site to place a whole collection of extra genetic gadgets for expression of heterologous genes, reporter systems and environmental markers at user's will. The second is the possibility of cloning large DNA fragments inside the mobile element for a final implantation of new traits into the chromosome of the target strain. Given the randomness and the high frequencies of such insertions, one can then select the insertion out of a large collection, which adjusts expression of the desired feature to the right level under the desired operation conditions. The ease of replacement of the antibiotic resistance marker (or any other functional part) allows the same transposition/delivery system to be reused for subsequent insertions. This shows the value of DNA synthesis and standardisation of functional modules for combining valuable properties in a single genetic tool that are otherwise scattered in various vectors and rendered useless for the lack of fixed assembly formats. We anticipate pBAM1 to become a frame of reference for the construction of a large number of vectors aimed at deployment of heavily engineered genetic and metabolic circuits.
UMIL also focused on P. putida, particularly on optimising the genetic background of these bacteria through the abolishment of physiological bottlenecks to the expression of the desired phenotypes. UNIL is developing a site-specific integration system that will enable to insert large DNA fragments into the genomes of re-engineered bacterial strains with synthetic constructs. UNIL has already been successful in producing artificial integration sites, which are targeted with a higher efficiency than natural ones. Nowadays, these target sites carry a conditional switch that lets the cell produce green fluorescent protein when the DNA is integrated in the correct site.
The substantial increase of knowledge about the genetics, regulatory processes and metabolism of microbial forms of life obtained during the last years is remarkable. Such information is being included in large databases, many of them of free access. Once combined with the vast number of scientific publications, a huge volume of data is deposited in the hands of researchers. SB aims the partial design of organisms with specific functionalities by a rational usage of this information. There is much room for improvement concerning the available computational tools and the limitation in storage capacity, coherence and interoperability of the existing databases. This is a main target of the international efforts in bringing SB to a mature state, capable of producing highly reliable organisms capable of performing all kinds of useful tasks. The work carried out during this project has the potential of modifying the state of the art in this realm substantially.
One of the fundamental principles of SB is the construction of biological standardised parts and devices, which are interchangeable. A proper characterisation of these parts and devices appears as a key issue in order to make them reusable in a predictive way. In the recent past scientists have witnessed several initiatives towards the design and fabrication of synthetic biological components and systems as a promising way to explore, understand and obtain beneficial applications from live. For instance, in the post-genomic era one of the most fascinating challenges is to understand how the phenotypic behaviour of living cells arises out of the properties of their complex network of signalling proteins. While interacting biomolecules perform many essential functions in these systems, the underlying design principles behind the functioning of intracellular networks still remains poorly understood.
Several initiatives have been reported to uncover key working principles of such genetic regulatory networks via quantitative analysis of relatively simple, experimentally well-characterised, artificial genetic circuits. The desired performance of these synthetic networks and in turn the resultant phenotype is strongly dependent on the expression level of the corresponding genes, which is further controlled by several factors such as promoter strength, cis- and trans-acting factors, cell growth stage, the expression level of various RNA polymerase-associated factors and other gene-level regulation characteristics. Thus, one important ingredient to elucidate gene function and genetic control on phenotype would be to have access to well-characterised promoter libraries. These promoter libraries could be in turn useful for the design and construction of novel biological systems.
Recently a methodology (see http://partsregistry.org/Measurement for details) has been reported to characterise the activity of promoters in the Registry of Standard Biological Parts (see http://partsregistry.org for details) by using two different cell strains. UPVLC proposed the use of a synthetic gene regulatory network as a framework to characterise different promoter specifications by using a single-cell strategy. In this context characterisation stands for evaluating the parameters of a query promoter as compared to a standard promoter acting as a 'scale'. A proper promoter characterisation is an essential step towards realistic standardisation. Once this is accomplished, SB will arrive to a new stage where simplicity will allow massive design of organisms. This transition will provide society with powerful tools that could be employed to address several important goals such as the massive production of biofuels or the reduction of atmospheric pollution.
HZI developed ToBiN, a collection of computational tools for the genome-wide study of microbial physiology. Currently, the platform ToBiN contains several modules articulated in a way that allows them to go far beyond the exposure of annotation flaws and to reach a transversal view of the interaction's hierarchy, from regulatory circuits to host-pathogen relationships. Among the several components supplied by the platform, the one most likely to be used by the highest number of modules is a visualisation engine able to render a representation of the genome-wide physiological organisation of the cell. This visual component can be panned and zoomed in a Google Maps fashion and, whenever connected to modules generating data sets with values mappable to a particular compound, reaction or gene, is able to overlay graphical representations of selected quantitative and/or qualitative data sets. An example of the utility of the visualisation engine would be the perception of the correspondence between a metabolic flux distribution and transcription levels for the various pathways.
The automatic design tools developed by CNRS will be an important component in the development of a language for SB. They have also developed computational tools to be added as part of the SynBio toolbox. CNRS created a working group and a workshop series on computational frameworks and tools for SB with the PROTDES, Genetdes and Asmparts tools. Finally, they contributed Desharky to automatically design biodegradation pathways using a database of enzymes with the ad hoc developed tool. The Kegg database has been used as a proof of concept, but the tool could be adapted to any database.
The Imperial College Group has been setting up a repository of modelling frameworks for SB and developing an open-source system for collaborative tool development and problem solving in SB. Their study shows that the genetic circuit model effectively describes the function of the system and its dynamics, providing a solid basis for a system understanding of the metabolism of important pollutants, such as toluene and xylene. On modelling, GA contributed with the development of foundational tools and concepts in accordance with its technical expertise in DNA synthesis methodology. Within this task, a number of straightforward developments and applications have been initiated and existing processes and tools have been expanded and adjusted to the requirements of environmental and general SB, which have immediately been implemented into the company's technology platform to expand the service portfolio. Partner GA's tightened strategic gene synthesis market position provided access to many industrial, academic and governmental SB stakeholders, opening opportunities for a continuous dialog with different experts from diverse disciplines.
As illustrated by the comprehensive list of dissemination activities, addressing the scientific community, the industry sectors, civil society, policymakers and students, this dialog has been maintained and is further continued in order to address and influence social, ethical and regulatory issues as well as to trace potential market opportunities in environmental SB. The dissemination activities were mainly related to inform scientific and industrial researchers and developers about technological opportunities and applications to discuss regulatory and financial issues and for teaching purposes. In addition, the engagement in synthetic biology resulted in participation in and even foundation of interest groups (IGSC, SBIA, BioM-WB, DECHEMA work group) involved in regulation, cooperation, information, funding and other related activities on national and international levels. The topics addressed within this project and the activities initiated and concluded represent an integral part of the ongoing development of SB in Europe that is far from being finalised. The development of SB not only provides new and exciting opportunities and research potential for science, but it also represents a promising economical prospect. The results of this project directly contribute to the technological basis required to take advantage of this prospect, although the actual demand and market for commercial SB projects is limited.
Databases are an integral part SB. CNIO has developed the Bionemo database, which manually stores curated information about proteins and genes directly implicated in the biodegradation metabolism. When possible, the database also includes information on sequence, domains and structures for proteins as well as regulatory elements and transcription units for genes.
CNIO accomplished the creation of a corpus of document relevant to biodegradation metabolism and regulation. This task was extended for the generation of a bibliome consisting in all the articles relevant for any given bacterium. This methodology will be tested in the contexts of the MICROME project. A database containing all knowledge on biodegradation reactions was created and resulted in the Bionemo database. Bionemo can be accessed via the website http://bionemo.bioinfo.cnio.es The server implements a simple search interface that allows simultaneously querying all the biological entities described. Results are shown categorised by tabs representing classes containing the entity types. From the results page, the user can easily access entity-specific pages, in which all information available is summarised. Links to external databases, including the original UM-BBD metabolic information, GenBank and Uniprot, the NCBI Taxonomy database for microbial species, and the PubMeb references to the original information sources, are provided. In addition to the website access, Bionemo can be downloaded as a SQL dump and installed locally.
A Perl API (application programme interface) is provided (see http://bionemo.bioinfo.cnio.es/api.html for details). A REST service (a key design idiom that embraces a stateless client-server architecture in which the web services are viewed as resources and can be identified by their URLs) is integrated in the biological web service proxy (see http://code.google.com/p/bwsproxy/ for details). This is a free resource which main goal is to speed up the responses from different web services related with biology, bioinformatics and SB. The proxy catches several operations that highly demand computational resources. Finally, a MaDAS system was implemented. The main goal of MaDAS (see http://madas2.bioinfo.cnio.es for details) is to allow users to add their own annotations. A project is a unit that typically stores different annotations related to one genome or stores annotations related to a particular issue across several genomes. This is the case of TARPOL where biodegradation related annotations were collected in several microbial species. A Bionemo plug-in that connect MaDAS with the Bionemo database was created. Through this plug-in the annotations stored in Bionemo are available in MaDAS and can be retrieved in DAS format using the embed MaDAS DAS server.
The design of metabolic pathways for 'Biochemical building blocks' has been identified as foreseen applications in SB by IDC. Instead of searching for particular biomaterials or compounds, IDC has suggested to look for useful biochemical 'building blocks' and their potential to serve as a starting material for a larger group of derivatives. Many of those building blocks have excellent potential to compete with petrochemical equivalents, as many new products are possible with novel functionality or new applications.
TARPOL is contributing to the exchange of knowledge among European researchers working on SB through the organisation of several workshops and conferences. Among these activities, CNRS-IHPST organised a two-day workshop in Paris gathering historians, philosophers and biologists to evaluate the place of SB among other biological disciplines, and its novelty; HZI organised a special session on the topic computational design tool for synthetic biology within the BioPathways meeting in Stockholm; and UPVLC organised a symposium on SB titled 'III Jornadas Internacionales de Biologia Sintetica' and UVEG in coordination with HZI is organising a workshop to foster the discussion on minimal cells and its applications to take place next November. CSIC will organise the Fifth Meeting of the Spanish Network of Systems Biology in December 2009 on the topic of 'Fostering systems and SB in Southern Europe.'
Potential impact
Training and dissemination activities on SB are important in the project. UMIL has scheduled a course entitled 'Evolution and design of biomolecular systems: Concepts and strategies for systems and SB' and many dissemination activities have been carried out by partners. These include participation in scientific meetings, peer-reviewed articles, conferences to the general public, and science popularisation articles, among others. The website for the project (see http://www.sb-TARPOL.eu/ online) is under continuous update and contains public as well as private sections. UPVLC has participated intensively in the organisation of dissemination activities. Institute Cavanilles (UVEG) and Intertech Group of UPVLC co-organised the Summer Course on SB. The participation in dissemination activities not directly supported by TARPOL consisted of the preparation of two courses on SB and the organisation of two-day conferences during the project.
The ethical, human practices and safety issues have been approached in an unprecedented way. The US Presidential Commission for the Study of Bioethical Issues held a meeting on the topic of SB. The work performed on economic, environmental and ethical implications of SB applications in environmental biotechnology has been reviewed in a comprehensive report, which is one of the most complete reviews on the societal and environmental implications of a novel technology.
Summary
The report led by IDC and prepared in collaboration with partners (BU, UVEG, UNIVE, CNRS-ENS, IP and CEA) tries to give a glimpse into the future of SB and its potential applications in the area of environmental biotechnology. There are a number of applications where SB could make a difference in order to transfer our society to become more economical and environmental sustainable. In this report, we have highlighted four major areas with a total of 20 specific applications where SB has a great likelihood to improve currently available technologies. Each of the applications has been assessed in detail in order to find out:
(1) to what extend SB could improve current technologies;
(2) what the economic impact of SB could be;
(3) what the environmental benefits and downsides could be and
(4) whether any social or ethical problems would be created, exacerbated or improved.
This assessment is intended to support national and international funding agencies in their decisions to allocate resources to SB-based biotech applications while taking into account any foreseeable economic, environmental and social/ethical issues. Our outlook is based on the current scientific state-of-the-art. However, there is a notable degree of uncertainty about future development paths which we have to acknowledge when giving recommendations for what we see as the most promising directions for SB in environmental biotech.
Biofuels
We are convinced that SB can help produce state-of-the-art and next generation biofuels. Current efforts are mainly targeted towards an improved production of bio-ethanol from agricultural products, although we see significant problems with this approach as ethanol exhibits technical problems. Other non-ethanol biofuels such as bio-butanol or biodiesel are much better suited to replace petroleum-based gasoline as their chemical properties resemble it much closer. SB could help to overcome current impasses in the production of butanol and other non-ethanol fuels. One problem that is faced by most biofuels produced from plant material is the limitation of the use of hemi- and lignocellulosic material. Improvement in that area would definitely increase economic feasibility of biofuel production. The problem that will arise when SB will be able to provide a solution to the technical problems just mentioned is that more and more agricultural land will be devoted to plant energy-crops instead of food crops. In order to avoid this, we suggest to also use non-food-competing biological resources such as perennial plants grown on degraded lands abandoned from agricultural use, crop residues, sustainably harvested wood and forest residues, double crops and mixed cropping systems, municipal and industrial wastes.
Next to agricultural-based ethanol, biodiesel and butanol, there are algae-based biofuels and biohydrogen. Current concepts foresee a significant advantage of algae-based biofuels over agriculture-based biofuels because of higher yield per area and the independence of arable land, and clean water. First calculations predict that future algae production systems will only be economically feasible if the price for one barrel oil is constantly above USD 70 and if the production systems entails at least an area of 200 ha. The capital costs of such large production facilities will probably lead to an exclusion of SMEs and play in favour of 'big oil'. Still, algae production systems could be a highly promising avenue of future fuel production, once major obstacles are solved dealing with algae genomics, metabolism and harvesting. Although biohydrogen has been praised as an extremely promising fuel by scientists, our assessment is more cautious. Hydrogen is only useful as fuel if large changes in infrastructure take place. We point to a more distant future beyond 2050, also termed as the hydrogen economy. Although SB could well contribute to improve yield of hydrogen producing cyanobacteria, the actual impact of hydrogen in society and economy depends much more on other areas such as infrastructure. Finally we analysed the prospects of microbial fuel cells (MFCs) as energy converter. Although we see MFCs as extremely promising and an area where SB could contribute a lot, it will most likely be applied in some niche markets and areas of application, rather than large-scale deployment due to the limited energy production.
Bioremediation
Bioremediation is an area with great potential of benefits provided by SB. Bioremediation is usually applied on materials with a massive occurrence such as solid (organic) wastes, sewage, industrial wastewater, contaminated soil or contaminated ground water, measured in millions of tons or cubic meters. We believe that SB has the potential to create tools to improve the treatment methods, saving costs and environmental resources. Moreover, it can provide methods to produce energy or valuable goods from waste or wastewater. It can also provide tools for making up fresh or drinking water either from contaminated water or seawater. Another possible field of application is the production of biosensors to monitor environmental goods and hazards. At a differentiated evaluation, we have concluded that biosensors provided by SB-tools would have a great positive effect on the environment since they will help to survey environmental hazards more precisely and effectively. However, their economic and social impact is rather low because they can be considered as niche products.
SB-based approaches may provide a way of capturing, storing and recycling carbon dioxide. This may be through re-engineering of existing organisms or the creation of novel carbon processes especially using bottom-up approaches where inorganic chemistry is linked to living processes through agents such as the emerging proto-cell technology. SB-based carbon capture may not be able to sink carbon dioxide to completely remediate the current escalating levels that are being released through fossil fuel consumption, as geo-engineering scale approaches are necessary. But they can offer the possibility of carbon capture and recycling, which current industrial scale processes cannot do. It is recommended that because of the scale of the problem with carbon dioxide emissions and the urgent need for remediation, that SB approaches are supported in order to develop the next generations of carbon capture technologies which will not only store the carbon dioxide, but also recycle it into fuels and biopolymers with positive environmental impact.
Another positive impact, particularly to the environment, can be expected for soil and ground water remediation, especially with regard to the enhancement of the clean-up efficiency and to the development of new methods. On this field, the economic and social impacts are rather moderate, since it is a specific field with a limited scope of time.
The strongest impact we expect is for solid waste and wastewater treatment and for water desalination. The importance of the latter cannot be overstated in a world where billions of people have no access to clean drinking water or to fresh water for agricultural use. Solid waste and wastewater treatment also bear a great potential for improvement by SB due to their sheer amount and to their considerable organic content. We therefore strongly recommend supporting the development of these 3 issues. However, a possible constraint is that solid waste and wastewater cannot be treated in sealed vessels or rooms, due to their huge amount. They have to be treated openly in piles or basins. Therefore, the use of engineered cells may create a problem of interaction with the environment, which has to be kept in mind. But we expect no limitations for the use of non-proliferative systems like enzymes or proto-cells created with the aid of SB.
Biomaterials
SB will have a significant impact on the biomaterials market, particularly in the areas of fine chemicals and bio-plastics. A toolbox of products that will act as biodegradable materials is recommended. The bulk chemicals industry will be significantly affected by the SB-based technology, but uptake and environmental impacts will be slower. However, when new practices are adopted changes will last longer and take place on a much larger scale. In the fine chemicals industry incentives for investment relate to the economic potential of the end product (in contrast to bulk chemical manufacturing). The payoffs could predominantly have environmental impacts although these may be significantly limited to more efficient use of energy since the core manufacturing practice relies on petrochemicals. There is limited potential for SB-based techniques to have impact on avoiding recalcitrant molecules in the production process. Nonetheless, investment in SB-based processes in bulk chemicals is likely to have a positive overall impact on the manufacturing systems used in the running of the plant and because of the scale at which these processes take place, small changes may have significant positive environmental impacts.
For both fine and bulk chemical production we recommend the deployment of the Chemical Building Block System such as, or similar to, the USDoE. The field of biopolymers and bio-plastics most urgently needs revisiting in terms of its current labelling for recycling purposes since categorisation of the various products is extremely complex with negative economic consequences because of this (bio-plastics are not necessarily biodegradable). We recommend application of a method through which those bio-plastics that need recycling and those that can be composted are clearly recognisable, before large-scale use of SB for production of bio-plastics take place. An urgent need to develop completely biodegradable plastics exists, which would benefit from focused SB research and development into this area. Additionally there is a pressing need for high performance structural bio-plastics for manufacturing coupled with completely biodegradable additives. Both of these growth areas in the bio-plastics industry could be greatly improved by SB-based research.
Investment is particularly needed in research and development of new methods and products that will expand and develop tools and manufacturing processes with reduced environmental impact compared with the current manufacturing approaches although adequate biosafety issues on large-scale manufacturing units need to be established. Cellulosomes (complex molecules that degrade hemi- and lignocellulosic material) possess high economic potential for biofuels, paper and waste processing. SB has the potential to design more efficient and completely new cellulosome complexes to make new, efficient cellulose digesting proteins. Open sourcing of the cellulosome technology is recommended owing to the justice of distribution issues involved in the technology.
Novel developments in SB
Protocell technology represents a bottom-up approach to SB bridging inorganic and organic processes. Protocell technology enables better understanding of SB as a whole to develop new technologies. Although the research is in an early stage, the development of potentially radically novel and significant environmental interventions seem feasible. Investment in basic science to underpin the research and support it whilst imminent private investment is happening, is strongly recommended.
Protocell technology has a huge potential to offer radically different tools and methods than previously encountered with SB-based approaches because of its bottom-up nature and because of its overlaps with basic chemistry. A toolbox of potential products and investigation of issues related to open sourcing the technology should also be looked into. Xenobiology (also known as chemical SB) is another bottom-up approach to design and construct radically new biological systems with properties not found in nature. Using alternative base pairs to enlarge the genetic alphabet, or different chemical backbones in a xeno-nucleic acid, these chemically modified organisms and systems will enable a much higher level of biosafety when using engineered bio systems for or in the environment. Organisms with an enlarged genetic alphabet or a DNA with a different chemical backbone could be designed by SB in order to impede horizontal gene transfer and genetic pollution between engineered and natural organisms. Similar to protocells, xenobiology is at a very early stage of development and requires increased support for basic research in order to be able to achieve radically new concepts and applications.
Project website:http://sb-tarpol.org
[i] The new field is still missing a comprehensive language and a shared conceptual framework for the description of minimally functional biological.
[ii] Scientists and technicians, particularly in Europe, have so far failed to recognise their latent capacity to shape a brand-new discipline within their scope.
[iii] Promotion of SB might touch upon social sensitivities related to recreating 'life-in-the-test-tube', which threatens a re-enactment of the controversy stirred up by GMOs. TARPOL, led by the Universitat de Valencia and composed of 18 partners (see http://www.sb-tarpol.eu/ for details), has developed a dynamic two-year programme of activities, run by a large collection of European stakeholders.
TARPOL has been successful in recruiting environmental competences from neighbouring disciplines and developing numerous material and computational resources for advanced refactoring of biological systems. TARPOL has progressed in laying the foundations of SB, particularly on genome reduction strategies including a novel view of living organisms as information traps and contributed with dozens of basic, conceptual and applied publications. The consortium has paid attention to education. A range of conferences, dissemination and training activities have been organised, including the support to a successful iGEM team and the organisation of two intensive SB summer schools in Valencia (Spain) and Basel (Switzerland).
Mobilisation, interdisciplinary collaboration and training were the objectives concerning SB converge into a single goal: to set up a brand-new discipline which may have revolutionary effects on the lives of people in Europe. TARPOL has significantly contributed to the objectives as a key step to implement a powerful, sustainable knowledge-based bio-economy in our old continent.
Project context and objectives
The basic premise of SB is that methods commonly used to design and construct non-biological systems, such as those employed in computational sciences and engineering disciplines, could also be used to model and programme novel synthetic biological systems. SB is intrinsically trans-disciplinary and draws expertise from biology, chemistry, physics, computer science, mathematics and engineering. Synthetic biologists are attempting to develop 'artificial life', for both its tremendous applications in biotechnology and as a proxy for shedding light into the question of the origins of life. This is attempted by taking two separate and competing routes: the 'top-down' and 'bottom-up' approaches to minimal cells. In the former, a primordial or minimal cell is generated by systematically reducing a biological cell's genome until it no longer functions. The bottom-up methodology seeks to assemble from scratch components or information units until an aspect of life emerges. The overall intellectual and experimental challenges of implementing artificial life remain relatively long-term goals. However, guiding principles, experimental methodologies and theoretical insights from biomimetic chemistry and SB can be adopted in new ways for practical applications on a realistic, yet not necessarily immediate, time-frame.
Based on SB achievements, there is a possibility of building a living being, or a part of it, which either already exists or is a non-natural entity. This view on SB represents a promising applied field to areas as different as biomedicine, bioenergy, environment, etc. This type of programme requires a serious reflexion on the acquired responsibilities, as a consequence of the new natures that humans may create. In addition, SB demands a philosophical thinking on the panoply of futures that, more than ever, are close to man-made reality.
The main objective is to catalyse the shaping of the European SB community for translating this new conceptual and technical field into relevant environmental applications.
This goal comprises several specific objectives:
- to foster the transfer of SB conceptual fields into technical applications directed to solve environmental problems;
- to brand the conceptual and material interfaces between various disciplines required for surfacing and establishment of a European community on SB focused on tackling, monitoring and preventing environmental problems;
- to empower the SB environmental biotechnology field with material, technical and web based resources;
- to ensure that all research areas of SB are coordinated at European level so there is a maximum exchange of knowledge;
- to enable the development of a conceptual frame in which ethical and safety aspects of novel SB-related technologies, especially those related with environmental applications, can be optimally integrated into an emerging community;
- to promote scientific training and specialisation on SB, especially among scientists in early stages of their careers;
- to identify European research and development (R&D) needs and priorities on SB and to make recommendations for future cooperation areas and innovative research activities to be launched in the EU;
- to create awareness at academic, industry, social, policy and decision-makers level on the economic and scientific potential of SB for environmental applications.
Project results
We believe that the project has led to important advances in the conceptual field of SB. For instance, the state of the art of cell engineering in the context of genome research has been reviewed by UVEG during the first year, paying particular attention to what was learned on naturally reduced genomes from either symbiotic or free living bacteria. The identification of a genomic core for a cyanobacterium (Synechococcus elongatus) was accomplished by UVEG. This analysis will set the basis for designing a minimal photoautotrophic system suitable for a plethora of biotechnological applications. During the whole project, UVEG focused on the general coordination of the project, management, dissemination activities, and conceptual frame and consensual language definitions. The latter task has been broadly covered with a range of peer-reviewed contributions on the definition of the minimal genetic array for life to exist; the evolution of prokaryote-animal symbiosis; and theoretical limitations of synthetic life. Examples of this production are a review paper dealing with the milestones and challenges of synthetic biologists, co-authored by several TARPOL partners and coordinated by UVEG several book chapters on minimal cells and a myriad of publications on the origin of life, ethics of teaching evolution and a range of conceptual aspects of SB.
Dissemination and training activities organised by UVEG were key points of the project, with activities including a summer course on SB and the international meeting on synthetic and streamlined genomes. Thinking of SB as a new field of technology in the intersection of biology and engineering, the importance of attracting and training new researchers in this discipline becomes evident. With this objective, the Cavanilles Institute (UVEG) and the Intertech Group (UPVLC) coorganised the summer course on SB. This activity was developed from two sides: training and motivational. For this reason, the programme offered combined expertise of the organisers to train scientists from an interdisciplinary perspective in order to offer a range of activities for young researchers from a wide range of fields. The workshop on streamlined and synthetic genomes was organised as part of the TARPOL and EMERGENCE programmes to promote SB in Europe.
The workshop joined worldwide-recognised experts. The objectives were to assess the status, identify constraints and discuss the potential of research on minimal/streamlined genomes from different perspectives. This included the minimal and sufficient features of life, the study of naturally evolved reduced genomes, the engineering of minimal cells from bottom-up and top-down approaches, as well as various practical applications derived from research on minimal living systems. The workshop consisted of four sessions:
- Synthetic and digital biology, chaired by V. Martins dos Santos;
- Streamlining genomes, chaired by I. Economidis;
- Building genomes, chaired by G. Posfai; and
- Circuit design and evolution, chaired by V. de Lorenzo.
A satellite meeting on insect symbiosis in the era of systems and SB was held and allowed many of the attendants to join the community of synthetic biologists and discuss topics at the interphase between these closely related disciplines.
UVEG-led initiatives also served as a forum on which partners had the opportunity to discuss and prepare further research projects. We also participated in a course hold at Spetses (Greece) focusing on how Bioscience will generate major advances in our understanding of how molecular systems can support all properties associated with living organisms. The key to progress here was concluded to be to increase the number of significant inputs from the physical sciences, engineering, computation and mathematics, leading to powerful new quantitative and precise methods of analysis and far deeper insight into the fundamental principles of living systems. Introductory parts of the course explored how one can build up a comprehensive picture of a living system starting with principles of macromolecular structure and function, how molecules in living cells self-organise, how macromolecules assemble to form complexes, pathways and subcellular structures, how they function in pathways, how these are all networked together and how they are controlled and regulated. Emphasis was placed on the full range of quantitative techniques required to study bio systems.
Different approaches to capture the kinetics of pathways using mathematical representations such as biochemical systems theory and metabolic control analysis were presented and the analysis of rate control and regulation discussed. Particular consideration was given to the challenges for modelling posed by gene expression systems and macromolecular assembly pathways. Following on from this, it was shown how the design, modelling, construction and testing of man-made bio molecular systems can be developed from a thorough understanding of naturally evolved bio molecular systems. This unique course was of value to talented young PhD students and postdocs who are keen to engage with opportunities provided by these burgeoning interdisciplinary areas of research. More information is available on http://www.mib.ac.uk/spetses2010/index.html
In addition to courses and meetings, the interaction between TARPOL members resulted in the possible germ of new European research projects. Organisational meetings took place in Kolymbari and Vienna in order to set up a promising SB-based engineering of endosymbionts with medical applications. In the intersection among research, collaboration, training and dissemination, a TARPOL-linked iGEM team, the Valencia team, attended the international genetic engineered machine competition and got a gold medal with an innovative project on the implementation of a prion-based control circuit for tuning the Martian climate. The project was entitled 'Mad yeasts on Mars?' and contains the project in which the students presented an intermediate scenario in the pathway towards the terraforming of Mars. The idea is that, after preliminary changes devoted to make Mars conditions suitable for life, it can be colonised by microorganisms that will accelerate changes to make the planet conditions acceptable for plant life, which then will be able to generate enough oxygen to eventually allow the colonisation by animals and humans.
The project also included a human practices report with the largest inquiry on SB ever made, with more than 1000 interviews (see http://2009.igem.org/wiki/images/0/0d/Sins,_Ethics_and_Biology.pdf online). The iGEM 2009 research project, describing the first biological lightning display with aequorin-expressing yeasts as living pixels reported (see http://2009.igem.org/Team:Valencia; http://en.wikipedia.org/wiki/ILCD online) had, besides the recognition of the iGEM organisation committee, an unprecedented impact on international media. The success of European teams in the competition demonstrates that the combined effort of European universities and research centres may be able to change the fate of the geographic location of the excellence core on SB.
iGEM might be called the major educational success in the area of SB. In our estimate, the following elements in iGEM together provide an essentially 'irresistible' attraction to students. Given the fact that iGEM projects are a rather expensive form of education that does not (yet) mix well with more traditional educational programmes in the life sciences, the consortium suggests that the European Commission (EC) takes the funding of iGEM projects under consideration under the auspices of its research or education funding schemes.
UVEG has focused on training and theoretical development. But it is obvious that, in addition to the theoretical framework defining how a basic artificial cell should be, a real chassis is needed. In order to achieve this, BCR-HAS has carried out an applied work by developing core-genome E. coli strains to serve as host cells for various SB applications. Reduced complexity of the cells allows for more precise control/programmeming of cellular processes and for increased stability of engineered, synthetic genetic circuits. A set of bacterial strains is now available for use. In order to construct streamlined genome E. coli strains, up to 75 deletions were combined in a single genome, representing all together a 25 % genome reduction. This reduced genome approaches the core genome size predicted by comparative genomics for intestinal E. coli strains. The strain series was characterised for growth, transformation efficiency, mutation rate and other practical parameters. Milestone strains with best phenotypic characteristics were further modified for specific tasks. These include stable maintenance of normally unstable DNA constructs, and inducible, high efficiency recombinant protein expression. Strains with enhanced mutagenic capability, serving as host for in vivo mutagenesis, are also available.
Specific attention was paid to reduced-evolvability variants. Evolvability is an intrinsic feature of all living cells. Newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches are installed in the cell. BCR-HAS has shown that delayed genetic adaptation of reduced-genome host cells, devoid of all mutation-generating mobile genetic elements, improve maintenance of unstable genetic constructs. To further reduce the mutability of the host, point-mutation rates were significantly reduced by specific gene deletions. It was shown that various stress conditions, including recombinant protein expression, induce mutation-generating mechanisms to a high level in regular E. coli hosts. In contrast, the modified multi-deletional strains display low mutation rates, even under stress conditions. These minimalised, genetically stabilised strains are suggested to be beneficial hosts (SB chassis / engine) in both laboratory and industrial settings. Through re-sequencing and re-annotating Bacillus subtilis, a better comprehension of the limits between the paleome and the cenome has been achieved by IP. A genomic database for B. subtilis has been created by IP partner (BacilluScope), and another one is in process (SubtiliCyc).
Regarding advances in molecular assets, CSIC partner has developed a fully synthetic mini-Tn5 delivery vector in the first year and has also developed an application of one Pseudomonas putida strain for detection of 2,4 DNT in soil. During the second year, CSCIC has focused on synthetic genetic and molecular tools. The functioning of complex regulatory networks, or even a single gene, is revealed only when perturbations are entered in the corresponding dynamic systems and the outcome monitored. These endeavours rely on the availability of genetic tools to successfully modify à la carte the chromosome of target bacteria. Key aspects to this end include the removal of undesired genomic segments, systems for production of directed mutants and allelic replacements, random mutant libraries to discover new functions and means to stably implant larger genetic networks into the genome of specific hosts. The list of Gram-negative species that are appealing for such genetic refactoring operations is growingly expanding. However, the repertoire of available molecular techniques to do so is very limited beyond Escherichia coli.
Utilisation of novel tools is described (exemplified in two plasmids systems: pBAM1 and pEMG) tailored for facilitating chromosomal engineering procedures in a wide variety of Gram-negative microorganisms. The way that goes from genetically engineered microorganisms to synthetic involves the stepwise replacement of growing portions or their naturally occurring genomes by rationally designed and chemically manufactured DNA. Although the current ability to synthesise long genomic segments is in the range of 1 Mb, the contemporary level of knowledge does not allow assembling new activities or genetic circuits involving more than 20-30 kb of engineered DNA. It is thus likely that most SB endeavours of this sort will focus on handling a relatively short range of DNA sizes, whether for deletions from existing chromosomes, for genomic replacements of alleles by designed variants, or by straight implantation of new sequences. Numerous genetic tools exist in E. coli to this end but the state of affairs for other biotechnologically relevant Gram-negative bacteria such as Pseudomonas putida is far less satisfactory. Two strategies are described for implementing a large number of genetic manipulations in the chromosome of a large variety of Gram-negative microorganisms. P. putida was used as the target bacterium and the constructs named pBAM1 and pEMG adopted to give details of the underlying concepts and their practical application. These plasmids are tailored for implantation/insertion of heterologous DNA segments in the genome of the targeted strain as well as for directed mutagenesis or deletion or pre-specified chromosomal regions. Even though the procedures are different for each plasmid system, their utilisation share a good deal of the biological materials listed in the corresponding section below. The organisation and properties of pBAM1 make it suitable to be used either for creating saturated random transposon mutant libraries or for stably introducing gene networks or functional cassettes into the genome of a specific bacterial host. Both of these properties are due to the special characteristics of mini-transposons.
The pBAM1 plasmid is composed of four blocks. The first segment corresponds to the plasmid selectable marker, ampicillin. Next there is an R6K origin of replication that makes its maintenance dependent of the trans-supply of the p protein (pir gene). Thus, pBAM1 must be replicated in specialised E. coli strains, which expresses the p protein from a lysogenic phage, such as E. coli CC118lpir. A Tn5 transposase borne by the same plasmid (tnpA) recognises the end sequences of the mini-Tn5 transposon module (ME-I and ME-O) and catalyses the random motion of the mini-Tn5 cassette the target genome. All features have been individually optimised by CSIC, cured of the most common restriction sites present within its sequence, and then assembled and chemically synthesised de novo.
The pBAM1 frequencies of transposon insertions when applied to P. putida are in the range of 10-3 when the plasmid is delivered to the recipient by mating, or 10-7 when electroporation is used as an alternative method of suicide donation. On the other hand, the pEMG plasmid is used to generate directed scar-less deletions, as well as allelic replacements in the genome (Martinez-Garcia and de Lorenzo in preparation). This genetic system is a recreation of the method developed before for the same purpose in E. coli. The procedure is based on the homologous recombination forced by the appearance of double strand breaks (DSB) in the genome of the target bacterium upon cleavage in vivo by I-SceI, a homing endonuclease from Saccharomyces cerevisiae that recognises an 18-bp DNA sequence.
The I-SceI recognition sequence is not present in any of the microbial genomes sequenced so far, as revealed by blastn search against the 1379 completed (see http://www.ncbi.nlm.nih. gov/sutils/genom_table.cgi for details). In this way, integration of pEMG into the chromosome of choice endows flanking I-SceI target sequences to the extremes of a lacZ-alpha pUC19-based polylinker. Intracellular expression of the I-SceI enzyme in live bacteria is brought about in vivo by the cognate pSW plasmid. Transient expression of the nuclease is tightly controlled in pSW by means of the 3-methylbenzoate-inducible promoter Pm. The steps of the deletion strategy process include:
[i] cloning regions homologous to those flanking the desired deletion / replacement into pEMG;
[ii] cointegrating the resulting plasmid into the genome of the target host;
[iii] introduction of pSW into cells bearing the cointegrate;
[iv] induction of the DSBs;
[v] selection of the deleted/replaced strain and
[vi] pSW curation.
For this deletion/replacement process, the I-SceI expressing plasmid (pSW) can be introduced before or after obtaining the co-integrate. The delivery of the pEMG plasmid could be done either by mating or electroporation.
To summarise the CSIC's work, a synthetic plasmid composed of multiple formatted and optimised functional parts that behave as predicted -both individually and as an integrated system- has been created. Since the 1990s this is the first report that describes a fully edited genetic tool optimised and streamlined for its final applications rather than relying on cutting and pasting naturally occurring sequences. In short, non-functional DNA sequences were trimmed-off, common restriction sites present outside the multiple cloning site inside the mobile element were eliminated and the plasmid was designed following a modular pattern in which each business sequence was flanked by non-frequent restriction sites. In this respect, the key features of pBAM1 include not only the removal of many bottlenecks that flaw utilisation of its predecessors, but also the incorporation of a fixed standard for physical assembly and exchange of new DNA pieces while maintaining its overall layout. The modularity of the design and the origin of the parts enable pBAM1 for two specific applications.
The first is the exploitation of the cargo site to place a whole collection of extra genetic gadgets for expression of heterologous genes, reporter systems and environmental markers at user's will. The second is the possibility of cloning large DNA fragments inside the mobile element for a final implantation of new traits into the chromosome of the target strain. Given the randomness and the high frequencies of such insertions, one can then select the insertion out of a large collection, which adjusts expression of the desired feature to the right level under the desired operation conditions. The ease of replacement of the antibiotic resistance marker (or any other functional part) allows the same transposition/delivery system to be reused for subsequent insertions. This shows the value of DNA synthesis and standardisation of functional modules for combining valuable properties in a single genetic tool that are otherwise scattered in various vectors and rendered useless for the lack of fixed assembly formats. We anticipate pBAM1 to become a frame of reference for the construction of a large number of vectors aimed at deployment of heavily engineered genetic and metabolic circuits.
UMIL also focused on P. putida, particularly on optimising the genetic background of these bacteria through the abolishment of physiological bottlenecks to the expression of the desired phenotypes. UNIL is developing a site-specific integration system that will enable to insert large DNA fragments into the genomes of re-engineered bacterial strains with synthetic constructs. UNIL has already been successful in producing artificial integration sites, which are targeted with a higher efficiency than natural ones. Nowadays, these target sites carry a conditional switch that lets the cell produce green fluorescent protein when the DNA is integrated in the correct site.
The substantial increase of knowledge about the genetics, regulatory processes and metabolism of microbial forms of life obtained during the last years is remarkable. Such information is being included in large databases, many of them of free access. Once combined with the vast number of scientific publications, a huge volume of data is deposited in the hands of researchers. SB aims the partial design of organisms with specific functionalities by a rational usage of this information. There is much room for improvement concerning the available computational tools and the limitation in storage capacity, coherence and interoperability of the existing databases. This is a main target of the international efforts in bringing SB to a mature state, capable of producing highly reliable organisms capable of performing all kinds of useful tasks. The work carried out during this project has the potential of modifying the state of the art in this realm substantially.
One of the fundamental principles of SB is the construction of biological standardised parts and devices, which are interchangeable. A proper characterisation of these parts and devices appears as a key issue in order to make them reusable in a predictive way. In the recent past scientists have witnessed several initiatives towards the design and fabrication of synthetic biological components and systems as a promising way to explore, understand and obtain beneficial applications from live. For instance, in the post-genomic era one of the most fascinating challenges is to understand how the phenotypic behaviour of living cells arises out of the properties of their complex network of signalling proteins. While interacting biomolecules perform many essential functions in these systems, the underlying design principles behind the functioning of intracellular networks still remains poorly understood.
Several initiatives have been reported to uncover key working principles of such genetic regulatory networks via quantitative analysis of relatively simple, experimentally well-characterised, artificial genetic circuits. The desired performance of these synthetic networks and in turn the resultant phenotype is strongly dependent on the expression level of the corresponding genes, which is further controlled by several factors such as promoter strength, cis- and trans-acting factors, cell growth stage, the expression level of various RNA polymerase-associated factors and other gene-level regulation characteristics. Thus, one important ingredient to elucidate gene function and genetic control on phenotype would be to have access to well-characterised promoter libraries. These promoter libraries could be in turn useful for the design and construction of novel biological systems.
Recently a methodology (see http://partsregistry.org/Measurement for details) has been reported to characterise the activity of promoters in the Registry of Standard Biological Parts (see http://partsregistry.org for details) by using two different cell strains. UPVLC proposed the use of a synthetic gene regulatory network as a framework to characterise different promoter specifications by using a single-cell strategy. In this context characterisation stands for evaluating the parameters of a query promoter as compared to a standard promoter acting as a 'scale'. A proper promoter characterisation is an essential step towards realistic standardisation. Once this is accomplished, SB will arrive to a new stage where simplicity will allow massive design of organisms. This transition will provide society with powerful tools that could be employed to address several important goals such as the massive production of biofuels or the reduction of atmospheric pollution.
HZI developed ToBiN, a collection of computational tools for the genome-wide study of microbial physiology. Currently, the platform ToBiN contains several modules articulated in a way that allows them to go far beyond the exposure of annotation flaws and to reach a transversal view of the interaction's hierarchy, from regulatory circuits to host-pathogen relationships. Among the several components supplied by the platform, the one most likely to be used by the highest number of modules is a visualisation engine able to render a representation of the genome-wide physiological organisation of the cell. This visual component can be panned and zoomed in a Google Maps fashion and, whenever connected to modules generating data sets with values mappable to a particular compound, reaction or gene, is able to overlay graphical representations of selected quantitative and/or qualitative data sets. An example of the utility of the visualisation engine would be the perception of the correspondence between a metabolic flux distribution and transcription levels for the various pathways.
The automatic design tools developed by CNRS will be an important component in the development of a language for SB. They have also developed computational tools to be added as part of the SynBio toolbox. CNRS created a working group and a workshop series on computational frameworks and tools for SB with the PROTDES, Genetdes and Asmparts tools. Finally, they contributed Desharky to automatically design biodegradation pathways using a database of enzymes with the ad hoc developed tool. The Kegg database has been used as a proof of concept, but the tool could be adapted to any database.
The Imperial College Group has been setting up a repository of modelling frameworks for SB and developing an open-source system for collaborative tool development and problem solving in SB. Their study shows that the genetic circuit model effectively describes the function of the system and its dynamics, providing a solid basis for a system understanding of the metabolism of important pollutants, such as toluene and xylene. On modelling, GA contributed with the development of foundational tools and concepts in accordance with its technical expertise in DNA synthesis methodology. Within this task, a number of straightforward developments and applications have been initiated and existing processes and tools have been expanded and adjusted to the requirements of environmental and general SB, which have immediately been implemented into the company's technology platform to expand the service portfolio. Partner GA's tightened strategic gene synthesis market position provided access to many industrial, academic and governmental SB stakeholders, opening opportunities for a continuous dialog with different experts from diverse disciplines.
As illustrated by the comprehensive list of dissemination activities, addressing the scientific community, the industry sectors, civil society, policymakers and students, this dialog has been maintained and is further continued in order to address and influence social, ethical and regulatory issues as well as to trace potential market opportunities in environmental SB. The dissemination activities were mainly related to inform scientific and industrial researchers and developers about technological opportunities and applications to discuss regulatory and financial issues and for teaching purposes. In addition, the engagement in synthetic biology resulted in participation in and even foundation of interest groups (IGSC, SBIA, BioM-WB, DECHEMA work group) involved in regulation, cooperation, information, funding and other related activities on national and international levels. The topics addressed within this project and the activities initiated and concluded represent an integral part of the ongoing development of SB in Europe that is far from being finalised. The development of SB not only provides new and exciting opportunities and research potential for science, but it also represents a promising economical prospect. The results of this project directly contribute to the technological basis required to take advantage of this prospect, although the actual demand and market for commercial SB projects is limited.
Databases are an integral part SB. CNIO has developed the Bionemo database, which manually stores curated information about proteins and genes directly implicated in the biodegradation metabolism. When possible, the database also includes information on sequence, domains and structures for proteins as well as regulatory elements and transcription units for genes.
CNIO accomplished the creation of a corpus of document relevant to biodegradation metabolism and regulation. This task was extended for the generation of a bibliome consisting in all the articles relevant for any given bacterium. This methodology will be tested in the contexts of the MICROME project. A database containing all knowledge on biodegradation reactions was created and resulted in the Bionemo database. Bionemo can be accessed via the website http://bionemo.bioinfo.cnio.es The server implements a simple search interface that allows simultaneously querying all the biological entities described. Results are shown categorised by tabs representing classes containing the entity types. From the results page, the user can easily access entity-specific pages, in which all information available is summarised. Links to external databases, including the original UM-BBD metabolic information, GenBank and Uniprot, the NCBI Taxonomy database for microbial species, and the PubMeb references to the original information sources, are provided. In addition to the website access, Bionemo can be downloaded as a SQL dump and installed locally.
A Perl API (application programme interface) is provided (see http://bionemo.bioinfo.cnio.es/api.html for details). A REST service (a key design idiom that embraces a stateless client-server architecture in which the web services are viewed as resources and can be identified by their URLs) is integrated in the biological web service proxy (see http://code.google.com/p/bwsproxy/ for details). This is a free resource which main goal is to speed up the responses from different web services related with biology, bioinformatics and SB. The proxy catches several operations that highly demand computational resources. Finally, a MaDAS system was implemented. The main goal of MaDAS (see http://madas2.bioinfo.cnio.es for details) is to allow users to add their own annotations. A project is a unit that typically stores different annotations related to one genome or stores annotations related to a particular issue across several genomes. This is the case of TARPOL where biodegradation related annotations were collected in several microbial species. A Bionemo plug-in that connect MaDAS with the Bionemo database was created. Through this plug-in the annotations stored in Bionemo are available in MaDAS and can be retrieved in DAS format using the embed MaDAS DAS server.
The design of metabolic pathways for 'Biochemical building blocks' has been identified as foreseen applications in SB by IDC. Instead of searching for particular biomaterials or compounds, IDC has suggested to look for useful biochemical 'building blocks' and their potential to serve as a starting material for a larger group of derivatives. Many of those building blocks have excellent potential to compete with petrochemical equivalents, as many new products are possible with novel functionality or new applications.
TARPOL is contributing to the exchange of knowledge among European researchers working on SB through the organisation of several workshops and conferences. Among these activities, CNRS-IHPST organised a two-day workshop in Paris gathering historians, philosophers and biologists to evaluate the place of SB among other biological disciplines, and its novelty; HZI organised a special session on the topic computational design tool for synthetic biology within the BioPathways meeting in Stockholm; and UPVLC organised a symposium on SB titled 'III Jornadas Internacionales de Biologia Sintetica' and UVEG in coordination with HZI is organising a workshop to foster the discussion on minimal cells and its applications to take place next November. CSIC will organise the Fifth Meeting of the Spanish Network of Systems Biology in December 2009 on the topic of 'Fostering systems and SB in Southern Europe.'
Potential impact
Training and dissemination activities on SB are important in the project. UMIL has scheduled a course entitled 'Evolution and design of biomolecular systems: Concepts and strategies for systems and SB' and many dissemination activities have been carried out by partners. These include participation in scientific meetings, peer-reviewed articles, conferences to the general public, and science popularisation articles, among others. The website for the project (see http://www.sb-TARPOL.eu/ online) is under continuous update and contains public as well as private sections. UPVLC has participated intensively in the organisation of dissemination activities. Institute Cavanilles (UVEG) and Intertech Group of UPVLC co-organised the Summer Course on SB. The participation in dissemination activities not directly supported by TARPOL consisted of the preparation of two courses on SB and the organisation of two-day conferences during the project.
The ethical, human practices and safety issues have been approached in an unprecedented way. The US Presidential Commission for the Study of Bioethical Issues held a meeting on the topic of SB. The work performed on economic, environmental and ethical implications of SB applications in environmental biotechnology has been reviewed in a comprehensive report, which is one of the most complete reviews on the societal and environmental implications of a novel technology.
Summary
The report led by IDC and prepared in collaboration with partners (BU, UVEG, UNIVE, CNRS-ENS, IP and CEA) tries to give a glimpse into the future of SB and its potential applications in the area of environmental biotechnology. There are a number of applications where SB could make a difference in order to transfer our society to become more economical and environmental sustainable. In this report, we have highlighted four major areas with a total of 20 specific applications where SB has a great likelihood to improve currently available technologies. Each of the applications has been assessed in detail in order to find out:
(1) to what extend SB could improve current technologies;
(2) what the economic impact of SB could be;
(3) what the environmental benefits and downsides could be and
(4) whether any social or ethical problems would be created, exacerbated or improved.
This assessment is intended to support national and international funding agencies in their decisions to allocate resources to SB-based biotech applications while taking into account any foreseeable economic, environmental and social/ethical issues. Our outlook is based on the current scientific state-of-the-art. However, there is a notable degree of uncertainty about future development paths which we have to acknowledge when giving recommendations for what we see as the most promising directions for SB in environmental biotech.
Biofuels
We are convinced that SB can help produce state-of-the-art and next generation biofuels. Current efforts are mainly targeted towards an improved production of bio-ethanol from agricultural products, although we see significant problems with this approach as ethanol exhibits technical problems. Other non-ethanol biofuels such as bio-butanol or biodiesel are much better suited to replace petroleum-based gasoline as their chemical properties resemble it much closer. SB could help to overcome current impasses in the production of butanol and other non-ethanol fuels. One problem that is faced by most biofuels produced from plant material is the limitation of the use of hemi- and lignocellulosic material. Improvement in that area would definitely increase economic feasibility of biofuel production. The problem that will arise when SB will be able to provide a solution to the technical problems just mentioned is that more and more agricultural land will be devoted to plant energy-crops instead of food crops. In order to avoid this, we suggest to also use non-food-competing biological resources such as perennial plants grown on degraded lands abandoned from agricultural use, crop residues, sustainably harvested wood and forest residues, double crops and mixed cropping systems, municipal and industrial wastes.
Next to agricultural-based ethanol, biodiesel and butanol, there are algae-based biofuels and biohydrogen. Current concepts foresee a significant advantage of algae-based biofuels over agriculture-based biofuels because of higher yield per area and the independence of arable land, and clean water. First calculations predict that future algae production systems will only be economically feasible if the price for one barrel oil is constantly above USD 70 and if the production systems entails at least an area of 200 ha. The capital costs of such large production facilities will probably lead to an exclusion of SMEs and play in favour of 'big oil'. Still, algae production systems could be a highly promising avenue of future fuel production, once major obstacles are solved dealing with algae genomics, metabolism and harvesting. Although biohydrogen has been praised as an extremely promising fuel by scientists, our assessment is more cautious. Hydrogen is only useful as fuel if large changes in infrastructure take place. We point to a more distant future beyond 2050, also termed as the hydrogen economy. Although SB could well contribute to improve yield of hydrogen producing cyanobacteria, the actual impact of hydrogen in society and economy depends much more on other areas such as infrastructure. Finally we analysed the prospects of microbial fuel cells (MFCs) as energy converter. Although we see MFCs as extremely promising and an area where SB could contribute a lot, it will most likely be applied in some niche markets and areas of application, rather than large-scale deployment due to the limited energy production.
Bioremediation
Bioremediation is an area with great potential of benefits provided by SB. Bioremediation is usually applied on materials with a massive occurrence such as solid (organic) wastes, sewage, industrial wastewater, contaminated soil or contaminated ground water, measured in millions of tons or cubic meters. We believe that SB has the potential to create tools to improve the treatment methods, saving costs and environmental resources. Moreover, it can provide methods to produce energy or valuable goods from waste or wastewater. It can also provide tools for making up fresh or drinking water either from contaminated water or seawater. Another possible field of application is the production of biosensors to monitor environmental goods and hazards. At a differentiated evaluation, we have concluded that biosensors provided by SB-tools would have a great positive effect on the environment since they will help to survey environmental hazards more precisely and effectively. However, their economic and social impact is rather low because they can be considered as niche products.
SB-based approaches may provide a way of capturing, storing and recycling carbon dioxide. This may be through re-engineering of existing organisms or the creation of novel carbon processes especially using bottom-up approaches where inorganic chemistry is linked to living processes through agents such as the emerging proto-cell technology. SB-based carbon capture may not be able to sink carbon dioxide to completely remediate the current escalating levels that are being released through fossil fuel consumption, as geo-engineering scale approaches are necessary. But they can offer the possibility of carbon capture and recycling, which current industrial scale processes cannot do. It is recommended that because of the scale of the problem with carbon dioxide emissions and the urgent need for remediation, that SB approaches are supported in order to develop the next generations of carbon capture technologies which will not only store the carbon dioxide, but also recycle it into fuels and biopolymers with positive environmental impact.
Another positive impact, particularly to the environment, can be expected for soil and ground water remediation, especially with regard to the enhancement of the clean-up efficiency and to the development of new methods. On this field, the economic and social impacts are rather moderate, since it is a specific field with a limited scope of time.
The strongest impact we expect is for solid waste and wastewater treatment and for water desalination. The importance of the latter cannot be overstated in a world where billions of people have no access to clean drinking water or to fresh water for agricultural use. Solid waste and wastewater treatment also bear a great potential for improvement by SB due to their sheer amount and to their considerable organic content. We therefore strongly recommend supporting the development of these 3 issues. However, a possible constraint is that solid waste and wastewater cannot be treated in sealed vessels or rooms, due to their huge amount. They have to be treated openly in piles or basins. Therefore, the use of engineered cells may create a problem of interaction with the environment, which has to be kept in mind. But we expect no limitations for the use of non-proliferative systems like enzymes or proto-cells created with the aid of SB.
Biomaterials
SB will have a significant impact on the biomaterials market, particularly in the areas of fine chemicals and bio-plastics. A toolbox of products that will act as biodegradable materials is recommended. The bulk chemicals industry will be significantly affected by the SB-based technology, but uptake and environmental impacts will be slower. However, when new practices are adopted changes will last longer and take place on a much larger scale. In the fine chemicals industry incentives for investment relate to the economic potential of the end product (in contrast to bulk chemical manufacturing). The payoffs could predominantly have environmental impacts although these may be significantly limited to more efficient use of energy since the core manufacturing practice relies on petrochemicals. There is limited potential for SB-based techniques to have impact on avoiding recalcitrant molecules in the production process. Nonetheless, investment in SB-based processes in bulk chemicals is likely to have a positive overall impact on the manufacturing systems used in the running of the plant and because of the scale at which these processes take place, small changes may have significant positive environmental impacts.
For both fine and bulk chemical production we recommend the deployment of the Chemical Building Block System such as, or similar to, the USDoE. The field of biopolymers and bio-plastics most urgently needs revisiting in terms of its current labelling for recycling purposes since categorisation of the various products is extremely complex with negative economic consequences because of this (bio-plastics are not necessarily biodegradable). We recommend application of a method through which those bio-plastics that need recycling and those that can be composted are clearly recognisable, before large-scale use of SB for production of bio-plastics take place. An urgent need to develop completely biodegradable plastics exists, which would benefit from focused SB research and development into this area. Additionally there is a pressing need for high performance structural bio-plastics for manufacturing coupled with completely biodegradable additives. Both of these growth areas in the bio-plastics industry could be greatly improved by SB-based research.
Investment is particularly needed in research and development of new methods and products that will expand and develop tools and manufacturing processes with reduced environmental impact compared with the current manufacturing approaches although adequate biosafety issues on large-scale manufacturing units need to be established. Cellulosomes (complex molecules that degrade hemi- and lignocellulosic material) possess high economic potential for biofuels, paper and waste processing. SB has the potential to design more efficient and completely new cellulosome complexes to make new, efficient cellulose digesting proteins. Open sourcing of the cellulosome technology is recommended owing to the justice of distribution issues involved in the technology.
Novel developments in SB
Protocell technology represents a bottom-up approach to SB bridging inorganic and organic processes. Protocell technology enables better understanding of SB as a whole to develop new technologies. Although the research is in an early stage, the development of potentially radically novel and significant environmental interventions seem feasible. Investment in basic science to underpin the research and support it whilst imminent private investment is happening, is strongly recommended.
Protocell technology has a huge potential to offer radically different tools and methods than previously encountered with SB-based approaches because of its bottom-up nature and because of its overlaps with basic chemistry. A toolbox of potential products and investigation of issues related to open sourcing the technology should also be looked into. Xenobiology (also known as chemical SB) is another bottom-up approach to design and construct radically new biological systems with properties not found in nature. Using alternative base pairs to enlarge the genetic alphabet, or different chemical backbones in a xeno-nucleic acid, these chemically modified organisms and systems will enable a much higher level of biosafety when using engineered bio systems for or in the environment. Organisms with an enlarged genetic alphabet or a DNA with a different chemical backbone could be designed by SB in order to impede horizontal gene transfer and genetic pollution between engineered and natural organisms. Similar to protocells, xenobiology is at a very early stage of development and requires increased support for basic research in order to be able to achieve radically new concepts and applications.
Project website:http://sb-tarpol.org