The U.S. has the biomass production potential to dramatically offset yearly petroleum consumption, but many efficiency barriers remain for developing enduring bioenergy sources. Syntheticbiology allows researchers to redesign energy-relevant organisms to increase the efficiency and lower the cost of bioenergy technologies. However, developing complex gene circuit behavior in new organisms or networks can result in unexpected complications and off-target effects. Since cellular structure and scale can affect gene expression dynamics, understanding how gene expression operates within the physiological context of the cell becomes important for developing robust gene circuits. Gene expression occurs in a highly crowded and confined (from about 1 fL to several pL) environment. Macromolecules occupy 5-40% of the intracellular environment, effecting changes in molecular transport, association, and reaction rates associated with gene expression. Gene expression also exhibits “bursty” patterns of expression, characterized by episodic periods of high activity between periods of low activity. These bursting patterns are shaped not only by molecular mechanisms but also by the global availability of resources within the expression environment, both of which may be further modulated by physical effects, like crowding and confinement. Since manipulating the physical conditions surrounding gene expression can be difficult to achieve in cells, cell-free systems are used to directly probe gene expression reactions. In this work, gene expression reactions in cell-free systems are modified to mimic physiological levels of crowding and confinement, revealing information about the interplay between expression bursting, resource sharing, and spatial ordering in
mechanisms. The protocol described here is originally based on Kigawa et al. (2004) and Liu et al. (2005), but has significant modifications. It utilizes Mg- and K- glutamate over Mg- and K- acetate for increased efficiency, removes 2-mercaptoethanol, and lyses cells using a bead- beater. 17,23,24 Bead-beating is chosen over homogenization, pressure-based methods, or sonication due to its lower cost and comparable yields to competing systems. 23 3-phosphoglyceric acid (3-PGA) is used as the energy source as it was found to give superior protein yields when compared to creatine phosphate and phosphoenolpyruvate. 4,25 Our system can produce up to 0.75 mg/ml of reporter protein using either a sigma70-based promoter with lambda-phage operators or a T7-driven promoter, similar to yields from other commercial systems. 4,6 Five days are required to produce all necessary reagents (Figure 1). Furthermore, it provides a 98% cost reduction compared to comparable commercial cell-free systems - material costs are $0.11 per 10 μl reaction, which rises to $0.26 with labor included (Figure 2).
An analogous system, termed RTRACS (reverse transcription and transcription-based autonomous computing system), that relied on reverse transcriptase, DNA polymerase, RNAP, and RNase demonstrated modularity and programmability [57,58]. The modules of RTRACS received specific RNA input sequences and produced an RNA output through programmed computation. Experimental operation of an AND gate was demonstrated with RTRACS, and the prospect of more complex functionality such as oscillations was reported. The polymerase exonuclease nickase (PEN) toolbox bypassed the transcription step and relied exclusively on DNA and DNA-modifying enzymes to construct desired circuits [59,60]. Single-stranded DNA templates served as network architecture and short ssDNA species took the role of dynamic species that functioned as activators and inhibitors of templates. Despite its simplicity, the PEN toolbox successfully demonstrated bistability [60,61], oscillations [59,60,62], and pattern formations  through rational design approaches and easy monitoring . An even more abstract approach is feasible with precisely programmed DNA sequences. Numerous studies demonstrated the power of DNA strand displacement circuits, including instructions, to create chemical reaction networks , logic circuits [66,67], neural networks [68,69], and oscillators  through toehold-mediated strand displacement . These theoretical and experimental developments will enable future works to further enhance the programmability and complexity of synthetic in vitro circuits to control nucleic acid nanorobots for in vivo applications . 4. RNA Regulatory Circuits for Cell-FreeSyntheticBiology
The de novo construction of cellular life requires, in part, the assembly of components that confer the ability to replicate. Herein we describe efforts to reconstitute parts of the Escherichia coli cell division machinery inside of water-‐ in-‐oil emulsion compartments and synthetic phospholipid vesicles. The system was built with DNA and purified transcription and translation machinery housed in a compartment. A particular emphasis was placed on FtsZ, a protein that oligomerizes into a ring at the midcell and splits the cell into two. FtsZ does not contain a membrane interaction domain. In vivo, FtsZ interactions with the membrane are mediated by FtsA and ZipA. Therefore, the influence of FtsA on the behavior of FtsZ also was investigated. Fluorescently tagged constructs were used to facilitate evaluation by microscopy. The data showed that FtsZ readily assembles into rings in the presence of FtsA, thereby suggesting that the Fts system can be exploited for building a genetically encoded, self-‐replicating, cell-‐ like system. We also explored additional methods of dividing compartments, such as the use of aqueous two and three phase systems.
Synthetic consortia have also been applied to other fields of bioprocessing, including bioremediation, detoxi- fication of byproducts of the chemical industry, food in- dustry and the synthesis of chemicals [106, 107]. In contrast to the intrinsic robustness of naturally occur- ring microbial communities, synthetic consortia often suffer from a lack of long-term stability that may prevent some of these approaches from entering industrial pro- cesses. Several strategies have been proposed to generate stable microbial communities, including the engineering of symbiotic interactions among the consortium members, e.g., by cross-feeding of essential nutrients [93, 94, 108] or by removal of deleterious metabolic waste products . Alternatively, external factors have to be applied, e.g., antibiotics [91, 92] or predefined oxygen tension , to maintain a synthetic consortium. Furthermore, spatial  or temporal [112, 113] separation as well as embedding of different species into biofilms  may provide tools to reach consortium stability. Alter- native approaches to control consortia have been re- ported, including flipping of DNA elements , fitness engineering of individual strains , or the use of synthetic toggle switches [117–119] that enable cells to switch between two alternative states in response to a
Autologous engineered cell therapies are a paradigm shift from conventional biologics such as pills, vaccines, small mole- cule inhibitor molecules, and antibodies in that the approach requires a patient-specific product. Some have dismissed adop- tive T cell immunotherapy as a fringe or boutique therapy that would be impossible to commercialize ( Baker, 2011 ). Indeed, several challenges must be overcome before this disruptive ther- apy can become broadly applicable and widely available. The barriers that we currently perceive fall into two areas. First, the cell culture systems must be robust and reproducible. The T cell engineering process that we and others have developed requires complex logistics. Some of the variables that need to be standardized in order to scale this out for widespread use include developing a leukapheresis network, standardizing and scaling up the manufacturing of lentiviral vectors, and devel- oping validated cell-shipping and chain-of-custody procedures. For example, the cell culture media that will be used for commer- cial scale must be serum free because there is an insufficient supply of bovine or human-derived serum to support large-scale manufacturing ( Brindley et al., 2012 ). Second, personalized cell therapies cannot become widely available if the cell culture process requires extensive manipulation by highly skilled scien- tists and technicians ( Mason and Manzotti, 2010 ). Therefore, automated culture systems need to be developed. There is pre- cedent in the automotive industry, where cars were initially man- ufactured in assembly lines, but manually. Today’s automobiles are assembled largely by robots and other forms of automation ( Michalos et al., 2010 ). As engineered T cell processing becomes more automated, cell products will be produced for greater num- ber of patients more efficiently. Given the recent entry of the
NEBExpress ® Cell-free E. coli Protein Synthesis System The NEBExpress Cell-free E. coli Protein Synthesis System is a coupled transcription/ translation system designed to synthesize proteins encoded by a DNA or mRNA template under the control of a T7 RNA Polymerase promoter. The system offers high expression levels, the ability to produce high molecular weight proteins, scalability, and is cost-effective for high throughput expression applications. The speed and robustness of the system facilitates protein synthesis in applications such as protein engineering, mutagenesis studies and enzyme screening.
In summary, we have developed, in two different systems, the ﬁrst synthetic biological generation of new-to-nature bromo-metabolites and their synchronous cross-coupling within a living aerated microbial fermentation, even at high dilution. As many natural products are generated at low concentration, this is essential. This pioneering approach and exceptionally mild conditions for Suzuki–Miyaura cross-coupling of challenging halo-metabolites have the potential to be broadly used for the GenoChemetic diversiﬁcation of halogenated natural products and new-to-nature halo-natural products. This approach is powerful as it enables the generation and diversiﬁcation of a natural product in the presence of the cells that produce it. Being able to perform this chemistry in the presence of living cells at 28 or 37 °C, including on as challenging a system as free halo-tryptophan, opens up new possibilities, for example: assessing and screening, in vivo, the directed evolution of the biosynthetic enzymes like halogenases or multifunctional enzymes for example polyketide synthases (PKSs) and non- ribosomal peptide synthetases (NRPSs). In such directed evolu- tion studies, being able to assay in vivo is time efﬁcient and maintaining the viability of the cells removes need for duplicate plating—a time consuming process when handling the large numbers of colonies required for such systems. Recently, Sewald reported the use of the ﬂuorescence modulating cross-coupling chemistry, which we have previously developed 25 , as a screen for the directed evolution of a thermophilic halogenase 43 . The cell compatible chemistry developed here has the potential to signiﬁcantly open up this area, making this approach applicable to aryl halogenases that do not originate from thermophiles. One might also envisage utilizing the combination of syntheticbiology and synthetic chemistry, which results in a change in ﬂuorescence to enable cell sorting of particularly productive clones 25 . A further advantage of carrying out the chemistry in the presence of the living cells is that, with the provision of additional media, the cells can continuously produce their halo-metabolites, such an approach could potentially be applied to bioﬁlms in ﬂow, or possibly have dual utility in diversiﬁcation and increasing metabolite ﬂux 44 . From a technical viewpoint the ability to carry out the diversiﬁcation without an additional puriﬁcation step, in the presence of a continuously producing biosynthetic system is attractive. Furthermore, we ﬁnd that the enhanced lipophilicity that the cross-coupling diversiﬁcation confers, signiﬁcantly simpliﬁes the otherwise challenging extraction and puriﬁcation from other media components.
SB is a rational attempt to understand the basic concepts of this apparent complexity. Using an engineer’s view in biology, SB designs biological devices (synthetic cells or cell-free system) to trigger a biological response with respect to input controlled signal (Figure 1). In DD, such devices would be used to activate gene expression of biosynthetic units to explore NP-like chemical space. In-cell synthesis has the advantage to make use of natural evolution to create compounds compliant to biological environment, which is part of the lead optimization process. Genome editing tools give the possibility of following through reporter genes the action of a particular output signal, which is very useful to validate drug target or disease models as well as merging con- straints from both DD and drug production. This “rational- based biosynthetic drug design” 34 approach is somehow the
There is rich diversity of CF systems, reaction modes, and preparation techniques that provide many options for applying CF-synbio. For example, there have been considerable well- described iterations towards streamlining preparation of Escherichia coli-based CF extract in the last 30 years [61,62] . Some preparations also facilitate special applications, such as S150 extracts that are free of ribosomes  . A lingering difﬁculty of preparing extracts is consistency of extract viability, which can be affected by cell growth rate, harvest time, gene content, media composition, and growth temperature. We have made recent steps towards optimizing these variables on individual systems, yet gen- eral fundamental understanding of the impacts of these variables is not fully characterized  .
From the early days of syntheticbiology, it was clear that there was great potential for synergy with the field of chemical synthesis. Metabolic pathways responsible for the synthesis of valuable compounds (e.g., drugs, scents, and flavors) were thus moved out of organisms that did not easily lend themselves to production and into heterol- ogous hosts, such as yeast. This microorganism-based ap- proach has been incredibly successful and has led to the assembly of genes from disparate sources to create engi- neered pathways. Enzyme-based catalysis has the advan- tage of allowing for stereo-selectivity in aqueous, low- energy reactions (e.g., green chemistry) . By lever- aging naturally occurring pathways, it has been possible to generate tremendous chemical diversity, as seen in isopre- noids, from simple precursors . An exemplar of this approach is the synthesis of amorpha-4,11-diene and arte- misinic acid, which are precursors to the anti-malarial compound artemisinin [154–157]. This process has been repeated for other pharmaceutical pathways, enabling the production of opioids [158, 159] and taxol , as well as for the generation of molecules for the energy industry and the agriculture sector [13, 161].
As synthetic biological systems have become larger and more complex, deciphering the intricate interaction of synthetic systems and biological entities becomes a challenging task. Cell-freesynthetic biological approaches, with the aid of rapid progress in its scope, and toolkits may provide the right platform for rapid design–build–test cycles. New technological breakthroughs for syntheticbiology, such as CRISPR-Cas systems, can also be elucidated in this simplified TXTL test bed . The ease with which to program nucleic acids has dramatically accelerated the structural and functional complexity of nucleic acid-based molecular devices. These new developments encompass simplified synthetic model dynamical systems and nucleic acid nanostructures, as well as synthetic RNA regulatory components, which form the core of practical tools for biomedical applications. Compartmentalization for synthetic cells opens up ways for scientific inquiry and enhanced functionality through networks of synthetic and natural systems. Data-driven model building needs to guide the research and development towards complex synthetic systems with prescribed dynamics in the future. In the coming years, we anticipate that the utility of cell-freesyntheticbiology will rapidly expand the scope of biotechnology and syntheticbiology, and it will provide innovative solutions in biomanufacturing therapeutics for biomedical applications and biologic products for industrial applications.
Streptomyces cell-free, the recently developed high-yielding Streptomyces lividans
and Streptomyces venezuelae host platforms [59,62] can potentially provide an
opportunity to access high G+C (%) enzymes from secondary metabolism directly within a test-tube for combinatorial biosynthesis. With further advances in efficiency and yield, Streptomyces cell-free could be used for incorporating non-natural or potentially toxic substrates into natural products, thus expanding the chemical space of biosynthesis. A proof of concept of how cell-free can be used to incorporate non- natural amino acids into protein backbones was demonstrated for creating modified forms of the model protein GFP in E. coli cell-free  . Whilst this technology is in its
Stochastic Petri nets are an established concept for performance and depend- ability analysis of technical systems, see [MBC + 95], [BK02], recently extended by probabilistic model checking [DDS04]. An excellent textbook for numerical solution of Markov chains is [Ste94]. An overview on stochastic issues for systems biology is given in [Wil06]. The approximation of continuous behaviour by the discretisation of species’ concentrations by a finite number of levels has been proposed in [CVGO06]. The application of stochastic Petri nets to biochemical networks was first proposed in [GP98], where they were applied to a gene regu- latory network. Further case studies are discussed in [SPB01], [MSS05], [ST05], [SSW05], [Cur06]. A precise definition of biochemically interpreted stochastic Petri nets has been introduced in [GHL07b].
• Invest in a network of multidisciplinary centres to establish an outstanding UK syntheticbiology resource
• Build a skilled, energised and well-funded UK-wide syntheticbiology community • Invest to accelerate technology responsibly to market
expression memory has been synthetically recapitulated 53,66,70 and could be used to record and respond to environmental events to which cells are subjected, such as exposure to antibiotics, heat shock or toxins, or changes in cell density in a bioreactor (FIG. 4Ab). Finally, given the many ways to now target, modulate and modify chromatin, these approaches are likely to be used to correct disease-associated chromatin states in cancer, neurodegenerative diseases and many other conditions, or to artificially alter chromatin changes that are involved in developmental and differentiation pathways (FIG. 4B). Given that several epigenetic therapeutics, typically small-molecule inhibitors of chromatin- modifying proteins, have already been used in the clinic 86 , more-specific strategies derived from syntheticbiology approaches to intervene in epigenetic states could become promising next-generation therapeutic avenues in the near future. All of these types of applications are theoretically only as far from the clinic or the pilot plant as our ability to integrate them into existing syntheticbiology frameworks. Initial applications of chromatin-based regulation in industry are likely to augment and enhance existing synthetic systems and organisms, and they could be closer to implementation than biomedical applications. Applications in biomedically relevant cell types will be slower to realize, as they are limited by our understanding of the role of chromatin in cellular regulation, as well as by the higher standard for off-target effects amid worries of the oncogenic potential of artificial factors. However, synthetic approaches will probably contribute substantially to our understanding of biomedical problems and present novels ways to address them.
Listeria has been extensively studied genus of bacteria that promotes direct cell killing of breast tumors and offers efficient bactofection . Sun et al. demonstrated that attenuated Listeria monocytogenes (LM)–based vaccine completely eliminates the metastases and induce regression of primary tumor in 4T1 aggressive mouse breast tumor model . This anti tumor activity has been thought to be mediated by two mechanisms; the release of excessive ROS level in tumor microenvironment and the generation of cytotoxic T lymphocytes response against Listeria antigens which these both subsequently promote direct cell lysis and tumor killing . In another report involving in vivo breast cancer model, a well-defined attenuated Listeria construct ADXS31-164 was found to be capable in breaking immune tolerance towards the HER2/neu self-antigen, which known to be overexpressed in breast tumors . Meanwhile, the affibody displayed on the surface of engineered E. coli BLD21 (DE3) resulted in the internalization of this strain into HER2/neu-positive SKBR-3 breast cancer cells . This non pathogenic tumor-targeting bacteria also carried phage fX174 lysin gene E-mediated autolysis system which promotes autolysis once successfully enter the tumor cells . This demonstrated the use of E. coli as an alternative gene therapy.
sensors to couple metabolite, metals, nanoparticles, toxins and other environmental or biomedical relevant molecules to sev- eral outputs and easier to detect .
But, what does syntheticbiology do? Using a broad defini- tion, syntheticbiology is in the quest to simplify our under- standing of biological entities (viral, bacterial and eukaryal organisms) by constructing biochemical or genetic pathways both in vivo and in silico and building up computational mod- els to simulate the behavior of those pathways with the ulti- mate goal of testing them in the real world  (Fig. 3). Syn- thetic biology also attempts to generate genetically recoded organisms or biological entities by using a design process more systematic and predictable and by analyzing models that use all the data available for that particular engineered process, robustness in the output of the designed organism, scalable to different niches or conditions, and ideally, more efficient than the wild type counterpart . These criteria cover basically the necessities for applied genetic engineering with the easy approach of designing modules or parts to do specific tasks, which in a complete organism is still unpredictable at a large scale due to problems of interacting protein networks or en- zyme cascades that are self-regulated or that interact with oth- er pathways.
Consistent with the postulate that past scientific achievements lay the foundation for future innovation, the results of the survey showed that many of the tech- nologies that enable research in syntheticbiology are well established and in the public domain. For many of these earlier technologies patent protection was either not sought or, even if patent protected, sufficient time has lapsed for the technologies to enter the public do- main. For example, the vast majority of survey respon- dents reported use of bacterial cell culture technologies such as LB medium or glycerol freezing (Figure 5), yet these technologies were published in the scientific litera- ture as early as the 1950’s [37-39] and are squarely in the public domain. Similarly, the vast majority of survey respondents reported use of PCR technology, yet ele- ments of PCR technology have entered the public domain or will do so shortly. Specifically, foundational patents covering amplification methods (e.g., US 4,683,195 and EP 0 200 362 B), thermal cycling instru- ments (e.g., US 5,038,852 and EP 0 395 736 B), and thermostable DNA polymerases (e.g., US 4,889,818 and EP 0 258 017 B) have now expired. Although patents continue to be filed on improvements to PCR technolo- gies, many subsequent patents such as those covering thermostable polymerases with enhanced activities (e.g.
In addition, the machine metaphor fits neatly into a larger story of what syntheticbiology is and what it is aiming at. Following this story, syntheticbiology constitutes the latest step along the line of developing scientific bottom-up explanations of macro-objects and their behavior (Church and Regis 2012). At the lowest level, physics analyzes the movement and structure of atoms by identifying and analyzing subatomic structures and parts. At the next level, chemistry analyzes complex molecules by scru- tinizing the simpler molecules and atoms of which complex structures consist. At yet another level, living molecules, which is to say organisms, become the objects of ana- lysis. This is the realm of “ analytic ” molecular biology research, which traces the behav- ior of organisms back to their inner molecular genetic structures. At each level, analytic knowledge allows one to intervene technologically, alter the objects in question and devise novel objects. This is the “ synthetic ” side of each analytic science. In chemis- try, for example, naturally occurring compounds such as sugar or vitamins can be pro- duced synthetically and, in addition, compounds not known from nature, such as plastics, can be synthesized. Syntheticbiology, it is expected, will lead to similar devel- opments with regard to organisms. Synthetic biologists will be able to rebuild naturally occurring organisms and to create novel organisms by means of DNA synthesis (Kas- tenhofer 2013).