What is Synthetic Biology? ...for advanced users

Synthetic Biology is an interdisciplinary research and technology field which strives to develop tools and methodologies that aid the design of biological systems and living organisms for useful purposes (Khalil & Collins, 2010). As opposed to traditional genetic engineering, synthetic biology places strong emphasis in engineering principles and methodology in the design and construction of biological systems (Church, Elowitz, Smolke, Voigt, & Weiss, 2014). In the past, biotechnology project designs tended to be ad hoc and this meant that there was little transfer of knowledge between projects. As a result, project timelines for the development of biotechnological applications were lengthy, time consuming and very costly. With synthetic biology, traditional molecular biologists, microbiologists and cell biologists come together with engineers, such as electrical and software engineers among others, to develop concepts and procedures relevant to the engineering of living matter. These concepts and procedures enable the realisation of project designs that build upon previous work as well as designs that allow for future work to build upon them (Endy, 2005).

Historically, the landmark work that established synthetic biology as a discipline was carried out at the beginning of the 21st century (Elowitz & Leibler, 2000; Gardner, Cantor, & Collins, 2000), but there were some earlier key discoveries and inventions that allowed for the emergence of the field. Such a key discovery was the elucidation of the regulatory mechanisms that allow a cell to respond to the perturbations and changes in its environment, and this was achieved by the study of the lac operon in E. coli (Jacob & Monod, 1961). Also, in the 1970s and 1980s the discovery of restriction enzymes and the development of molecular cloning techniques were central to the ability of scientists to produce recombinant/synthetic DNA for design purposes (Danna & Nathans, 1971; Roberts, 2005; Smith & Wilcox, 1970), and this was complemented by the invention of the polymerase chain reaction (PCR) that allowed the easy amplification and isolation of DNA sequences in molecular biology research (Mullis et al., 1987). Later, automated DNA sequencing and computational tools made whole microbial and eukaryotic genomes available for the scientist to use as a pool of available biological parts, diverse enough in functionality to carry out any purposeful task imaginable (Blattner et al., 1997; Fleischmann et al., 1995; Goffeau et al., 1996). At the same time, studies in the field of systems biology, that used computational tools to analyse myriads of interactions between cellular components, revealed hierarchical structures within cellular networks of biomolecules that although vast and “messy” resembled man-made engineered systems (Barabasi & Oltvai, 2004; Hartwell, Hopfield, Leibler, & Murray, 1999). As previously mentioned, in early 2000 two scientific papers were published in which the authors used engineering approaches to build genetic circuits that carried out a designed, purposeful function and now are widely regarded as marking the beginning of the field of synthetic biology. Both papers used mathematical modelling to describe and tune an interacting system of biomolecules, not normally encountered together in natural systems, which they viewed as a biological circuit to produce a toggle switch device (Gardner et al., 2000) and an oscillator device (Elowitz & Leibler, 2000). Following these initial publications, synthetic biology principles were widely adopted to produce biological circuits of genetically encoded, synthetic logic gates (Guet, Elowitz, Hsing, & Leibler, 2002), cell-to-cell communication systems (Tamsir, Tabor, & Voigt, 2011) and event counters (Friedland et al., 2009) among others. Notable achievements in the field include the heterologous production of antimalarial drug artemisinin from a metabolic pathway engineered in yeast instead of its low yield natural production from Artemisia annua plant (Ro et al., 2006) and the recreation of a viable Mycoplasma mycoides bacterial cell controlled entirely by synthesised DNA created chemically from digitized genome sequence information (Gibson et al., 2010). More recently, synthetic biologists have constructed the first designed eukaryotic chromosome in yeast (Annaluru et al., 2014) and efforts are on the way for constructing a designer yeast genome, while there has been great successes in the development of genome engineering and editing tools such as MAGE (Gallagher, Li, Lewis, & Isaacs, 2014) and CRISPR (Mali et al., 2013).

Although synthetic biology is mostly concerned with the design of biological systems and living organisms, the field is strongly influenced by engineering concepts such as standardisation and abstraction, and practises such as computer aided design (CAD). The reason for this influence is that such concepts and practises enable the scale-up of project design complexity and transfer of knowledge between projects. Standardisation is the process of developing and implementing technical standards or technical norms which allows for increased compatibility between projects developed separately. The iGEM Registry or Registry of Standard Biological parts is an example of the concept of standardisation as this applies to the field of synthetic biology. This particular registry promotes the use of a technical standard for the process of DNA assembly in which particular restriction digestion sites are found on each site of each biological part DNA sequence. These sites make use of a set of particular restriction enzymes to enable the assembly of biological parts into larger constructs. The adoption of this technical standard allows for cost reduction and efficient use of time. These benefits arise due to the fact that all registered biological parts can be assembled together using a small set of enzymes, and the circuit does not need to spend time to check or refactor DNA sequences of biological parts against assembly procedure incompatibility. Nevertheless, the popularity of iGEM standard is declining due to newer DNA assembly technologies -such as Gibson assembly (Gibson et al., 2009) and Golden Gate assembly (Engler, Kandzia, & Marillonnet, 2008)- that offer other procedure advantages and due to the affordable prices of large DNA fragments chemical synthesis. Abstraction is another concept adopted from engineering that allows managing complexity of very complicated systems, by dividing the system into levels of hierarchies. (Endy, 2005). A usual abstraction hierarchy used in synthetic biology includes the abstraction levels of “DNA sequence”, “Part”, “Device” and “System”. For example, a synthetic biologist with the task of designing an oscillator device will be working on the “Part” level of the hierarchy. The relevant information that the designer will have to consider are transcription factors (repressor proteins) with appropriate repression function characteristics; but the complexity of DNA sequence for the coding of the protein product or of the operator site functionality is hidden (or abstracted) from him, as this problem is dealt at the DNA sequence hierarchy level. Lastly, the field places a strong emphasis to the development and use of CAD tools. These tools can help reduce time and costs towards a functional prototype device by running test simulations in silico rather than the lengthier wet lab experiments. RBScalculator is one CAD tool that is widely used in the synthetic biology community and it allows the in silico design of ribosome binding sites to tune protein expression at the desired levels (Salis, Mirsky, & Voigt, 2009).

Synthetic biology is considered an emergent and enabling technology with applications in many sectors such as the healthcare, environmental protection and bioenergy (Synthetic_Biology_Leadership_Council, 2016). For the healthcare sector, designed biological systems could be used for drug discovery applications. In one such example, a chemical compound library was screened with the use of a drug responsive transcription factor that aided in the identification of an anti-tuberculosis drug (Weber et al., 2008). Additionally, designed biological organisms can be used to fight diseases, and one such application was demonstrated with the use of engineered therapeutic E. coli cells that invaded cancer cell lines when they encountered hypoxic environment or high cell density (Anderson, Clarke, Arkin, & Voigt, 2006). Another use of designed organisms in the healthcare sector is for the production of pharmaceutically active chemicals in a cost- efficient manner. This concept was demonstrated by the production of the anti-cancer drug taxol in engineered E. coli (Ajikumar et al., 2010) and by the production of the anti-malarial drug artemisinin in engineered yeast (Ro et al., 2006).

Environmental protection has attracted a lot of interest from synthetic biologists and a large number of applications have been demonstrated targeted for this sector. Engineered biological systems have been used to monitor the environment for pollutants, as in the case of engineered P.putida cells that utilised non-natural, directed evolution derived transcription factors that recognised nitrotoluenes for the purpose of tracing explosives in the soil (Garmendia, de las Heras, Galvao, & de Lorenzo, 2008). Furthermore, in addition to monitoring and measuring chemicals in the environment, designed organisms were engineered to degrade pollutants in order to clean up a polluted site, as in the case of an engineered E. coli that was designed to seek and destroy the atrazine herbicide (Sinha, Reyes, & Gallivan, 2010). Moreover, engineered organisms have been designed to relieve the strain put on the environment by human activity by producing commodity chemicals from renewable resources. For example, plastics produced from mineral oils (distillate of petroleum) have a multifaceted negative impact on the environment, and in order to try to address those problems a study designed the biomass crop switchgrass to produce a biodegradable chemical with plastic like characteristics (Somleva et al., 2008).

Synthetic biologists have also shown a strong interest in developing solutions for the bioenergy sector. High energy density alcohols, such as butanol and derivatives, have been produced using engineered cells that make use of feedstock from renewable carbon sources such as glucose (Atsumi, Hanai, & Liao, 2008), from non-edible plant parts (Higashide, Li, Yang, & Liao, 2011), or even from carbon dioxide (Atsumi, Higashide, & Liao, 2009).

In addition to providing practical solutions, the engineering of biological systems can be used to enable basic research by helping to elucidate the mechanisms of various biological phenomena (Cagatay, Turcotte, Elowitz, Garcia-Ojalvo, & Suel, 2009).

Text from Nicolas Kylilis PhD dissertation, 2017 "Synthetic biology biosensor design for medical diagnostics"