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The mainstream fronts of Synthetic Biology: Guest post

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This is a guest post from M. A. Loera Sánchez from the iGEM team UANL 2012. I have carried out a few small grammar edits but otherwise the essay is all his work, and I would like to thank him for the opportunity to host it on my blog. All references are below the main text.

The mainstream fronts of synthetic biology

“What I cannot build, I cannot understand”.

This phrase by the genius physicist Richard Feynman is cleverly encrypted into the genetic code of the first bacterial cells with an artificial genome that have ever existed.

Actually the quote says “what I cannot create…”, but maybe the scientists at the JCVI –who are behind this tremendous breakthrough- preferred to save some base pairs to avoid the use of the word “create” and its tricky implications. They published this work in 2010 and opened a whole new world of possibilities and made it completely clear to anyone what we mean when we talk about Synthetic Biology and what its ultimate purpose should be: to understand life by building it.

Although the term “Synthetic Biology” has been around since the mid-1970s, the definition of it has been very vague: some people would call Synthetic Biology anything related to general genetic engineering procedures; others, perhaps more rightfully, would claim to be doing Synthetic Biology because of working with DNA synthesis or making bacteria behave like tiny computers. Even the 2010 report by the US Presidencial Comission for the Study of Bioethical Issues has to define the term considering different points of view (that of the molecular biology, the chemist and the engineer) and states that the activities related to Synthetic Biology are considered by some to be just extensions of already existing fields, like Molecular Biology, Genetic Engineering and Microbiology.

I remember (oh, the shame!) being skeptic about the possibility of something so oxymoronic being, well… true. I still turn red when I recall that I kind of corrected the person who first said “Synthetic Biology” to me by telling her that what she wanted to say was maybe Systems Biology.

So what is it really?

Well, my work in Bio! has been devoted to dig into the deeps of Synthetic Biology and the iGEM competition, and throughout this time I began to notice what I would call “the mainstream fronts of Synthetic Biology”. These are the main orientations that so called Synthetic Biology projects would take and by enlisting them, I think it will be easier to clarify the distinctive characteristics of this field.

Front 1: DNA synthesis

The development of DNA synthesis techniques allows us now to synthesize DNA sequences larger than just PCR oligos; genes and genomes are being fully synthesized; difficult constructions are eased by just synthesizing the needed sequences, without needing sub-clonations to gain a useful restriction site. One of the most awesome accomplishment is the generation of the first replicating cells with a fully synthetic genome, which by the way was dubbed “Synthia”. Moreover, one of the most amazing applications of synthesized DNA has little to do with living cells: they are used as mechanical pieces, because after all DNA is a physical entity and can be used as such, giving place for the field of DNA Nanostructures.

A particular technique called “DNA origami” has been really fruitful (ref1) Since the publication of its invention in 2006, DNA origami custom structures have been employed as mechanical and computational devices; there is software available for generating the DNA sequence needed to construct a wanted shape. More recently, DNA origami structures have been employed as intelligent devices that recognize and deliver drugs to cancerous cells.

Front 2: The standardization school and the iGEM competition

The iGEM logo

One of the characteristics of gene cloning is the messy and sui generis protocols that vary from lab to lab and from construction to construction. What would happen if these procedures were standardized, so that any construction needed could be accomplished by anyone with the same steps and same restriction enzymes?

A group at MIT has been dedicated for the last years to standardization in the biological field. The main drive is to accelerate the development of Biology by mimicking the development ofthe engineering fields, i.e. to have a consensus analogous to what mechanical engineers have with the size of materials and tools. Moreover, if we are using standard parts, the quantitative characterization of them would be also facilitated, making mathematical models and simulations useful as predictive tools.

Accordingly, there is the Registry for Standard Biological Parts, which is basically a collection of DNA sequences that share a particular set of rules that ease their combination with each other. Each of these sequences is a biological part and among the existing parts are promoters, ribosomal binding sites, coding genes and also composite parts made of the combination of single parts; the parts are also referred as “BioBricks” and the organism in which they are meant to work is termed the “chassis”.

The fundamental characteristic of BioBricks is the set of rules that ease their combination; these rules allow the concatenation of DNA sequences by iteratively using the same restriction enzymes in the same order. The basics of this technique are the design of idempotent vectors and were first presented by the Knight group at MIT.

The huge potential of the use of BioBricks is reflected by the number of awesome and creative projects that have been presented at the International Engineered Machine competetion (iGEM), which began at the MIT too. In this competition, undergraduate students are challenged to make the most innovative biological machine by using BioBricks. Among the projects that have been presented since the first iGEM edition are biosensors, bioremediators, light-controlled cells, metabolically engineered cells, synthetic genetic circuits and many others, along with their corresponding mathematical models. Students are also encouraged to make a contribution for the humanities side of the project.

Front 3: Genetic code expansion

There are many amino acids that would give proteins many useful characteristics, but in nature, only twenty amino acids are commonly encoded by the 64 codons found in living beings, plus some organisms that also encode some rare amino acids like pyrrolysine and selenocysteine.

What can be done to exploit all the chemical potential found in the so called unnatural amino acids? One approach would be to chemically incorporate the unnatural amino acids into already existing proteins, but this method is highly unspecific and may have a low efficiency; the ideal way would be to incorporate those unnatural amino acids at the very moment when the wanted protein is translated from its mRNA.

This is actually possible by using orthogonal translation systems and expanded genetic codes. The common 64-codon/20-amino acids genetic code has been expanded to 64-codon/21-amino acids and even 65-codon/21-amino acids, with one codon adapted for an unnatural amino acid. (refs 2 and 3)

The system behind the incorporation of this unnatural amino acid is the presence of a tRNA and an aminoacyl tRNA-transferase (AARS) enzyme from a phylogenetically distant organism; both the tRNA and the AARS have been modified to recognize the unnatural amino acid by directed evolution. Because of the phylogenetic distance with the host machinery, the AARS would charge the unnatural amino acid into the tRNA without interfering with the native tRNAs and AARS from the host, i.e. they would be an orthogonal translation system.

The remaining piece is the codon that would code for the unnatural amino acid. In Escherichia coli, the amber codon (UAG) is the least frequent codon found in its genes, so it has been commonly employed to be recognized by the orthogonal tRNA without having a gross effect on the growth rate of the bacteria. Nevertheless, custom codons have also been generated; most interestingly, four-nucleotide codons have proven to be functional.

Unnatural amino acids in proteins have been used as sites for chemical synthesis (the presence of some chemical groups in the unnatural amino acid increases the efficiency and specificity for some procedures, like click chemistry), so that many different active compounds can be conjugated. In this way, proteins conjugated with fluorescent probes, FRET pairs and poly ethylene glycol have been generated. Also, by incorporating phosphorylated residues, it has been possible to hack the normal signal transduction systems in cells. Finally, recently, genetic code expansion has been used to make “oligobodies”, a mix of DNA oligos and antibodies.

Front 4: Synthetic genetic circuits

If the intricate regulation of gene expression can be broken down to a number of basic processes, then by understanding these basic processes, we should be able to eventually understand what happens when they are combined and how they can make a full gene regulation mechanism.

The generation of synthetic circuits like switches and oscillators in prokaryotic and eukaryotic cells has been an exciting matter of study that has brought together mathematicians, computer scientists, molecular biologists and engineers. Switch-like genetic circuit behavior has been attained by using riboswitches, engineered promoter sites and transcription factors that respond to a particular stimulus, like light or some chemical compounds.

The conception and mathematical study of synthetic oscillating genetic circuits have been active since the early 1960s, but it has not been until recently that in vivo experiments have validated those idealizations. A considerable amount of effort has been dedicated to the mathematical characterization, simulation and in vivo implementation of oscillating genetic circuit topologies.

Synthetic genetic circuits have been employed to elucidate natural circuits by rewiring them or inducing “short circuits” into signaling cascades; properly functioning basic synthetic genetic circuits are the key for composite synthetic systems, like logic gates and synthetic signalling cascades.

Front 5: Metabolic Engineering

The realization that for Metabolic Engineering it was not enough to just mix existing genes to rewire biosynthetic pathways is perhaps the main reason why some Metabolic Engineering studies are considered to be also part of Synthetic Biology. The holistic understanding of metabolic flux, promoter activity, preferential codons and scaffold proteins are the basics for Synthetic Metabolic Engineering.

One of the seminal studies was that from the Keasling group, who rewired E. coli with asynthetic metabolic pathway that leads to the production of a precursor of an anti-malarial drug, artemisinin. The field has developed to include also biofuel biosynthetic pathways. (ref 5 and 6)

Another approach for Synthetic Metabolic Engineering, also developed by the Keasling group, is the use of synthetic protein scaffolds. These synthetic scaffolds are made by natural protein binding domains from phylogenetically distant organisms; a synthetic scaffold contains a number of different binding domains. When a set of enzymes from a particular metabolic pathway is genetically conjugated with a ligand domain – each enzyme with a different ligand domain- they will attach to the synthetic scaffold in an ordered pattern. The synthetic scaffold acts then as a spatial anchor for the metabolic pathway that can greatly alter the metabolic flux towards a desired product.

One interesting derivation of the scaffold concept was developed by team Slovenia in the iGEM 2009. Instead of using proteins as scaffolds, the team used a DNA molecule to recruit the enzymes of the metabolic pathway; this was possible by genetically conjugating the enzymes to a DNA binding domain from a phylogenetically distant transcription factors. Thus, the DNA-binding enzymes would attach to their recognition sites in a synthetic DNA construct and enhance metabolic flux through a biosynthetic pathway.

Closing remarks

With the enumeration of the main Synthetic Biology fronts:
1) DNA synthesis
2) biological parts standardization
3) genetic code expansion
4) synthetic genetic circuits
5) metabolic engineering,
it can be seen that something that all the projects share is the drive to gain an increasingly precise control over cellular processes, considering ground elements –i.e., custom DNA sequences, custom proteins produced by genetic code expansion, standard biological parts and basic synthetic circuits- but also higher-order elements –composite synthetic circuits and engineered metabolic pathways.

The degree and the tools with which Synthetic Biology intends and is actually beginning to control cellular processes are what distinguish it from any other field of the biological sciences.

Synthetic Biology is a fast growing and productive field. There is indeed a lot to do, particularly regarding to the precise quantitative characterization of biological parts and circuits; but since the field is attracting many smart and active young minds from different disciplines, the growth and innovation rate will likely increase in the years to come.

Mar 18, 2012.

1) Douglas, S., Marblestone, A., Teerapittayanon, S., Vazquez, A., Church, G., & Shih, W. (2009). Rapid prototyping of 3D DNA-origami shapes with caDNAno Nucleic Acids Research, 37 (15), 5001-5006 DOI: 10.1093/nar/gkp436
2) Young, T., & Schultz, P. (2010). Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon Journal of Biological Chemistry, 285 (15), 11039-11044 DOI: 10.1074/jbc.R109.091306
3) Kazane, S., Sok, D., Cho, E., Uson, M., Kuhn, P., Schultz, P., & Smider, V. (2012). Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR Proceedings of the National Academy of Sciences, 109 (10), 3731-3736 DOI: 10.1073/pnas.1120682109
4) Moon TS, Clarke EJ, Groban ES, Tamsir A, Clark RM, Eames M, Kortemme T, & Voigt CA (2011). Construction of a genetic multiplexer to toggle between chemosensory pathways in Escherichia coli. Journal of molecular biology, 406 (2), 215-27 PMID: 21185306
5) Nielsen, J., & Keasling, J. (2011). Synergies between synthetic biology and metabolic engineering Nature Biotechnology, 29 (8), 693-695 DOI: 10.1038/nbt.1937
6) Jarboe, L., Zhang, X., Wang, X., Moore, J., Shanmugam, K., & Ingram, L. (2010). Metabolic Engineering for Production of Biorenewable Fuels and Chemicals: Contributions of Synthetic Biology Journal of Biomedicine and Biotechnology, 2010, 1-19 DOI: 10.1155/2010/761042

S.E. Gould About the Author: A biochemist with a love of microbiology, the Lab Rat enjoys exploring, reading about and writing about bacteria. Having finally managed to tear herself away from university, she now works for a small company in Cambridge where she turns data into manageable words and awesome graphs. Follow on Twitter @labratting.

The views expressed are those of the author and are not necessarily those of Scientific American.

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