Fighting Infection
Cancer
Vaccines
Friendly Bacteria
Cellular and Regenerative Medicine
Conclusion

The Clinical Applications of Syntheticology – Toby Murcott

Introduction.

One way to look at medicine is that it attempts to repair malfunctions of the human body, be they internal errors such as genetic diseases or invasions of parasites and pathogens. Synthetic biology aims to re-engineer biological processes, and to do so in such a way as to produce specific, targeted outcomes. It’s no accident, then, that a great deal of hope and effort is being invested in developing synthetic biology for many difficult to treat diseases such as cancers, antibiotic resistant infections or genetic disorders. Even though the discipline is in its infancy, there are already some promising approaches emerging from labs around the world.

In a recent review published in Science, Ruder et al. [Ruder, et al. Science 333, 1248 (2011); DOI: 10.1126/science.1206843], described a number of ways in which synthetic biology may help human health. These approaches are universally elegant and demonstrate an impressive diversity of ideas. This breadth is one of the challenges for the SYBHEL project in that each approach is likely to produce a different set of legal and ethical questions.

Fighting Infection.

The discovery and development of antibiotics has had a profound effect on human health. Many previously deadly diseases are now easily cured. But, inevitably, their impact is starting to wane as bacteria fight back and acquire resistance. Drug development has slowed and new approaches to treating antibiotic resistant infections are needed. Enter the engineered bacteriophage, variations on viruses that infect only specific bacteria.

The first approach described by Ruder et al tackles persistent bacterial infections that hide themselves inside a mucous-like protective coat called a biofilm. A bacteriophage can be engineered to force infected bacteria to do two things. The first is, on infection, to quickly produce large quantities of new virus, burst open and die. This kills the bacterium, helping to reduce the infection and spread the virus rapidly to the remaining bacteria. The virus itself poses no risk to human health. The second is to produce an enzyme that breaks down the biofilm, exposing the bacteria to both antibiotics and the body’s immune system. Lab tests showed this eventually killed 99.997% of biofilm bacteria.

A second study described by Ruder et al produced bacteriophages that, once inside an infected bacterium, turned off the mechanisms that protect it from antibiotics. Treating E. coli infected mice with both antibiotics and the engineered bacteriophage resulted in an 80% survival rate, compared to 20% with antibiotics alone.

A very different approach to infectious disease is seen in a synthetic biology approach that could be used to tackle malaria. The parasite that causes this disease has to spend part of its life cycle inside a mosquito. Genetically modified mosquitoes can be engineered to block this stage, but that will only work if the genetic alterations are spread quickly to the whole mosquito population. A piece of syntheticDNAhas been constructed that should, in principle, encourage another set of genes to spread rapidly throughout a breeding population, almost like an infectious genetic disease. And in laboratory tests it did indeed spread throughout a caged population very quickly. This was a proof of principle, and did not carry any anti-malaria payload, so to speak. The next step would be to add an anti-malaria genetic modification to this system, producing, in theory, a genetic construct that would reduce or even eradicate malaria transmission.

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Cancer.

One of the greatest challenges for cancer treatments is to eliminate cancerous cells in the human body without damaging associated tissue. Ruder et al describe two synthetic biology approaches which have produced promising results in the lab.

The first involved engineering bacteria to produce a protein that makes them stick to human cells, but to produce that protein only in the absence of oxygen. Tumours often grow in low levels of oxygen as their blood supply is poor. The hope is that when the bacteria encounter a low oxygen tumour, they will stick to and invade the cancerous cells. The engineered bacteria were shown to invade human cells in the test tube only when oxygen levels were low. A potential problem with this approach, though, is that it would rely on the blood supply to deliver the bacteria to the cancer. However, tumours often have poor blood supply, making them low in oxygen, which is why this approach works in the first place. A lack of blood makes them susceptible, but also makes delivering the treatment a challenge.

This obstacle has been circumvented in a different approach that tackles one of the genes involved in turning cells cancerous in the first place. The CTNNB1 gene is involved in many colon cancers when it behaves in an abnormal fashion. Reducing the output of this gene when this occurs could potentially stop colon tumours from growing and spreading. The researchers engineered a bacterium that could penetrate colon cancer cells and interfere with the workings of the CTNNB1 gene and demonstrated that it could indeed target colon cancer cells growing in laboratory mice.

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Vaccines.

Synthetic biology has also been turned onto the challenge of developing new vaccines. A vaccine works by offering the body’s immune system a sample of the disease causing organism, known as an antigen, so that it can remember it and attack it if it invades in the future. An established way of doing this is called attenuation, in which a virus or bacterium is crippled so it cannot cause disease, but retains enough identifying features for the immune system to recognise it. This is, however, a delicate balancing act that doesn’t always work.

One rather elegant piece of synthetic biology has been to produce an artificial cell-like body that can alert an immune system but not cause disease. It works by wrapping some genes and the mechanism to translate them in a membrane similar to our own cell membrane. The genes included can, in theory, be changed for those from any disease causing organism. This will result in little fatty bubbles, called liposomes, that resemble part of the virus or bacterium. However, because they contain nothing else they are not able to produce disease, removing at a stroke the balancing act of attenuation – that tricky process by which an infectious organism is deactivated but not totally destroyed..

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Friendly Bacteria.

We carry inside us 10-100 times more bacteria than we do human cells. These are essential for life, helping us to digest food, produce some vitamins and ward off invading pathogenic bacteria. These bacteria, collectively called the microbiome, live happily inside us and are potentially excellent targets for synthetic biologists.

One example is the development of E. coli, the common gut bacterium, that can help fight off cholera, at least in mice. The E. coli were engineered to produce a chemical signal that cholera bacteria use to coordinate infection. The signal effectively swamped and so jammed the cholera bacteria’s signals. Baby mice given these engineered bacteria followed, 8 hours later, by cholera bacteria had an increase in survival rates of 80% over mice given cholera alone.

A second approach to utilising the microbiome is to engineer bacteria to produce useful drugs inside the body. The trick here would be to make sure that they are produced in the right amounts at the right place in the body, relying on the ability of synthetic biology to build in mechanisms to respond to external stimuli. These would turn on when they detect pathological conditions (rather like the cancer targeting bacteria), and turn off again when the disease has passed.

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Cellular and Regenerative Medicine.

The final topic Ruder et al discussed was the engineering of human cells to behave in specific, useful, ways. The challenge here is that the majority of synthetic biology to date has been done in bacteria. Mammalian cells like ours are significantly more complex and therefore significantly harder to engineer. However, it has been done.

Mice have been engineered to contain a synthetic biological mechanism that is turned on by the presence of uric acid, the chemical responsible for gout, and produce enzymes that remove the uric acid. Once it is all gone then the mechanism turns off. This mimics the behaviour of many different natural cellular processes, miniature thermostats turning off and on in response to a myriad of chemical signals in the body. The system was tested in mice genetically engineered to over-produce uric acid. This new system succeeded in reducing their uric acid levels and the clinical problems such as gout and crystals in the kidneys associated with excess uric acid.

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Conclusion.

The review authors conclude by arguing for much more work to be done in mammalian cells, developing systems such as the uric acid removal mechanism. Without them it will be impossible to move synthetic biology from the laboratory into the clinic. The examples here demonstrate that it is possible to build synthetic biological systems that can effectively seek out and treat disease in the body. The potential is enormous, but real clinical treatments are still a long, long way away.

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Zsófia Clemens’ monthly report on the state of the art in synthetic biology

Synthetic Toxicology: Where engineering meets biology and toxicology.
Schmidt M, Pei L.
Toxicol Sci. 2010 Nov 10. [Epub ahead of print]

The article delineates implications of synthetic biology for toxicology. In the second part of the article ramifications of synthetic toxicology are explored with an emphasis on potential applications and their risks. Applications include: constructing toxicity testing platforms, biotransformation of toxins, designing biosensors for environmental toxicology. For example widespread arsenic contamination and its detoxification is a great challenge. A potential synthetic toxicology application might be to couple innate arsenic detoxification pathways with synthesized arsenic detection pathways. The article also discusses issues related to cell-free synthesis of toxins and toxic use reduction through synthetic biology and implications of non-natural proteins for toxicology.

Genes that move the window of viability of life: Lessons from bacteria thriving at the cold extreme: mesophiles can be turned into extremophiles by substituting essential genes.
de Lorenzo V.
Bioessays. 2011 Jan;33(1):38-42.

Recent evidence suggests that a few genes might suffice for adaptation to extreme environmental conditions. For example, genes from cold-loving bacteria expressed in E. coli allow them to grow at 5°C or even lower temperature. This feature might be exploited to develop vaccines used in warm-blooded animals, including humans. The rationale beyond is that such pathogens possess the entire antigenic repertoire but will be killed at higher temperature. So far attempts to increase heat-sensitivity have been less successful. Heat-tolerance is known to rely on heat-shock proteins but expression of these proteins in E. coli failed to augment heat tolerance. Presently heat sensitivity is one of the bottlenecks to use microorganisms in industrial processes. Other environmental extremities to be aimed by synthetic biology include humidity, surface tension, UV, radiation, pressure etc. In an application E. coli was made tolerant to the toxicity of by-products of biofuel production.

Biology by design: from top to bottom and back.
Fritz BR, Timmerman LE, Daringer NM, Leonard JN, Jewett MC.
J Biomed Biotechnol. 2010;2010:232016. Epub 2010 Nov 2.

This review article summarises basic principles of synthetic biology. Considerations regarding ”bottom-up” and “top-down” approaches are discussed and illustrated. Three topical areas of application are discussed: biochemical transformations, cellular devices and therapeutics, and approaches that expand the chemistry of life. This latter approach is aimed at designing nonnatural compounds that behave predictable and envisions components such as peptide nucleic acids or xenonucleic acids replacing DNA. These artificial molecules have the potential to expand biopolymer information storage.

Environmental biosafety in the age of synthetic biology: do we really need a radical new approach? Environmental fates of microorganisms bearing synthetic genomes could be predicted from previous data on traditionally engineered bacteria for in situ bioremediation.
de Lorenzo V.
Bioessays. 2010 Nov;32(11):926-31. doi: 10.1002/bies.201000099. Epub 2010 Oct 8.

This article suggests that fears and reservations about environmental risks of synthetic biology are exaggerated. There are examples in the article to illustrate that genetically engineered organisms (GMO) are less fit and less likely survive when competing with natural counterparts. It is highlighted that GMOs have never caused any environmental harm. Reprogramming bacteria is hindered by our limited understanding of live systems. Difficulties are also reflected in failed attempts to reprogram pathogens for fighting cancer cells. It is suggested that organisms with synthetic genomes do not represent additional risk.

A comparative analysis of synthetic genetic oscillators.
Purcell O, Savery NJ, Grierson CS, di Bernardo M.
J R Soc Interface. 2010 Nov 6;7(52):1503-24. Epub 2010 Jun 30.

This review presents state of the art in the design and construction of oscillators comparing the features of the main networks highlighting their advantages and disadvantages. Starting with the simplest Goodwin oscillator the article considers repressilators and several types of activator–repressor networks and most recent oscillators constructed in mammalian cells. Very recent oscillators include Fussenegger oscillators, Smolen oscillator, variable link oscillators and the metabolator. Features of each network, models used for their in silico design, mathematical background, validation and in vivo data are presented.

Synthetic circuits, devices and modules.
Zhang H, Jiang T.
Protein Cell. 2010 Nov;1(11):974-8. Epub 2010 Dec 10.

This mini-review summarises recent advances in designing basic building blocks for synthetic biology applications. These include artificial gene control elements such as the zinc finger protein which provides a powerful tool to modulate gene expression. Other elements include synthetic RNA for post-transcriptional regulation (such as artificial riboswitches) and synthetic proteins (such as multi-domain binding protein or scaffold proteins). The construction of higher-order genetic circuits and devices allow more sophisticated control. These artificial elements can be combined into synthetic biological modules such as in the case of an engineered cyanobacterium producing isobutyraldehyde and isobutanol directly from CO2.

Synthetic biosensing systems.
Marchisio MA, Rudolf F.
Int J Biochem Cell Biol. 2010 Nov 23. [Epub ahead of print]

This review summarises synthetic biology of sensing systems. These systems are in continuous evolution. Compared to the early synthetic gene circuits more advanced systems are constructed by coupling artificial and natural pathways. At present numerous components such as receptors, adapters, scaffolds and their interaction with ligands have been characterised. In addition, based on cell–cell communication mechanisms more complex networks such as cell phones and ecosystems have been modelled. The review describes cell-cell signalling mechanisms, post-translational biosensors, transmembrane signalling and intracellular signal transduction for biosensor applications.

Characterization of engineered actin binding proteins that control filament assembly and structure.
Brawley CM, Uysal S, Kossiakoff AA, Rock RS.
PLoS One. 2010 Nov 12;5(11):e13960.

Cytoskeleton found in eukaryotic cells is built from actin filaments. The network of actin filaments is strictly regulated in response to various stimuli. Therefore it is an ideal target for reengineering the cytosceleton. Normally there are over a hundred distinct actin binding proteins that modulate establishment of actin filaments. The actin filament is polar, with distinct ends known as the barbed and pointed ends maintaining distinct polymerization rates. Here the authors produced new artificial proteins that unlike the majority of actin binding proteins bind to the pointed end of actin filaments and regulate polymerization. These artificial proteins were generated by phage display mutagenesis. Effective strategies to select and screen for proteins with desired properties are described.

Biomaterials. 2010 Dec;31(36):9395-405. Epub 2010 Oct 8.
Recombinant self-assembling peptides as biomaterials for tissue engineering.
Kyle S, Aggeli A, Ingham E, McPherson MJ.

Synthetic self-assembling structures mimicking the natural extracellular matrix is an approach used in tissue engineering applications. The self-assembly process relies on peptides since they can be easily synthesized. P(11)-4 is a 11 amino acid peptide with well-known characteristics. The authors used simple site-directed mutagenesis to produce a series of other P(11)-family peptide expression vectors. Here they report improved recombinant expression and a new purification strategy for the self-assembling peptides. The purified peptides were analysed, characterized and tested for cytocompatibility.

J Mol Biol. 2010 Oct 28. [Epub ahead of print]
Multichromatic Control of Gene Expression in Escherichia coli.
Tabor JJ, Levskaya A, Voigt CA.

The authors recently constructed a red light-sensitive E. coli transcription system based on a cyanobacterial phytocrome and E. coli signaling pathway. Here, they expand light regulation with the development of a green light-inducible transcription system in E. coli based on a photoswitchable system from cyanobacteria. Transcriptional output in this system was shown to be proportional to the intensity of green light applied. Expression of both sensors in a single cells allows two-color optical control of transcription in engineered cells. Such a system would allow expression of different genes to be controlled with different colors of light.

Biotechnol Bioeng. 2010 Oct 21. [Epub ahead of print]
An active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells.
Bagh S, Mandal M, Ang J, McMillen DR.

One of the future goals of synthetic biology is to create genetically programmed agents that are able to fight disease. The authors here designed a system featuring several key properties that will be required for a future intracellular disease-prevention mechanism. The system detects the onset of the lytic phase of bacteriophage lambda in E. coli., responds to prevent its lethality and it can be deactivated externally by a temperature shift when desired. The authors have also formulated a mathematical model that explained the behavior of the engineered system.

Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production.
Tsai SL, Goyal G, Chen W.
Appl Environ Microbiol. 2010 Nov;76(22):7514-20. Epub 2010 Oct 1.

Cellulosomes are extracellular complexes of cellulolytic enzymes of bacteria capable of degrading cellulose. In this paper a functional minicellulosome was assembled using a synthetic yeast consortium. The design consisted of four different engineered yeast strains capable of displaying different proteins of the cellulosomes such as a trifunctional scaffoldin carrying three different cohesins and corresponding dockerin-tagged cellulases. Based on the specific cohesion-dockerin interactions the secreted cellulases were docked in a highly organized manner. By expoiting the modular nature of each population it was possible to fine-tune and optimize cellulose hydrolysis and ethanol production.

Methods Mol Biol. 2011;692:235-49.
Design of synthetic mammalian quorum-sensing systems.
Weber W, Fussenegger M.

Bacterial quorum-sensing components have previously been engineered in some applications such as for controlling population density. Here the authors provide a detailed protocol of a mammalian cell-to-cell signaling device. This system comprises a sender and a receiver cell line. The sender was engineered for expression of alcohol dehydrogenase converting ethanol into acetaldehyde while the receiver for the dose-dependent translation of the acetaldehyde concentration into transgene expression. It is suggested that this design can be adapted to various cell types and transgenes for mammalian cells-based quorum-sensing systems.

Curr Opin Genet Dev. 2010 Oct 7. [Epub ahead of print]
Watch the clock-engineering biological systems to be on time.
Aubel D, Fussenegger M.

Oscillations can be found throughout in nature. Synthetic biologists have assembled a variety of synthetic clocks mimicking the dynamics of various biological processes. The review reports the repressilator, the first synthetic oscillator design, the first synthetic clock combining positive and negative feedback loops as well as the metabolator that integrates metabolism into transcriptional regulation to generate oscillation. Another synthetic clock is the mammalian intron clock referring to manipulating intron lenght and thereby manipulating oscillations. Other advanced synthetic clocs include the sense–antisense pendulum clock and low-frequency mammalian oscillators. The review also highlights major challenges such as synchronization of individual engineered cells across the population, difficulties with engineering multiple oscillator components and prevent interference between different oscillation components.

October 2010

 

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

Gao X, Wang P, Tang Y.

Appl Microbiol Biotechnol. 2010 Sep 19. [Epub ahead of print]

Polyketides constitute a diverse family of compounds synthesized by bacteria, fungi and plants. They include clinically useful drugs such as antibiotics, antifungals, cytostatics, anticholesterolemics and antiparasitics. Enzymes that synthesize polyketides are referred to as polyketide synthases (PKS). Since many of the natural hosts of PKS are difficult to culture establishing a universal heterologous host has become an important goal. The review summarizes recent advances in engineering E. coli for the biosynthesis of PKS of different types.

A nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum

Nijland JG, Ebbendorf B, Woszczynska M, Boer R, Bovenberg RA, Driessen AJ.

Appl Environ Microbiol. 2010 Sep 17. [Epub ahead of print]

Production levels of penicillin by Penicillium chrysogenum have markedly increased by classical strain improvement methods. These high yielding strains contain multiple copies of the penicillin biosynthetic gene cluster. Authors of the article investigated the effect of increasing number of gene clusters on the level of penicillin production. It was found that penicillin production increased but saturated at high copy number. Remarkably, the acyltransferase enzyme located in peroxisomes saturated already at low cluster numbers. It was suggested that acyltransferaseactivity is limiting for penicillin biosynthesis at high biosyntheticgene cluster numbers.

Engineered photoreceptors as novel optogenetic tools

Möglich A, Moffat K.

Photochem Photobiol Sci. 2010 Oct 28;9(10):1286-300.

Optogenetics is a new tool in cell biology denoting the use of genetically encoded, light-gated proteins that is photoreceptors which control cellular behavior. Engineered photoreceptors resemble fluorescent reporter proteins that are designed to monitor cell processes but in addition they are able to modulate activity and therefore offer control over cells. Engineering is based on naturally occurring photoreceptors with fusing light absorbing sensor domains with an effector domain. The article summarizes basic principles and application of such engineered photoreceptors.

Using light to control signaling cascades in live neurons

Rana A, Dolmetsch RE.

Curr Opin Neurobiol. 2010 Oct;20(5):617-22.

Light as a controlling signal has several advantages including non-invasiveness, high specificity and spatio-temporal control. These two latter features are of crucial importance in the nervous system where timing of events is essential. Recently photo-activable proteins have been developed to control and manipulate cell processes. These artificial photoreceptor proteins (APP) consist of a light-sensing and an effector domain. The article summarizes potential use of APPs in the nervous system research and its challenges in practice.

A synthetic-natural hybrid oscillator in human cells

Toettcher JE, Mock C, Batchelor E, Loewer A, Lahav G.

Proc Natl Acad Sci U S A. 2010 Sep 28;107(39):17047-52.

In this study a tunable oscillator was constructed based on the p53 signaling pathway in mammalian cells. The authors reduced this circuit to contain a single feedback loop. In contrast to natural cells, the reduced circuit exhibited damped oscillations with amplitude that depends on input strength. By constructing other variants of the circuit the authors demonstrated that important features of oscillation dynamics such as the amplitude, period, and the rate of damping can be controlled.

Using synthetic biology to understand the evolution of gene expression

Bayer TS.

Curr Biol. 2010 Sep 14;20(17):R772-9.

 

Evolution of phenotype is thought to rely on changes in gene regulation rather than changes in encoding proteins themselves. For example there are many proteins that show remarkable sequence conservation over a large evolutionary time-span. This aspect of evolution can be stated in analogy with synthetic biology which aims to manipulate gene expression basically through their regulation. This article highlights cases where synthetic biology was used to rewire regulatory networks to understand their functional advantages. Synthetic biology also allows for testing evolutionary paths not taken by currently existing organisms.

Microfluidic approaches for systems and synthetic biology

Szita N, Polizzi K, Jaccard N, Baganz F.

Curr Opin Biotechnol. 2010 Aug;21(4):517-23. Epub 2010 Sep 9.

Microfluidics deals with the behavior of fluids of small amounts. Advantages of microfluid approaches include reduction of sample volumes, shorter analysis time and increased sensitivity. The review summarizes how these advantages can be harnessed for synthetic biology. So far microfluidic chips have been developed for oligonucleotide synthesis and for facilitating electroporation, a technique used to input new DNA into cells. Microfluidic devices are currently used for circuit expression, storage and analysis. Future aims include integrating several processes in a single device allowing fully automated cell analysis and assembly of synthetic systems.

Transgenic biosynthesis of trypanothione protects Escherichia coli from radiation-induced toxicity

Fitzgerald MP, Madsen JM, Coleman MC, Teoh ML, Westphal SG, Spitz DR, Radi R, Domann FE.

Radiat Res. 2010 Sep;174(3):290-6.

Trypanothione is a unique compound found in trypanosomes which are parasitic protozoa. The major function of trypanothione is the defense against radiation and oxidative stress. Therefore trypanothione was suggested to act as a radioprotective agent when heterologously expressed in bacteria. To test this trypanothione synthetase and reductase genes from T. cruzi were introduced into E. coli. The transgenic E. coli was able to produce trypanothione and compared to control bacteria was found to be 4.3-fold more resistant to killing by gamma radiation. These results point to the possibility of using trypanothione as a novel radioprotective agent.

September 2010: Synthetic biology for translational research. Burbelo PD, Ching KH, Han BL, Klimavicz CM, Iadarola MJ. Am J Transl Res. 2010 Jul 20;2(4):381-9.

The review focuses on translational applications of synthetic biology. One application is to develop diagnostic serologic immunoassays against different pathogens. Advantage of artificial gene synthesis for antigens is that the corresponding DNA is not required. Another application is to design artificial, multi-epitope genes that can be used as vaccines. A powerful application is the synthetically attenuated virus engineering (SAVE), a procedure based on codon deoptimation in order to attenuate pathogenic viruses. Other applications include developing new drug screens, modify cells and engineer new synthetic pathways to treat cancer, neurodegeneration and infection. Another ultimate goal is to develop genetically-modified organisms to fight disease such as genetically modified bacteriophages against antibiotic resistant bacteria or engineer bacteria to invade cancer cells.

Structural synthetic biotechnology: from molecular structure to predictable design for industrial strain development Chen Z, Wilmanns M, Zeng AP. Trends Biotechnol. 2010 Aug 18. [Epub ahead of print]

Structural synthetic biotechnology is a new field with great promise for industrial biotechnology. It combines synthetic biology and structural cell biology. The latter concerns to a field designing and assembling cell components as well as revealing details about macromolecules and bioreactions at the atomic level. The review discusses developments of structural synthetic biology and its application in metabolic engineering in industrial strain development. Specific applications include: programming metabolic pathways, engineering allosteric regulation of enzymes and designing scaffold proteins that enhance the efficiency of the metabolic pathway and engineering cellular signaling pathways.

Challenges in synthetically designing mammalian circadian clocks. Susaki EA, Stelling J, Ueda HR. Curr Opin Biotechnol. 2010 Aug 12. [Epub ahead of print]

Engineering approach is typically applied to simple biological systems. This review overviews the synthetic biology approach of a complex and dynamic system, the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Complex transcriptional and post-transcriptional properties of the SCN have previously been described. The synthetic approach of the circadian clock requires implementing the signal transduction network, electrophysiological network and intercellular circuits of the SCN. Rational synthesis of circadian system properties will be important in understanding the biological clock and associated clinical problems such as sleep disorders.

Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Callura JM, Dwyer DJ, Isaacs FJ, Cantor CR, Collins JJ. Proc Natl Acad Sci U S A. 2010 Aug 16. [Epub ahead of print]

The authors previously described a synthetic, riboregulator system that controls gene expression posttranscriptionally through highly specific RNA–RNA interactions. In the present study a series of experiments are conducted to demonstrate the advantageous features of this system. These features include: component modularity, leakage minimization, rapid response time, tunable gene expression and independent regulation of multiple genes. These features make this system an ideal synthetic biology platform for interfacing with different microbial systems.

The synthetic integron: an in vivo genetic shuffling device. Bikard D, Julié-Galau S, Cambray G, Mazel D. Nucleic Acids Res. 2010 Aug 1;38(15):e153. Epub 2010 Jun 9.

The article describes generation of large number of genetic combinations of the tryptophan operon in E. coli. Recombination was carried out using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. The generated operons varied in their fittess and tryptophan production capacities. Several assemblages required six recombination events and produced 11-fold more tryptophan than the natural gene. Selection of optimal arrangements from randomized libraries might be an alternative of rational design of metabolic pathways.

Oscillations by minimal bacterial suicide circuits reveal hidden facets of host-circuit physiology. Marguet P, Tanouchi Y, Spitz E, Smith C, You L. PLoS One. 2010 Jul 30;5(7):e11909.

 

A synthetic genetic circuit is presented that causes unexpected oscillations in bacterial population density over time. Contrary to expectations, oscillations did not require the quorum sensing genes. Instead, oscillations were likely due to density-dependent

plasmid amplification and parallel increase in expression of a plasmid-borne toxin that established a population-level negative feedback. A mathematical model that captured the plasmid copy number and circuit dynamics was validated by the experimental results. The results point to the importance of plasmid copy number and potential impact of interactions on the behavior of engineered gene circuits.

Synthetic gene networks in mammalian cells. Weber W, Fussenegger M. Curr Opin Biotechnol. 2010 Aug 4. [Epub ahead of print]

 

Following initial synthetic biology work that was mostly conducted in prokaryotes, there is now increasing interest towards mammalian systems given its direct link to biomedical applications. The review summarizes recent efforts in mammalian synthetic biology in the past two years. Recent advances include approaches following the classical small-molecule-based designs as well as new principles such as those using light as a trigger for controlling cells. The article also highlights synthetic gene networks exhibiting oscillating behaviour.

An engineered mammalian band-pass network. Greber D, Fussenegger M. Nucleic Acids Res. 2010 Aug 6. [Epub ahead of print

In the formation of embryonic patterns morphogens carry information depending on its concentration. In the present study the authors engineered and optimized a mammalian genetic circuit capable of sensing a specific concentration within morphogen gradient. The components involved linked mammalian transactivator and repressor control systems to detect and respond to low-treshold and high-treshold concentration levels of tetracycline. A mathematical model was also derived to simulate interactions between various modular elements. These results have implications for future tissue engineering, gene therapy and biosensing applications.

A synthetic riboswitch with chemical band-pass response Muranaka N, Yokobayashi Y. Chem Commun (Camb). 2010 Aug 19. [Epub ahead of print]

A riboswitch is an mRNA molecule that can regulate its own activity through binding small target molecules in bacteria. Rarely natural riboswitches have two binding domains, called tandem riboswitch. The authors engineered such a tandem riboswitch containing an ON and OFF riboswitch unit. The resulting complex riboswitch functions as a chemical band-pass filter circuit. It is remarkable that such a function is achieved without regulatory proteins.

Synthetic analogs tailor native AI-2 signaling across bacterial species Roy V, Smith JA, Wang J, Stewart JE, Bentley WE, Sintim HO. J Am Chem Soc. 2010 Aug 18;132(32):11141-50.

Quorum sensing (QS) is bacterial cell-cell communication brought about by the secretion and reception of small signal molecules, called autoinducers (AI) Elements of the QS system can be used to interrupt communication. Anti-QS agents that quench QS communication but are otherwise harmless to cells are thought to pose less evolutionary pressure on bacteria and therefore believed to prevent the emergence of new antibiotic-resistant strains. The authors developed agonists and antagonists of the AI-2 that mediates the QS response in multiple bacterial species and also demonstrated the biological basis of this action. The results suggest new modalities to interrupt bacterial communication and an alternative approach to treat bacterial infection.

April-July 2010

 

Recognition, neutralization, and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles: a plastic antibody

Hoshino Y, Koide H, Urakami T, Kanazawa H, Kodama T, Oku N, Shea KJ.

J Am Chem Soc. 2010 May 19;132(19):6644-5.

Hoshino et al report a new technique to create plastic antibodies that are able to clear a target peptide toxin in the bloodstream. These organic antibodies are artificial versions of natural antibodies produced by lymphocytes with comparable binding affinity and selectivity. When injected into mice plastic antibodies diminished the toxic effect of the target peptide and enhanced survival. It is suggested that the reported technique could be used to generate plastic antibodies against a range of antigens and might be a cheap and simple alternative of natural antibodies that are produced slowly and in complicated ways in living mammals.

Synthetic biology of protein folding

Moroder L, Budisa N.

Chemphyschem. 2010 Apr 26;11(6):1181-7.

This article highlights the concept of using synthetic amino acids that are not found in nature for in vivo protein synthesis. This method does not alter the DNA sequence itself but produces variation at the level of protein translation. Such genetic code engineering is a useful tool to study protein folding. The article highlights two examples of alternative protein folding resulting in novel structural properties of the synthesized protein. One of the examples is the prion protein where structural conversion of the protein is thought to result in prion protein aggregation leading to a devasting neurodegenerative condition.

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An inactivated West Nile Virus vaccine derived from a chemically synthesized cDNA system

Orlinger KK, Holzer GW, Schwaiger J, Mayrhofer J, Schmid K, Kistner O, Noel Barrett P, Falkner FG.

Vaccine. 2010 Apr 26;28(19):3318-24

The article reports the first chemical synthesis of a flavivirus, the West Nile Virus. The synthesized virus contained no undesired mutations and exhibited undistinguishable properties compared to the wild-type virus. This demonstrates that chemically synthesized viruses might be also used for vaccine production or research.

Synthetic biology approaches in drug discovery and pharmaceutical biotechnology

Neumann H, Neumann-Staubitz P.

Appl Microbiol Biotechnol. 2010 Jun;87(1):75-86.

This review gives an insight into biological components available to the synthetic biological approach of pharmaceutical biotechnology. Bioactive compounds, such as cyclic peptides can be maintained in the form of genetically encoded library. Combining modules of multi-enzyme complexes might result in novel properties of the enzyme such as in the case of polyketide synthases. De novo creation of metabolic pathways might be used to expand biosynthetic activity e.g. to produce terpenoids that do not naturally occur. Well-known examples of terpenoids are the anti-malarial drug artemisinin and the anti-cancer agent paclitaxel. Optimizing metabolic pathways e.g. by feeding cells with synthetic oligonucleotides, was shown to fine-tune ribosomal binding and leading to increased production of lycopene in an engineered E. coli. Recombining individual modules has been employed to design proteins that act as biosensors (such as an E. Coli strain responding to light) or to design bacteria that deliver chemotherapeutic drugs to tumour cells.

Strategies for protein synthetic biology

Grünberg R, Serrano L.

Nucleic Acids Res. 2010 May;38(8):2663-75.

This review presents state of the art of protein synthetic biology. Compared to gene engineering, design and engineering of complex protein systems lags behind due to the versatility and interactions of proteins at the modular level. The review outlines industrial and biomedical applications of protein synthetic biology and discusses potential new avenues.

Creation of a bacterial cell controlled by a chemically synthesized genome

Gibson DG et al. Science. 2010 Jul 2;329(5987):52-6.

The group of J. Craig Venter announced the creation of a synthetic cell controlled only by a synthetic genome. Creation of the synthetic cell relied on their previously established procedures including synthesis, assembly, cloning, and transplantation of a bacterial genome. Although the cytoplasm of the recipient cell is not of synthetic origin the authors refer to such a cell as “synthetic cell” since the only DNA in the cell is the synthesized DNA and phenotype of the recipient cell is diluted with protein turnover. The synthesized cell has expected phenotypic properties and capable of self-replication. As in previous studies the investigators watermarked the synthetic chromosome to distinguish it from naturally occurring cells.

Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study.

Lee CH, Cook JL, Mendelson A, Moioli EK, Yao H, Mao JJ.

Lancet. 2010 Aug 7;376(9739):440-448.

The study reports an alternative approach of tissue regeneration. Instead of using stem cells or direct implantation the authors demonstrate that the rabbit synovial joint can regenerate with a biological cue. The articular surface of the humeral condyles was excised and replaced by an anatomically correct bioscafford infused with transforming growth factor ?3 (TGF?3). Four months after surgery, TGF?3-infused bioscaffolds were fully covered with hyaline cartilage. Compared to TGF?3-free bioscaffords, TGF?3-infused bioscaffords showed greater thickness and density. These findings suggest that the synovial joint can regenerate without cell transplantation.

The art of reporter proteins in science: past, present and future applications.

Ghim CM, Lee SK, Takayama S, Mitchell RJ.

BMB Rep. 2010 Jul;43(7):451-60.

Reporter genes are commonly used in many laboratories to study gene expression. The first reporter gene, the lacZ was published 30 years ago. The review discusses the three reporter systems: ?-galactosidase, luciferases and fluorescent proteins, their past and current applications. Novel applications include studying bacterial populations within smaller scale systems such as biofilm and microfluids. The review suggests the new generation of reporter genes might used to characterize and fine-tune biological parts to be applied in synthetic biological applications.

Synthetic biology of minimal living cells: primitive cell models and semi-synthetic cells

Stano P

Syst Synth Biol 2010

DOI: 10.1007/s11693-010-9054-3

The article discusses synthetic biology approaches to study minimal cells and synthetic cells. The articles describe attempts to define minimal life in the framework of the autopoietic (self-reproducing) theory and shows a report on autopoietic chemical systems based on fatty acid vesicles, a model which might be relevant for primitive cells. The article also contains a review of the four most advanced studies implementing some simple functions in the synthetic cell. It is concluded that semi-synthetic cell models can provide insights into the nature of cellular life and might be useful for biotechnological applications.

A comparative analysis of synthetic genetic oscillators.

Purcell O, Savery NJ, Grierson CS, di Bernardo M.

J R Soc Interface. 2010 Jun 30. [Epub ahead of print]

This review presents a comparative analysis of the main synthetic oscillators constructed in vivo or studied theoretically. A wide range of synthetic oscillators is considered starting from the simplest Goodwin oscillator, repressilators, several types of activator-repressor networks and the Fussenegger oscillators, the only oscillator that has been implemented in a eukaryotic system. This review might be used as a guideline to synthetic biologists and engineers wishing to use existing synthetic oscillator models and designs.

Ligand-dependent regulatory RNA parts for Synthetic Biology in eukaryotes.

Wieland M, Fussenegger M.

Curr Opin Biotechnol. 2010 Jul 15. [Epub ahead of print]

A variety of artificial regulatory RNA parts have been developed that are capable of controlling gene expression in eukaryotes. The genetic switch relies on a specific ligand binding to a RNA domain. Depending on the surroundings, this can affect transcription, translation or RNA interference. The article provides a summary of the main controllable RNA parts developed so far in eukaryotic systems.

 

Update on designing and building minimal cells.

Curr Opin Biotechnol. 2010 Jul 15. [Epub ahead of print]

Jewett MC, Forster AC.

This review presents the major advances occurring in the design and synthesis of minimal cells during the past few years. Minimal cell projects are proceeding in two different directions: top-down referring to in vivo reduction and bottom-up meaning in vitro construction. Major progresses include: minimization of the Escherichia coli genome, sequencing of minimal bacterial endosymbionts and identification of essential genes. The review proposes an RNA-based and a protein-based in vitro model for minimal cells and discusses their potential advantages and practical hurdles.

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