Pushing the frontiers

31 October 2016 - By: Caroline Wood

Pushing the frontiers 

Nicotiana benthamiana
Nicotiana benthamiana plant growing under green LEDs. Photo: Martin Battle, Jones Lab, University of Essex


By Caroline Wood

Artificial life has traditionally belonged to the realm of science-fiction yet it is fast becoming a reality as synthetic biologists go boldly where no other biologists have gone before. From gene circuits to carbon capture to light-activated organisms – the pioneers of this brave new world were in full force at our Annual Meeting in Brighton. 

Green for Go

What if we could activate biological systems with literally a flick of the light switch? PhD student Martin Battle (University of Essex, United Kingdom) is investigating the possibility of doing just this using the green-light photoreceptor CcaS from the freshwater photosynthetic cyanobacterium Synechocystis. Normally, the CCAR response regulator of the gene responds to green light by producing an antenna complex which transfers light energy to the photosystems. “But if you replace the coding sequence for this antenna protein with something else, then essentially this will be expressed in response to green light” says Martin. Whilst this has already been demonstrated in E.coli, Martin hopes to use this system to introduce green-light regulated processes in plants. Although plants depend on light for photosynthesis, they generally use only the red and blue ends of the spectrum, reflecting green light back. This offers the potential to introduce new signalling pathways which can be activated without affecting other processes. “Many researchers already grow their plants under red and blue LEDs only to save energy” Martin says. “So it would be easy to supplement them with green light when you want to switch the system on”. To see if this is feasible, Martin is expressing synthetic constructs linked to reporters such as yellow fluorescent protein in Arabidopsis and Nicotiana benthamiana. “At the moment we have had some problems with low or oscillating expression” he says. This may be because bacterial and plant phytochromes are related and have similar structures, hence the endogenous plant light receptors may be interfering with CcaS function. However, this technology is not limited to plants; Wilfried Weber (University of Frieburg, Germany) has demonstrated that optical control can also be applied to mammalian cells. “By functionally rewiring plant and bacterial photoreceptors to growth factors, synthetic polymers, kinases or transcription factors, we achieve optical control along the whole signal transduction cascade” he says. “We apply these optogenetic tools to control cell migration, mechano-signalling as well as cell differentiation”.  

Expressing design

Synthetic versions of biological systems are increasingly used to help us understand how their natural counterparts work in vivo. One of the most complex cellular processes is glycosylation; the addition of oligosaccharide chains to protein backbones that determine their structure and function. These chains are added and modified sequentially as proteins progress through the endoplasmic reticulum and Golgi body. “Glycosylation affects a wide range of processes and characteristics including blood types, immune responses and hormone signalling,” says Dr Kate Royle (Imperial College, United Kingdom). “Due to the numerous enzymes involved, their overlapping distributions, and substrate promiscuity this complex process can generate hundreds of different glycans”. Disentangling this web could enable new, therapeutic glycoproteins to be engineered and cast light on the mechanism of glycosylation disorders. As such, Kate and her colleagues have embarked on an ambitious project to build a synthetic Golgi body reactor. “By disengaging the glycoenzymes from the cell, we can linearize the network into individual, sequential modules and characterise them as parts” she says.  Their strategy is to express the glycoenzymes in E.coli before immobilising them in sequence to form the synthetic reactor. This will either be done using affinity-tag immobilisation of the enzymes onto support pellets of a packed bed reactor or attaching the enzymes with non-specific surface chemistry to a microcapillary.  Meanwhile, the model yeast Pichia pastoris will be used to express a core glycan composed of mannose residues, which can be built up to form the target substrates. 

The group are currently working towards the production of their first prototype: G2, a complex human glycan with a two-branched structure. “Our first step will be to use the range of therapeutic targets we have generated to test the limits of the system,” says Kate. “Ideally, we will be able to produce different glycoform versions of antibodies to test which is the most efficient”. 

Doing it our (path)way 

All of our food ultimately originates from CO2 fixed into organic compounds, yet this process is highly inefficient in plants. “To meet our energy and food needs by photosynthetic CO2 fixation, we will need an estimated three worlds by 2050” says Tobias Erb (Max Planck Institute for Terrestrial Microbiology, Germany). “Our strategy is construct artificial CO2-fixation pathways to create novel CO2 -fixing microorganisms.” Tobias is particularly interested in a group of highly-efficient CO2 fixing enzymes called enoyl-CoA carboxylases/reductases (ECRs), recently discovered in the Alphaproteobactera species Rhodobacter sphaeroides and the Actinobacteria Streptomyces. “ECRs are an order of magnitude more efficient in converting CO2 than RuBisCO, the key carboxylase of plants” he says. Using ECRs as a starting point, Tobias has constructed several theoretical CO2-fixation cycles, combining known reactions of enzymes from across the biological kingdoms. “Our drafts are not based on existing biochemical pathways, but on what is biochemically plausible, so they are not constrained by natural evolution” says Tobias. The enzyme candidates can be further engineered to improve their performance: “We opened up the substrate-binding pocket of ECRs so that they can now carboxylate over sixteen different substrates, which allows us to draft many different CO2-fixation cycles” says Tobias. One of their most promising synthetic cycles, containing 17 enzymes from nine different organisms, including three engineered enzymes, is theoretically more efficient than the plant Calvin cycle.

carbon fixation pathways
Constructing artificial carbon fixation pathways requires cloning genes from multiple different organisms.Photo: Tobias Erb


But theory doesn’t always translate into reality and much optimisation needs to be done. Exposing enzymes to novel substrates frequently causes unwanted side-reactions, requiring them to be engineered further or the addition of ‘proofreading’ enzymes. Even when the system works in a reaction tube, introducing the pathway into microorganisms is challenging as we have little idea of how it will interact with endogenous metabolism. Tobias argues that a “bottom-up, reductionist approach”, rather than a ‘top-down’ one, is essential to fine-tune these cycles. “For instance, a cycle of 17 enzymes at just three possible expression levels (low, medium, high) would have 129,140,163 (317) possible test combinations - without the guarantee that it would operate in the complex background of an organism” he says. Nevertheless, his group has already begun to demonstrate proof-of-principle by introducing the plant Calvin cycle into non-CO2 fixing microorganisms. “I think that within the next decade we will see the transplantation of artificial CO2-fixation cycles into living organisms or even synthetic chloroplasts” he concludes. 

Rewiring the system

Given that so much of our DNA is shared with other organisms, it is how our genome is “wired up” that makes us truly different. One of the most potent applications of synthetic biology is to introduce novel ‘gene circuits’ into micro-organisms. These have been used to develop sensory systems for toxic compounds, yet these can be activated by other substances besides the target. However, Baojun Wang (University of Edinburgh, United Kingdom), has demonstrated that we can learn a thing or two by borrowing concepts from electronic circuits. “We used the concept of a double-input AND gate to act as a filter for nonspecific signals” says Baojun. These systems contain two sensors which must both be activated to produce the output. A circuit to detect zinc ions, for instance, may have two input sensors, one of which also responds to lead ions, and the other to cadmium ions. The gene for the output protein (e.g. Green Fluorescent Protein, GFP) is under the control of the AND gate output promoter which contains binding sites for each cooperative activator protein expressed from the cognate input sensor. Hence both input sensors must be activated before output gene transcription occurs. “If only lead or cadmium ions are present, only one sensor is activated and GFP is not produced” says Baojun. 

The concept of sensors can be taken further to improve the design of gene circuits that induce microorganisms to produce useful products. “These systems are highly efficient, but sometimes you want to tone production down” he says. In his “dynamic adaptive circuits”, sensors are introduced that respond to inputs such as light intensity or nutrient level by altering enzyme activity or turning on downstream genes that reduce metabolic flux.  They can also be engineered to respond to the accumulation of toxic intermediates, either by reducing production or activating ‘clean up’ enzymes. “These gene circuits can thus help to build more adaptive and balanced microbial factories” says Baojun. 

One of Baojun’s most exciting projects is seeking to engineer bacterial viruses (phages) with circuits that recognise pathogenic microorganisms in the human gut (such as E.coli and Salmonella) but not the benign species which digest food1. The phages will deliver CRISPR-Cas9 nucleases that will cleave specific sequences in the host bacteria’s genome. “Potentially, this could be developed into an oral administration to treat diarrhoeal diseases, particularly in resource poor areas” says Baojun. This could help avoid the side-effects of antibiotic treatments, which can affect beneficial gut bacteria and make nutritional disorders worse.  

Artificial Life

But perhaps the ultimate expression of synthetic biology is to completely engineer artificial cell systems. Ambitious as this sounds, Oliver Castell (Cardiff University, United Kingdom), 2016 SEB Cell President’s Medallist, has demonstrated that even simple approaches can go far. He focuses on mimicking the phospholipid cell membrane that surrounds cells and controls the flux of materials in and out. When a glass slide is coated with a hydrogel, then immersed in a lipid solution, the lipids will arrange as a monolayer at the water/lipid interface. If an aqueous drop is then added to the solution, a ring of lipids also forms around it. As the droplet sinks down through the solution, the lipids surrounding it contact the monolayer and become a bilayer. The inside of the droplet now resembles the cytoplasm of a cell, surrounded by a membrane. Transmembrane proteins can be inserted into the bilayer by including them in the drop’s contents. Because the system is based on glass slides, biological processes can then be measured optically. Ion flux through transmembrane pores for instance, can be quantified by filling the droplets with fluorescent dyes, such as the Ca2+ sensitive Fluo-8. As Ca2+ ions in the surrounding solution enter the droplet through the pore, they react with the dye, causing the pore to appear as a bright spot. 
This is a powerful tool to understand membrane kinetics, but Oliver has taken it further to inspire a novel technique for DNA sequencing.

A synthetic membrane bilayer
A synthetic membrane bilayer made using the droplet interface method.Photo: Oliver Castell


In conventional nanopore sequencing, an ionic current is passed through a protein nanopore embedded in a polymer membrane. “DNA is then ratecheted through the pore and this disrupts the ion flow” says Oliver. Because the four different bases alter the flow to different extents, the DNA molecule can be sequenced as it passes through the pore. “The main disadvantage of this system is that only one nanopore can be measured at a time, so only small genomes can be sequenced” says Oliver. “Our idea was to use our optical system to sequence DNA in a similar fashion but using multiple pores”. In his approach, the aqueous droplet contains DNA constructs tethered to bacterial streptavidin protein, Fluo-8 dye and nanopores from Staphylococcus aureus yeast. Once the droplet has docked and formed a bilayer-pore system, a current is applied to drive Ca2+ ions inside where they fluoresce with Fluo-8. The same charge also drives negatively-charged DNA out through the pores in the opposite direction, however, the streptavidin tether causes the DNA molecule to get stuck within the pore. The trapped DNA makes the pore more narrow, reducing the flow of the Ca2+ ions". “The residual fluorescence shows the flow of Ca2+ through the pore, and by that we can infer the base sequence as each has a distinct optical signature” says Oliver. Furthermore, the process can be scaled up to sequence many pores at once. 

Oliver is already looking beyond to new applications, including encapsulating stem cells in droplets to protect them from the immune system. “We’re not saying that you shouldn’t study cells anymore” he concludes. “We are simply building parallel systems to help us ask smarter questions”. 

As our technology and knowledge improve further, the future looks set to see even more innovative scientific advances. No doubt our synthetic biologists will be back with more wondrous innovations at the 2017 SEB meeting in Gothenburg. To view the complete list of abstracts from the SEB Brighton session ‘Synthetic Biology: Systems Design and Rewiring’, visit:

http://www.sebiology.org/docs/default-source/Event-documents/cross-disciplinary-abstracts.pdf?sfvrsn=0

http://gcgh.grandchallenges.org/grant/missiled-bacteriophage-enabling-controlled-infant-gut-health






 

 

 

 

 

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Caroline Wood

Caroline Wood was the SEB’s 2014 science communication intern. Since then, Caroline has been a regular contributor the SEB, reporting on events and writing insightful features for our members.
Caroline has an undergraduate degree from Durham University in Cell Biology and is currently a PHD student at Sheffield University studying parasitic Striga weeds that infect food crops. You can read her blog here.