The BioMachine

How simple can useful RNA be? Very simple

2010-02-24T20:49:21Z

Since the discovery that RNA molecules can act as catalysts in chemical reactions, they have become prime candidates for being the kick start that life on Earth need billions of years ago. Although the conventional explanation is that proteins were cooked up in the primordial oceans and ultimately became parts of living organisms, there is no clear way to get from proteins appearing randomly to self-organising systems based on proteins.

Although proteins can make efficient catalysts, they have no clear way to reproduce. RNA is now a much more likely candidate as it can pass on genetic information and catalyse the reactions needed to do it. Although proteins are now responsible for most of the body’s catalysis, RNA ‘ribozymes’ are still in active and can be found at the heart of what are understood to be some of the most ancient cellular functions.

]]> RNA plays a key role in the translation process used to make proteins from a template encoded in RNA. The chances are, according to the ‘RNA World’ theory, that RNA made more RNA templates before life became complex enough to depend on a mixture of RNA and proteins. But this leaves the question of how a sufficiently complex ribozyme came about that could catalyse production, at the very least, copies of itself.

But, what if a self-replicating ribozyme could be very simple? How simple could it be? Publishing in PNAS, a team from the University of Colorado at Boulder has found a very simple ribozyme. It has just five bases but is able to catalyse reactions with other segments of RNA. It’s not very quick, but it works and, unlike previous work on simple RNAs, does not depend on metal ions for the catalytic activity.

The work by Professor Michael Yarus hints that RNA may put together proteins quite readily. “Essential intermediates in protein biosynthesis arise surprisingly easily in the presence of very short RNAs,” they write.

“The ultimate importance of these observations may lie partly in the unknown number of other reactions that can be accelerated by comparably small RNAs. This is because for each such minuscule RNA reaction, there is a prima facie case that it would become accessible even after the most primitive ribonucleotide polymerisation.”

It’s possible that this simple catalyst is a one-off, Yarus and colleagues warn. “On the other hand, it will be extraordinarily important to look for other tiny RNA active centres, now knowin they can exist.”

It makes the RNA World hypothesis look more likely and may help in the development of very simple synthetic cells for bioproduction.

GM problem? Add more evolution

2009-12-08T21:51:38Z

Amid the excitement around being able to synthesise and edit DNA directly, it's easy to forget about some of the older methods for altering a genome. Methods that are more acceptable in places such as Europe when genetic modification has an extremely bad image.

For a new breed of potatoes designed to only produce a starch suitable for treating paper as well as foodstuffs, a team at the Fraunhofer Institute in Germany used good old-fashioned breeding to get the job done. Well almost. Good old-fashioned selective breeding with the foot hard on the accelerator pedal, using directed-evolution techniques to speed up the process of producing viable variants.

There's nothing particularly new about speeding up the process. Although protesters look upon direct genetic modification as 'unnatural', it's worth noting that plant breeders have used chemical and radiation treatment for some years to induce mutations that speed up the process of moving the candidate genomes into new territory. However, because these techniques do not carry the tag 'GM', nor do they carry the stigma.

"We are working here with natural principles. In nature, sunlight triggers changes in the genome, With chemistry, we accomplish the same thing - only faster," said Jost Muth of Fraunhofer IME in the release put together by the institute.

Normally, you have to wait after a burst of cross-breeding and mutation to see what crops develop. Not in this case. As soon as the seeds germinated, samples of the leaves were taken and their genomes analysed directly to see which mutants had picked up desired traits.

The researchers analysed 2748 seedlings to find a genome that had the genetic profile they were aiming for: the ability to produce amylopectin exclusively. Luckily the potato already has an amylopectin-production gene, which reduces the amount of mutation that the potato has to go through. However, the aim was to find a mutant that could shut off the production of sister starch amylose, which the 'Tilling' potato could. This avoids the need to purify the starch after harvesting and separation.

This autumn, the team grew about 100 tonnes of potatoes with the required genome, and without specialised GM trials. "Special measures aren't necessary, because the Tilling potatoes are totally normal breeds that contain no genetically modified material," said Muth.

The name Tilling is derived from the name the team gave to the process: targeting induced local lesions in genomes, a technique developed in the late 1990s in Seattle.

In an artificial cell, what is a gene?

2009-10-21T20:34:57Z

The Innovation, Universities, Science and Skills Select Committee has published a set of UK government responses for its 'Evidence Check' programme on topics ranging from homeopathy to synthetic biology. Although the response from the Department of Health on the status of homeopathy in the health service is a little disconcerting, the most interesting response is from the Health and Safety Executive on possible future regulation over synthetic biology, particularly artificial cells.

The executive takes the view that most of the existing work on synthetic biology is already covered by regulations on work with recombinant DNA, which seems fair enough. However, the response adds:

"Future work may involve the creation of artificial cells, which would not fall within the scope of existing legislation. Consequently, a minor amendment is being proposed to the definition of GM as part of the development of a single regulatory framework for work with human and animal pathogens and GMOs. This will enable the regulations to cover artificial cells, should the technology develop in that direction. This change will be consulted on prior to the implementation of the new regulatory system."

The amendment for the single regulatory framework, the executive adds, is likely to extend the definition of genetic modification to include the "introduction of genetic material into a cell artificially created for that purpose, where the cell is then capable of replication or of transferring genetic material".

I can see two potential issues with this. One is what happens if the definition of artificial cell extends to bioreactors? Will that make the operation of biobreweries more difficult? And, more fundamentally, what does 'genetic material' mean in this context? Early artificial cells will doubtless use standard DNA but does the definition of gene cover synthetic nucleic acid strands, such as peptide nucleic acid or alternative 'genetic' systems based on alternative nucleic acids, or molecules with a similar function?

Two grants for Wilson Center

2009-09-23T06:47:44Z

The synthetic biology project based at the Woodrow Wilson International Center in Washington DC has picked up two grants from the US National Science Foundation.

One grant is to support a workshop that will host EU and US researchers looking at what synthetic biology can do, if anything, for sustainability. The University of Virginia will work with the Wilson Center on organising the workshop.

The second grant is to explore how online prediction markets - which have controversially been touted as ways to predict things from terrorist attacks to financial meltdowns - can be used to tap into the public mood on synthetic biology.

"Although online prediction markets have attracted significant interest from scholars and increasing application in corporate environments, little work has been done to apply these markets to critical issues in science and technology," said David Rejeski, director of the project at the Wilson Center in a statement.

Pointers - 6 August 2009

2009-08-06T09:29:54Z

Bio-design automation and synbio tools - The Design Automation Conference (DAC), held recently in San Francisco, concentrates on electrical engineering. But there are a lot of parallels between electrical design and the current state of synthetic-biology design. Melanie Swan reports on the design-automation tools that appeared at the First Workshop on Bio-Design Automation that was held on the first day at DAC.

I missed this in June when it first turned up but you can browse BioBrick parts on your iPhone with software from OpenWetWare. It's in the App Store here.

Pointers - 29 July 2009

2009-07-29T10:42:02Z

Ethical perspective defending the patenting of DNA - some interesting thoughts but I'm a bit concerned that the bit about public libraries doesn't mention the fact that the Registry of Standard Biological Parts contains patented material and so often only gets used with a research exemption.

Harvard hits the fast-forward button on directed evolution

2009-07-28T14:51:33Z

The Multiplex Automated Genome Engineering (MAGE) technique developed in George Church's lab at Harvard Medical School provides a promising alternative to whole-genome synthesis. It's certainly going to work out a lot cheaper than writing a million base-pair sequence from scratch and can cope with situations where the changes needed to something such as E coli involve edits throughout the chromosome.

In their paper for Nature that was published online over the weekend, Harris Wang, Farren Isaacs and their co-authors describe how they used MAGE to essentially perform rapid prototyping on a genome and increase the amount of the chemical lycopene that E coli could produce. It takes advantage of an aspect of MAGE that lets you tune how much variation out of the edits the system makes to a genome.

Talking about MAGE last year at the Royal Society, Isaacs explained how it was being used to perform hundreds of edits on the E coli genome in a bid to rework the genetic code itself to build a wider range of proteins than natural genetic systems can today. The first step was to remove one of the stop codons used by E coli to terminate translation. All of the 300 or so instances of the sequence TAG would be replaced by the more common TAA variant of the code. This allows the three-base TAG codon to be redeployed as the code for a non-natural amino acid. Isaacs said initial results from this project are nearing completion.

Full-scale codon replacement is the kind of thing you can do if you push MAGE through many cycles. The process starts off with a single, isogenic genome. As MAGE proceeds, the replacement pieces of DNA do not supplant the original sections all at once. So, if you have a lot of simultaneous replacements to make, there will be a lot of variation between the genomes of individual cells. But, as you push MAGE further, ultimately all the sections get replaced and you wind up with a new isogenic genome. To get many different mutants, you simply stop the MAGE process in the middle and analyse what comes out. In the latest experiment, the process came up with more than 4 billion genomic variants each day. They then isolated variants that showed a significant increase in lycopene production - finding one that had a five-fold increase over the wild type in just three days.

The core of the MAGE techique is a protein used by the λ-Red virus to introduce its own changes into a bacterial genome with a little help from genetic engineering. The β protein binds to oligos that are intended to replace sections of DNA in the actual genome. The protein helps the sections displace the Okazaki fragments that the cell's own machinery uses to build complementary DNA strands on the lagging strand during DNA replication. Each end of the section provides a match to the original DNA to let it stick, with the new 30 base pairs or so of DNA lying somewhere in the middle. Normally, the cell's mismatch repair proteins would spot this alien DNA - because it does not marry up with the original complementary sequence - but one of the key genes for the repair mechanism has already been knocked out.

When the DNA replicates again, the new DNA is copied and becomes fully part of the genome. Some of the replacement fragments don't make it or, in the case of this experiment, are replaced by other near matches, which gives rise to the huge genetic variation at the edit points.

The edit points are not picked at random. Isaacs explained that some of them are simple knock-outs, putting stop codons in the middle of genes that might divert feedstock chemicals away from the pathway that produces lycopene. The other target was the gene that codes for the protein that makes lycopene itself. One of the directions of Isaacs' research is on the efficiency of translation as mRNA is used to produce the final proteins.

The Shine-Dalgarno sequence (TAAGGAGGT) in the section of messenger RNA where the ribosome first attaches generally boosts translation efficiency but it does not work in all cases. The structure of the messenger RNA seems to have an effect. Rather than try to design the most-efficient sequence a priori, Wang and Isaacs decided to let evolution have a go. They developed a variety of oligos that contained subtly different variants of the Shine-Dalgarno sequence and added them to the MAGE pool. They could then pull out the one that worked the best at the selection stage. The first selection of variants were made by looking for colonies that produced an intense red pigmentation on Luria-Bertani agar plates with the best performers screened from the ten thousand or so that process identified.

Isaacs thinks the technique can go much further and use evolution to pick out winners from more extensive reworkings of the genome, acting on promoters as well as ribosome binding sites. It will, in a sense, press the fast-forward button on evolution by letting biology explore genetic states that might be inaccessible because single changes on the way to them would kill off the candidate cells. Experiments such as the one performed by Mark Isalan and Luis Serrano and the team at CRG in Barcelona have shown how swapping bits of gene promoters can alter the fitness of bacteria. And these techniques could show how regulatory networks of genes affect cell behaviour.

"We want to think of general strategies that we can use with this method to introduce diversity with a universal oligo pool, and allow the cells to explore new genetic landscapes that will confer new properties. And then come up with screens and selections that let us pull out cells with specific behaviours," said Isaacs.

The aim is to target all 4000 genes of E coli with on the order of 15 000 oligos. "We could target every known coding and regulatory locus. You are seeing just the beginning of what we are trying to do," said Isaacs.

The core MAGE technique could work in other cell types, such as yeast, Isaacs claimed: "The mechanism that we use is something that is conserved across biology."

Pointers - 14 July 2009

2009-07-14T14:23:04Z

The New York Times reports that Exxon and J Craig Venter's Synthetic Genomics are to work on biofuel-producing algae - presumably using some of the genetic components that Venter's researchers have been capturing from a wide variety of organisms on their bulk genome-sampling missions.

Lloyd's has taken a look at synthetic biology and decided insurers need to wise up over "systemic risks". According to Insurance Daily, the report says: "The enormity of some adverse scenarios suggests the inclusion of various forms of sub-limit in the future."

The Lloyd's report itself is available alongside the news release.

Pointers - 11 July 2009

2009-07-11T09:12:08Z

Nature blogs on concerns over the public acceptance of synthetic biology at the recent Washington DC conference organised by the OECD. And whether the technology needs a new name, like "shiny, happy biology". The blog post doesn't mention it but Drew Endy was referring to a joke made at the SB 2.0 conference in 2006 over whether "synthetic biology" was the right name.

Local news for Bostonians on an MIT competition for clean energy. Cambridge, MA-based InAct Labs, which is working on microbial fuel cells, is one of the semi-finalists.

Protein design: why copy nature?

2009-07-10T21:12:06Z

Nature, 19 March 2009: Design and engineering of an O₂ transport protein by Ronald Koder, JL Ross Anderson, Lee Solomon, Konda Reddy, Christopher Moser & P Leslie Dutton

Most protein design consists of tweaking an existing, natural structure, often using directed evolution with the occasional bit of deliberate peptide insertion so that it will bind to a specific molecule. Take the work of Angela Belcher's team at MIT, for example, where the coat protein was altered to include an amino acid known to bind to carbon nanotubes or to iron phosphate.

The idea of deliberate protein design suffered a serious blow when Professor Homme Helliga and researchers in his lab had to withdraw a couple of papers. Collaborators were unable to repeat Hellinga's results - instead of creating a structure from scratch that could act as a ribose binding protein, some of the wild-type protein wound up in the experiment and was responsible for all the desired activity.

One big problem is that proteins are complex structures. But much of that complexity comes from the way evolution works, with bits of genes getting copied, inserted, knocked out and mutated.

"This complexity frustrates biochemists seeking to understand structure and function and presents an extraordinary challenge to protein engineers who aim to reproduce or create new functions in proteins," argue the authors of the Nature paper.

If you design a protein from scratch, is it worth following the example of the natural world? "However common it may be in nature, we maintain that complexity is not an essential feature of protein as a material, nor is it an essential feature of catalysis, as shown by synthetic chemical systems," the authors reckon.

Eric Drexler of nanomachine fame, agrees: "...close adherence to natural models is often intellectually and technologically crippling."

The idea behind this paper is to use protein structures that are much simpler than those employed by nature, and more akin to regular catalysts.

How big is your pond?

2009-07-09T17:31:47Z

Lingchong You and colleagues at Duke University claim that, although there are many variants of quorum sensing in bacteria, there seems to be one constant: the total volume of bacteria in relation to the volume of their environment is a key to quorum sensing, no matter what kind of microbe is involved.

"If there are only a few cells in an area, nothing will happen," said Anand Pai, who has worked with You on the project. "If there are a lot of cells, the secreted chemicals are high in concentration, causing the cells to perform a specific action. We wanted to find out how these cells know when they have reached a quorum."

The researchers write about the project in the July 2009 issue of Molecular Systems Biology.

Synthetic biology in Second Life

2009-07-09T17:15:34Z

Forget furry encounters and virtual people glued to unreal fruit machines, members of the iGEM team based at Calgary in Canada have decided to make a real use of the virtual-reality space Second Life. And it looks as though the work by Patrick King and colleagues will make use of the scripting and visualisation features of Second Life as well as the meeting-space environment that most people (as least those that have stayed) use:

"The most often touted feature is that SL can offer a classroom-like environment for people at any distance from one another. While the iGEM Calgary island will make an excellent hangout for idle igemmers the world over, our focus is less on creating a classroom, and more on presenting concepts directly. We want to make it easier for new students to grasp the basics of synthetic biology by making it accessible and interactive. This is where SL's object creation and scripting facilities come into play: we can create anything we want, from molecules to cells to lab equipment, and then make it behave like the real thing."

The environment will not just be for the Calgary team, King writes:

"My number one goal for this project is for it to be useful to others, especially early university or high school students just beginning with iGEM, but also biology students in general, and the public. For it to be useful, it must be used; feedback on the accuracy of our work is essential! I hope that Lindsay Island will be open to the public near the end of the summer, but the real test will not come until iGEM 2010, when we will meet our first batch of fresh students."

SBGN goes synthetic

2009-07-05T19:20:22Z

One of the papers in the Royal Society Interface special issue on synthetic biology takes a look at the potential for a standard graphical notation for engineers working on system design. As one of the authors is Hiroaki Kitano, the proposal, naturally is for the Systems Biology Graphical Notation (SBGN) coupled with the Systems Biology Markup Language (SBML).

The authors concede that SBGN may need extensions to handle the constructs that synthetic biology engineers want to use but argue:

"Sharing of symbols representing identical biological elements would further help in developing a common graphical lingua franca for biological engineering, on the same lines as in electrical circuit diagrams and other advanced engineering disciplines.

"We strongly believe that careful collaboration on the visual as well as model representation aspects between the two communities would foster the development of a standard graphical notation schema and accelerate the application of computational techniques."

However, one lesson from electrical engineering is that graphical languages do not last long. Circuit diagrams cope well with simple analogue and digital circuits in electronics but as soon as things get complex, text tends to win out. Just look at the way in which textual languages such as Verilog and VHDL pushed graphical schematics to the margins. The textual representations also deal with the idea of parallel operation better. And in synthetic biology, it all happens in parallel.

Synthetic biology's ethical catch-22

2009-07-05T17:05:04Z

Writing for a special issue of Royal Society Interface on synthetic biology, Steven Yearley, a member of the Genomics Policy and Research Forum sponsored by the UK's Environmental and Social Research Council, claims the regulatory and ethical concerns around the technology go hand-in-hand with the hype. And, to a certain extent, agrees with the idea that Big Promise technologies, by having bold claims made for them, wind up the concern to the point that extra regulation becomes inevitable:

"...once these assertions about far-reaching novelty or widespread applicability are made the regulatory implications are hard to avoid. The more strongly the claims are put forward, the more powerful the apparent regulatory logic.

"Proponents of synthetic biology need to make claims about its startling novelty and wide-ranging implications if they are to win support, yet they cannot make these claims without simultaneously raising questions about suitable safety and regulatory standards."

I'm not entirely convinced by this. Although you can see the effect proposed by Yearley reflected in the concern over nanotechnology, I think synthetic biology poses greater ethical and regulatory concerns to people because of the issue of dealing with life. You also have the shadow of genetically modified organisms hanging over it, which has encouraged government-funded organisations to focus very much on ethics and regulation in the hope of heading off another GMO crisis.

However, it is interesting to consider how things might have gone if J Craig Venter had not beaten the drum so hard on his lab's work.

Yearley's main point is that any ethical review of synthetic biology has to dispense with the kinds of framing used for bioethics so far. In other words, bioethics is not up to the job of determining the ethics of a synthetic biology. This is not as bizarre as it sounds: Yearley's argument is that bioethics narrowed its focus because that was what the main players wanted. Yearley does not make the argument directly but mentions an issue raised by the 1999 paper by Cho and Caplan, which was the first foray into this area, that because synthetic biology could challenge the popular view of what gives the living life any ethical debate has to take that into account. This is not something that traditional bioethics has had to deal with much, other than deciding at which point an organism has a distinct identity.

Synbio focus group fun

2009-06-25T14:37:38Z

It sounds as though there was a good crowd who took part in the extended discusssion sessions for the latest Royal Academy of Engineering (RAE) survey on attitudes to synthetic biology:

"...but you don't know where these things will end up...maybe creating babies...in two weeks..."

"There are nicer ways to create babies."

"Not at my age!"

Exchange between female participant in her fifties and an observer.

Intriguingly, the participants felt that totally artificial organisms would be safer or better than modifying existing creatures, which is a bit of a problem for just about any synthetic biology programme underway right now. The core reason seemed to be the feeling that totally artificial organisms would be easier to control. Although this smacks of Jurassic Park at work, it's easy how people might see modified organisms as more problematic based on earlier bioremediation plans going awry - such as the introduction of cane toads to Australia.