Life, but not as we know it

To paraphrase Star Trek medic Dr James McCoy: it's life, Jim, but not as we know it. It's obviously intelligently designed.

Professor Chris Voigt's living inventions include microorganisms programmed with DNA-coded logic and operational circuits. He has equipped bacteria with genetic machinery to synthesise and secrete super-strong spider silk, and developed bacterial compounds to seek out, invade and destroy cancers.

Voigt's laboratory at the University of California, Berkeley, is at the forefront of a new era in genetic engineering, an era that goes far beyond simple one- or two-gene additions or deletions. He and his co-workers are collecting a constructor kit of genes from many different species, and interconnecting them to perform complex, multi-stage biosynthetic processes. They use accelerated mutation to boost their output, and recombination to fine-tune the function of their proteins.

Voigt, a plenary speaker at this year's Lorne Conference on Protein Structure and Function, predicts that within two decades, biotechnologists will be custom-designing microbes from scratch, to synthesise industrially valuable products or perform tasks like bioremediation or carbon sequestration.

It's not blue-sky dreaming - Voigt says some projects, like the Salmonella bacteria that spin spider silk, are "going pretty well", and are approaching commercialisation.

Voigt and his colleagues started out with what they describe as 'toy tricks' - for example, bacteria that synthesise compounds that darken on exposure to light. A bacterial lawn on an agar plate can then be used to take a photograph when light is shone on it through a mask-type negative.

Another trick is to insert a genetic logic gate in the gene circuits that detects edges between light and dark, sharpening the image. Similar circuits can be designed to perform logical operations and simple calculations, to enhance control of synthesis processes.

Like industrial production lines, synthesis processes must be performed in a set sequence, so the biochemical circuits also require inbuilt timers.

The Berkeley team has now moved on to more complex and practical applications, like the spider-silk project.

"Our idea was to program the bacteria to perform a series of processes, in much the same way that assembly lines build complex products, to make fibres out of spider-silk proteins," Voigt says.

"We've given the bacteria the ability to make spider-silk proteins, put them into the growth medium, and cut the fibres to form threads. We can encode all these steps in the same cell."

Although the resilient silk of a species like the golden orb spider, Nephila, which is stronger than Kevlar, might seem ideal for weaving a super-strong, lightweight fabric, Voigt's team has refined natural selection's handiwork for a novel medical application: making new arteries.

"With gene-synthesis technology, we don't need to go out into the wild and collect spiders for their silk genes," he says.

"We use databases to search for silk genes from spiders around the world, and recombine selected sequences to create synthetic genes for silks with the properties we want."

The beauty of spider silk, he says, is that it is non-immunogenic in the human body, and combines great strength, low mass, durability and elasticity. It looks an ideal material for replacement arteries. Synthetic arteries must be compliant - capable of stretching and contracting to accommodate sudden, large pressure changes as the heart beats.

Genes for human proteins can also be inserted into silk-gene sequences to add functionality - for example, to enhance colonisation by human cells.

---PB--- Protein export

Currently, vascular surgeons perform heart-bypass operations with arteries taken from the legs, whose diameters are usually an imperfect match for the coronary arteries to which they have to be joined.

The more familiar applications for super-strong fabrics woven from synthetic spider silk include bullet-proof vests more resistant to penetration than Kevlar, super-lightweight parachutes that could fold up to pocket size, or near-indestructible lightweight sails for high-performance racing yachts.

Voigt's team chose Salmonella bacteria because they have syringe-like transmembrane channels that inject toxins into the cells of their mammalian hosts. These structures can be co-opted to export proteins from the cell interior into the growth medium.

As a toolkit component, the syringes can be employed for any biosynthetic process requiring the protein product to be exported.

They can also modify genes in a way that allows the host microbe to produce the "right stuff" from animal genes - normally, bacteria are unable to perform processes like glycosylation, the addition of sugar molecules that fine-tune protein function.

Voigt's team is collaborating with another group at Berkeley that is designing microfluidic devices to mimic spider spinnerets - the tiny nozzles that secrete silk protein fibrils, to be woven into super-strong threads.

Bacteria are preferred to yeast as biofactories. Voigt says their simpler, more stable genetic architecture is more accommodating of transgenes. Yeasts tend to eject transgenes from their chromosomes.

As for applications, blue sky is the limit. "Biofuels is a big one, and a big part of what we're doing," Voigt says.

"We're trying to develop cells capable of secreting cellullase enzymes to digest waste agricultural products and convert them into fuels like alkanes and longer-chain alcohols, which have greater energy density than ethanol."

There is also potential to create designer algae or bacteria to capture carbon dioxide from coal and oil-fired fired power stations and synthesise it into biofuels - effectively using greenhouse gas emissions from fossil fuels as a renewable energy source.

As an example of the potential of the new technology, Voigt and his colleagues described a potentially revolutionary cancer therapy, based on re-engineered E. coli bacteria, in the Journal of Molecular Biology in 2006.

They engineered the microbes to express the invasin protein from Yersinia pseudotuberculosis, a cousin of the plague bacterium Y.pestis. Invasin allows Yersinia to invade its host's cells.

They propose to place expression of the invasin gene under the control of environmental sensors selected to distinguish between normal and cancerous cells by detecting biochemical cues exclusive to cancerous cell lines - when the microbe detects the cancer, it activates the invasin gene and penetrates the cancerous cell.

They also equipped the E. coli with a quorum-sensing biochemical circuit from the luminescent bacterium Vibrio fischeri lux, and a hypoxia-sensitive promoter from the fdhF (formate dehydrogenase F) gene from E. coli.

They also experimented with an arabinose-inducible araBAD promoter sequence, as an alternative to the hypoxia sensor.

The quorum-sensing circuit allows the bacteria to congregate in large numbers, while the fdhF promoter activates synthesis of a cytotoxic agent under hypoxic conditions - tumour tissues are typically hypoxic because of inadequate blood supply.

The engineered bacteria invaded the cancerous cells at densities greater than 108 bacteria per millilitre, after activation in an anaerobic growth chamber. Where the arabinose-inducible promoter was used, activation was via growth in a 0.02 per cent arabinose solution.

Voigt says that this approach could be used to engineer bacteria to sense the microenvironment of a tumour, and respond by invading the cancerous cells and releasing a cytoxic agent - presto, a highly selective tumour-killing therapy, with none of the toxic effects of chemotherapy or radiotherapy.