January 30th, 2012 | Category: B. subtilis, Beginner's Guides, E. coli, iGEM, Information processing, Materials science, Media coverage, Synthetic Biology, Video | Leave a comment

iGEM showcase part 2: Materials Science

In the first post of this series I looked at the last three winners of the iGEM Grand Prize: University of Washington, Team Slovenia and the University of Cambridge. In the second post I shall cover three imaginative iGEM projects which set out to produce useful materials in novel ways; namely Blood, Bone, and Concrete. These projects were the work of the Berkeley 2007 team, the 19-strong TU Munich 2011 team and Newcastle 2010 respectively. These projects are good examples of the ambition and imagination that is so often present in the work of the iGEM teams; even if the final devices are not fully assembled, scientific and social ideas are explored and new parts are generated for future teams to use and exploit.

Blood.

The rationale behind the Berkeley Bactoblood project was straight forward, they envisaged an inexpensive, disease-free and universally compatible blood substitute,

There are currently no blood substitutes approved for use in the US or the UK, and whole blood is almost always in short supply. Developing countries have the greatest need for blood transfusions, yet many lack the necessary donation and storage infrastructure and the required pool of healthy donors.

E. coli as a cost-effective, red blood cell substitute! I am not sure if the team investigated the popularity of injecting genetically modified E. coli into the blood stream with the wider public, but a quick straw poll here (Fig. 1) suggests some reticence for the idea. Even the presence of a genetic self-destruct button for the E. coli didn’t help persuade anyone. However, the competition is about exploring ideas and generating technologies so we shouldn’t be squeamish.

Fig. 1. Biologists 'decode' DNA for the first time.

Fig. 1. The idea behind Bactoblood is explained in our office.

In order to prepare Bactoblood, the team sought several features for their E. coli: it had to be capable of transporting oxygen; the system had to be stable and inducible to a high degree; the cell could not induce toxic responses in the blood; the cell should be capable of surviving long periods of storage without refrigeration and there should be a fail-safe, self-destruct button.

It is worthwhile looking through their pages in more detail (as with all the projects). Here, for the sake of brevity, I will only briefly outline some of the devices they built. These are the devices for generating the oxygen transport capacity and the genetic kill-switch, in case anything should go wrong (heaven forfend).

To generate the oxygen carrying capacity the team needed several elements. The primary aim was to generate human haemoglobin, but they knew that this had to be processed properly (something E. coli couldn’t do on its own) and that it required extra help to fold and to remain stable. They therefore created:

  • A haemoglobin generating device comprising two human haemoglobin genes (HbA and HbB) plus the gene coding for the enzyme methionine aminopeptidase (required for correct amino acid processing).
  • A chaperone device, comprising the gene for the alpha haemoglobin stabilising protein, required for correct folding and stabilisation of haemoglobin.

Haem is a critical component of haemoglobin, and while E. coli already possesses a haem biosynthesis pathway they knew that they would need to boost production in the cell to achieve their aims. They were also aware of the molecular dangers of handling oxygen and the damage that this can do to haemoglobin molecules. To address this issue they needed parts to repair oxidative damage. They therefore created:

  • A haem biosynthesis device, comprising a four gene operon, allowing production of the enzymes which catalyse the first four steps of an eight step pathway to haem. The team reasoned that producing a haem precursor molecule was preferable to the full biosynthesis of haem due to the potential for deleterious effects.
  • Finally they prepared a four gene device to repair autoxidation damage to the haemoglobin as a result of its interactions with oxygen.

All genes for all parts were under control of the inducible T7 promoter.

In total, to provide the oxygen carrying capacity they desired, the team required 12 different genes to be expresses simultaneously, which is no mean feat. I believe from their wiki-pages that the devices were not stacked up in one chassis at a time. However, each part was tested to a greater or lesser extent, and the haem generating device worked sufficiently well to turn E. coli red-brown (Fig. 2). These devices should be available in the registry, (for example BBa_I716376) for others to investigate further.

Lysis

Fig. 2. The implications of this project are complex.

Generating a genetic kill switch was in many ways a simpler feat of engineering. The team made E. coli cells with the inducible ability to produce proteins toxic to E. coli (for example barnase and the restriction endonuclease BamHI).

The toxic proteins were present under control of the inducible PBAD promoter (described in use by Grand Prize winners Cambridge here). In the presence of arabinose, gene expression from the PBAD promoter results in the presence of barnase or BamHI in the E. coli cells.

The team tested how expression of these toxic components affected the ability of the E. coli to grow and importantly, the phenotype of the cells. They found that in the presence of the toxin, the cells lost their ability to reproduce. However, the cells remained intact and the proteome was undamaged. The oxygen carrying capacity of the cells should therefore not be be lost.

This part may well prove a useful addition to other teams building further safety nets into their designs.

Bone.

By their own admission, the TU Munich team last year,

…brought up a lot of creative solutions, which the world should have, but from our point of view, is yet still not ready for.

With great power comes great responsibility.

In fact, the concept was incredibly ambitious and futuristic: 3D tissue engineering. The team foresaw a future in which engineered bacteria are capable of precise, three dimensional and inducible production of bone.

The team understandably broke the project down into more manageable (yet still incredibly ambitious) chunks. They identified two components such a technology would require: 1) the bacteria would need to be embedded in a suitable, light transmitting matrix in which genes could be induced, and 2) precise control over which cells would express the required genes, and which would not, is essential.

For the first aim they required media that,

1. Allowed a good transmission of light to enable precise gene expression in immobilized cells.

2. Allowed the even distribution of cells within the matrix.

3. Provided an environment that ensured the survival of the bacteria.

Sadly they did not embed their cells in carbonite but instead settled for an M9 minimal media with GELRITE as the gelling agent. The media thus provides all the support the cells require. However, to get an even distribution of cells, E. coli had to be added prior to gelatinisation of the media. Temperature was critical, and if the media was too hot, the cells would be killed. They found that a heat-resistant strain of E. coli was capable of surviving the embedding process and moreover, fluorescence microscopy indicated that cells embedded in such a manner could produce GFP or RFP upon induction.

For the second part they realised that a logical AND-gate was required. Having just one wavelength of light switch on your collagen producing genes would not give the precision required … but two might:

Our most convincing approach was to design a logical AND-gate which converts two inputs in one output. In our case, it has been suitable to use two different wavelengths as inputs to induce our output – gene expression. Only when a bacterium is hit by both wavelengths it expresses a protein, e.g. a coloured molecule, to generate a three-dimensional picture inside the gel block. Our AND-Gate is built upon light sensor systems developed and optimized by Edinburgh’s iGEM-Team from 2010 and on recent results of the Voigt lab at UCSF

The design is based on the following principal, with the two wavelengths coming from two different directions precise control over gene expression could be achieved:

Fig. 2. The optogenetical AND-gate

Fig. 3. The optogenetical AND-gate.

The team embarked on a lot of cloning and construction work, including sequencing, but sadly the parts did not work as expected. However, this is the iGEM competition, and the parts (e.g. the optogenetical AND-Gate: red/blue light sensitive) have been submitted to the Registry, including thoughts as to what was not successful (is the red-light sensitive promoter to blame?). Future teams may well take these constructs further and (maybe) such devices will one day be marketed directly to the consumer?

The team also put a lot of effort into the human practises aspect of the iGEM competition and produced various outreach resources, including this introductory SynBio video:

Concrete.

The wonderfully titled BacillaFilla stepped outside of the normal iGEM model organism E. coli and into Bacillus subtilis. The aim of the project was to use an engineered B. subtilis to swim into cracks in concrete where it would produce CaCO3, filamentous B. subtilis cells and a levansucrose glue. In doing so this biological repair mechanism would help repair cracks in concrete that can lead to structural failure (Fig. 4).

Fig. 3. Catastrophic failure.

Fig. 4. An example of catastrophic failure.

Cracks in concrete allow water into the structure, which corrodes steel reinforcements, weakens the concrete structure itself and is difficult to repair. The team had identified other projects in which biological means are being deployed to help repair or maintain concrete structures, these were bioconcrete and self-healing concrete. However, they reasoned that these projects only use bacteria to repair cracks in specialist concrete in new buildings. They sought a solution in which cracks in existing structures could be tackled.

To do this they turned from the usual iGEM workhorse E. coli to B. subtilis. Why?

Firstly, B. subtilis is a soil bacterium, rather than an inhabitant of the gut, and so is better suited to living outdoors. Filamentous B. subtilis cells also have similar properties to the synthetic fibres used in reinforced concrete. B. subtilis cells plated on sucrose media produces levan, a long chain polysaccharide that acts like a glue, something that they could use to their advantage. And finally, the team knew that,

Bacillus subtilis produces urease, which catalyses the hydrolysis of urea into ammonium and carbonate (CO32-). Since the cell walls of the bacteria are negatively charged, they draw cations from the environment, including Ca2+, to deposit on their cell surface. The Ca2+ ions subsequently react with the CO32- ions, leading to the precipitation of CaCO3 at the cell surface…CaCO3 expands at the same rate as concrete, making it an ideal filler.

Furthermore, the strain of B. subtilis that they chose (168) sporolates, making it easy to store and transport.

The team embarked on the construction of several parts to achieve these aims. They used a modelling approach to determine how to boost precipitation of CaCO3 at the cell surface. They determined that in order to boost urea hydrolysis they would need to increase arginine and arginase production. They built a two parts: the first comprising a regulatory RNA responsible for repressing the activity of a protein that itself is responsible for repressing arginine biosynthesis and up-regulating arginine breakdown; and the second comprised a gene for the enzyme arginase, which breaks arginine into ornithine and urea. These parts were subsequently combined into one device and deposited in the parts registry though further characterisation is required.

The team were successful in converting B. subtilis into an inducible filamentous former,

B. subtilis cell division is dependent on FtsZ. FtsZ forms a … ring at the midpoint of the cell and contracts. YneA indirectly stops the formation of the FtsZ ring. In nature, yneA … allow[s] the cell to repair DNA damage before continuing with the division cycle … By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.

The team successfully expressed YneA, (BBa_K302012) allows the formation of filamentous B. subtilis cells.

The teams efforts were covered well by the media, most notably a positive piece from synthetic biology champion the Daily Mail, though it has to be said the science/futurism blog io9.com managed to get the best tabloid angle on the story with the delightful: the bacteria that could make you grow concrete in your mouth.

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