January 6th, 2012 | Category: iGEM, Information processing, Multimedia, Synthetic Biology, Video | One comment

iGEM showcase part 1: Grand Prizes

The iGEM competition has grown dramatically in the first ten years of its existence. The competition, which is driven by the enthusiasm of the undergraduate students taking part, has accomplished many amazing things. I will be posting a series of pointers towards fascinating projects for those keen to take part or for those simply interested in the potential of synthetic biology. In this first part I thought I would take a look back at the teams awarded the grand prize in the last three years. The projects involved health, fuel security and foundational technologies of synthetic biology.

2011

Last year the Grand Prize went to the 23 strong team from the University of Washington for their two projects Make it or break it: diesel production and gluten destruction, the synthetic biology wayThe break it part of the project took a remarkable experimental approach to solving a significant health issue.

Fig. 2. An opportunity to show some plants (even if they are the bad guys).

Fig. 1. Barley looking harmless.

Gluten is a protein present in wheat, barley and rye (Fig. 1). In people suffering with Coeliac’s disease, gluten in the diet results in inflammation of the gut and a decrease absorption of nutrients. Symptoms range widely in severity from diarrhoea, fatigue and weight loss through to abdominal distension, anaemia and neurological symptoms. There is no treatment except for the elimination of glutens from the diet.

In many patients the immune response appears to be triggered by proline/glutamine rich regions of the gluten (known as gliadins) and the students reasoned that a protease capable of breaking such bonds (whilst remaining active within the stomach) would be a good starting point for drug design. One candidate enzyme capable of digesting gliadins is already undergoing clinical trials, however the enzyme’s optimal pH of 7 is a long way from the pH in the stomach. Attempts to enhance its activity at gastric pH have not had great success and this is where the students felt they could act.

The approach they used was remarkable. They turned the problem on its head and instead of looking for enzymes that would cut gliadins, they looked for enzymes capable of performing protein hydrolysis at gastric pH, regardless of substrate specificity. They then sought to engineer substrate specificity for gliadins post-hoc.

The team identified Kumamolisin-As as a likely candidate for engineering. They then sought to alter the properties of the enzyme using a combination of computer assisted mutation design (foldit) combined with the generation and screening of over 100 different mutations to the Kumamolisin-As gene sequence. This resulted in a low pH, gliadin cleaving enzyme with activity seven times greater than the control enzyme (which just happens to be the enzyme currently under trial). The part was submitted to the Registry and iGEM teams in the future may take this work on even further.

As if tackling a serious health issue was not enough of a challenge, they also took on climate change and fuel security. Make it saw the students following the method published by Andreas Shirmer et al. (2010) for the microbial biosynthesis of alkanes. In their own words,

Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols and biodiesel … However, current biofuels consist of drastically different compounds from those found in petroleum … Current biofuels contain either alcohols or long chain esters. Both of these molecules contain oxygen, which dramatically changes chemical properties … [they] are more corrosive than unreactive alkanes … both in pipelines, and in engines. … The fatty acid methyl esters in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids. Biodiesel has a higher freezing point than diesel, causing engine fuel filter clogging at low temperatures.

The team’s objective was to generate BioBrick parts for submission to the registry that were capable of producing drop-in fuels (alkanes) in E. coli. The availability of such parts would allow teams in subsequent years to develop this technology further. As a result of their efforts they succeeded in making the ‘PetroBrick’, a BioBrick part capable of generating alkanes in E. coli. Importantly, they were also able to demonstrate that optimising the growth and induction conditions dramatically improved yield. The PetroBrick has been submitted to the registry and the availability of these parts should provide a platform for subsequent improvements.

2010

In 2010 a team from Slovenia revolutionised the cellular function of DNA. They decided to move beyond DNA as a means of information storage and envisaged its use as a molecular scaffold, providing order and structure for biosynthetic pathways. The team hypothesised that biosynthetic pathways would operate more efficiently if the enzymes were arranged in an assembly line. Tethering enzymes to scaffolds has been shown to improve yields previously, either by fusing enzymes directly to each other, or by utilising a protein scaffold.

The students utilised different DNA binding proteins (zinc finger motifs) fused to the proteins of their choice. In this instance they used violacein biosynthetic proteins (more on these earlier later in 2009). In the presence of the DNA scaffold the biosynthetic proteins arrange themselves in biochemical order. Such scaffolding was shown to increase yield but also prevented side reactions and the accumulation of toxic intermediates. Given that the sequence of the DNA program defines the arrangement of the functional proteins, the students were able to modify the DNA sequence and re-arrange the enzymes to alter the final product.

The tools they developed were also deployed to generate and model oscillators. Oscillators are an important mechanism for processing and integrating information and maintaining homeostasis within a system. Preparing oscillators genetically can be tricky and sometimes it is easier to get a computer to do the work. The Slovenian team were able to use the binding of the zinc finger domains as artificial repressors of DNA transcription where each zinc finger protein acts as repressor to the next gene in a sequence, forming a circular repression scheme.

2009

This was the year that the team from Cambridge were awarded the grand prize for E. chromi an information processing device that detects environmental signals, processing the information to ensure a particular threshold concentration is present, and then returns the result in a format detectable with the naked eye. Such a device could be used to detect water pollutants or diagnose disease (see video below). As ever the parts from this project have been submitted for use in future competitions (e.g. 2010 winners Slovenia) and likewise, the team relied on parts previously submitted.

The students designed and characterised two sets of parts, sensitivity tuners and colour generators. These parts are used to tune the sensitivity of a particular promoter to detect an appropriate concentration of the target compound and to report the presence in a cheap, user-friendly fashion. As the team explain,

The Parts Registry’s repertoire of input-sensitive devices is incredibly varied. Teams have engineered E. coli to be sensitive to a wide range of environmentally significant compounds, including arsenic, mercury, lead, cyanide, etc., to genetically engineer biosensors as an alternative to other technologies. The Cambridge 2009 iGEM team identified two stumbling blocks to biosensor design.

1. Output: Previous iGEM biosensor projects have used pH, electrical conductance, and fluorescence as output. However, these reporter mechanisms require further steps to read the output. While this is acceptable for First World applications, for biosensors to have true Third World applications, a simpler output is necessary.

2. Response to Input: By utilizing an input-sensitive promoter, the biosensor is limited by the sensitivity of the promoter. For example, the promoter might be sensitive to input concentrations which have no real world meaning. The promoter’s sensitivity could be too high, so it reports concentrations below levels of real-world interest. Alternately, the promoter’s sensitivity could be too low, so it reports concentrations above those which mark the boundary between “safe” and “dangerous.” A second limitation is the the behavior of the PoPS output from the promoter; for example, output may vary linearly with input. This type of response is incompatible with a digital “safe” or “dangerous” output.

For the sensitivity tuners the team characterised how phage activator and promoter combinations downstream of a sensor promoter altered the level of expression from a second inducible promoter. The concept is that the sensor promoter, in response to external stimuli, generates an activator molecule. These activators in turn interact with an activator sensitive promoter which drives expression of the output genes (Fig. 2). By characterising the response of different combinations of phage activators and promoters the team were able to dictate the rate of transcription of the output genes in response to the external stimuli.

Fig. 2. The concept behind Cambridge 2009's pigment generating device.

Fig. 2. The concept behind Cambridge 2009's pigment generating device. Image: Cambridge iGEM 2009.

For this system they built on work from Cambridge iGEM 2007, who generated a PoPs amplifier device: a device designed to set the rate at which RNA polymerase molecules pass along DNA (PoPs roughly meaning the ‘current’ for gene expression). Their sensor promoter was the araBAD promoter (used in pBAD vectors) which is is positively and negatively regulated by the transcription factor AraC and AraC’s interaction with arabinose (Schleif 1992).

For the output, the team relied on generating in-house pathways (violacein and melanin and the references therein), and was derived from work of previous iGEM teams (carotenoid biosynthesis from the work of Edinburgh 2008 and Guelph 2008). The genes required for the synthesis of these pigments were then place downstream of the PoPs device giving an expression level of their choice.

This project was also notable because of the involvement of designers Alexandra Daisy Ginsberg and James King, who worked with the team to explore the potential of this new technology whilst it was being developed in the lab. They designed a timeline proposing ways that a foundational technology such as E. chromi could develop over the next century. Not all of them good.

I shall leave you with their video (warning: contains coloured poo).

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