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From 2010.igem.org

Water Sanitation

Quite early in the project the idea of water sanitation was put forward. We initially collected ideas about different substances, such as heavy metals, and pathogens we might be able to detect using relatively simple biosensors. In this list different parasites and bacteria were included.

Pathogen detection via quorum sensing

We later considered detecting the quorum sensing molecule N-acyl homoserine lactone produced by V. cholera for example. This molecule could than activate a simple colour output in our bacteria and thus exploit pathogen functions for detection. We later dropped this idea because, although it has a powerful application, it was too similar in design to biosensors that had been designed previously in iGEM projects. There were also a number of additional problems with this idea that made us not pursue this idea further.

Detection via receptors for bacteria-parasite interactions

A step further towards our final project was the idea to detect water borne parasites. Initially we did much research on parasite-bacteria interactions in order to find a sound mechanism we could implement in our system and link to a output. Even though we found some bacteria that interact with parasites, such as Salmonella with Schistosoma in the human gut, these we more often than not non-specific interactions and thus could give rise to false positive activations of our system.

Population control

This concept aimed to keep the total number of bacteria in equilibrium below the number they would naturally achieve, making the population number oscillate around this arbitrary threshold. This might be done by some clever use of quorum sensing systems acting on survival, growths or resistance genes to influence the performance of bacteria depending on the population density. However this construct would be evolutionarily unstable, as bacteria that lost the ability to self regulate growth would inevitably proliferate and outcompete the functional bacteria.

Safe biofilms

We came up with similar approaches to making biofilms more safe, for example by making the bacteria interdependent. This might be done by promoting cell survival via quorum sensing systems, so bacteria that become detached from the biofilm would die shortly after. This might make them more safe for the environment, limiting their spread, as well as for use in medicine, preventing tissue invasion.

Cell states reporters

This idea was to monitor cell activity which might be useful for scientists in many situations. We proposed the use of cell state reporters to quickly give visual information of the activities or phase the cells are undergoing. This might help to maintain a healthy population and to determine whether population grows anaerobically or aerobically. Furthermore it could be used as a warning signal when cells undergo undesired/stressful phases and to examine if the cells grow in synchrony. Many different regulon systems could potentially exploited for this purpose including without limitation:

• Ntr and Nif system that detect ammonia limitation,
• Arc and Fnr system that detect anaerobic conditions,
• the SOS and response to UV light,
• sporulation in response to starvation and the heat shock response.

Use of system specific promoter, enhancer or repressor elements could be used to create a system that gives feed back in form of different fluorescent proteins to the degree to which the system is activated and by extension about the environment the cell is in.

Rapid response system

Another threat that continued to keep our attention was the idea of a fast response. Most products of synthetic biology require a long time for gene expression until an output such as a pigment can be seen. We considered many different approaches, basically all involving and input to stimulate a two component system. The signal transducer was to activate a previously inactive enzyme and act on a substrate. the substrate was either to be colourless and only become visible after the enzyme acted on it, or be concentrated, such as a tight chain of pigments, that are than cleaved apart to colour the whole cell. We later settled for more simplified, yet still highly efficient variant of this system to make our sensor more robust.

Fv-fragment guided input module

In order to detect molecules for which no natural receptor exists, we considered using antibody based receptors, either fused to the signal transduction portions of bacterial receptors, or by introducing the whole Fc-binding receptor of the mammalian immune system into bacteria and combining it with whole antibodies. However antibodies are complex, glycosylated proteins that cannot be expressed by bacteria easily and supplying antibodies to each detection kit would be very expensive. Furthermore introducing genes essential to the human immune system into bacteria, such as E. coli was considered a very risky idea and the human practices side of our project would be weakened.