Introduction

We have decided to solve both these problems by designing and engineering a bacterial system able to control fruit's ripening in response to different stimulations: B. fruity.

Furthermore, we have planned two different commercial products in order to eliminate waste of food and to make the consumption of these fruits accessible even in unusual places like schools and offices: the "B. fruity Vending Machine" and the "B. fruity Home Edition".

We have designed an started to build a genetic circuit that activates fruit maturation thanks to ethylene production, a molecule produced by climateric fruits that affects growth, development, ripening, and senescence (C. J. Brady, 1987). However, B. fruity does not exploit the complicated ethylene production pathway of plants, because of the undesirable production of hydrogen cyanide (Shang Fa Yang et Al., 1984)!!! We instead, decided to follow a different metabolic pathway that is present in Pseudomonas syringae which involves a single enzyme: 2-Oxoglutarate Oxygenase/Decarboxylase, commonly named the Ethylene Forming Enzyme (EFE).

To inhibit maturation we selected methyl salicylate, an ester also known as wintergreen oil, that is produced by many plants, as a defense mechanism, and was shown to slow down, if used at high concentration (5 mM), the ripening process in tomatoes (Chang-Kui Ding et Al., 2002). To achieve methyl salicylate production we were lucky to use many of the parts submitted by the 2006 MIT iGEM team, as well as others built by us.

We have coupled this system to a blue light photoreceptor successfully used by other labs and iGEM teams in the past. Our system in the OFF state (no blue light) will produce methyl salicylate and stop unwanted ripening, while in the ON state ( Blue light exposure) it will produce ethylene and repress methyl salicylate production, thus promoting fruit ripening.

You can check our DATA page for a full description of the circuit.

We engineered the full system and characterized each component of the system in Escherichia coli. We have also tried to demonstrate the functionality of the enzymes involved in Bacillus subtilis.

In order to develop a possible commercial product it is more desirable to use a chassis capable to resist for a certain amount of time without nutrients. So we thought that Bacillus subtilis could fit perfectly our purpose! It can make spores and it is easy to re-activate by removing the source of stress and adding, for example, water/nutrients. Moreover, B. subtilis is not a human pathogen. It can, however, degrade or may contaminate food, but rarely causes food poisoning. Therefore, with the right precautions and attention, this chassis appear to be the best system for our project.