Introduction

Have you ever thrown away some bananas because they were too ripe? Are you one of many that hates waiting ages to eat the immature kiwis that you find at the supermarket? Do you know how bananas and other fruits are picked unripe from the tree and arrive to the supermarket ready to be sold and eaten? Have you ever thought about how much fruit is wasted in restaurants, markets, and industry?

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 stimuli: B. fruity.

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

How does B. fruity work?

We designed and started to build a genetic circuit that allows our bacteria to synthesize ethylene in order to boost fruit maturation. Ethylene is a molecule naturally produced by fruit and it affects growth, development, ripening, and senescence. (C. J. Brady, 1987) However, B. fruity does not exploit the complicated pathway present in plants, because of the undesirable production of hydrogen cyanide!!! (Shang Fa Yang et Al., 1984) Instead, we decided to follow a different metabolic pathway, present in Pseudomonas syringae, which involves only one enzyme: 2-Oxoglutarate Oxygenase/Decarboxylase, an Ethylene Forming Enzyme (EFE).

As ripening inhibitor, we went for methyl salicylate (MeSA): an ester also known as wintergreen oil and naturally produced by many plants as a defense mechanism. It was previously shown to slow down the ripening process in tomatoes, at high concentration (5 mM). (Chang-Kui Ding et Al., 2002) To achieve its production we used parts submitted by the 2006 MIT iGEM team, as well as others which we built ourselves.

How is B. fruity activated?

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, in the absence of ethylene, stop unwanted ripening, while in the ON state ( Blue light exposure) it will produce ethylene and repress methyl salicylate production, thus promoting fruit ripening.

Fig. 1: A schematic rappresentation of Ethylene production regulated by a photo-inducible circuit. The inverter ensures that in presence of Blue Light, 2-Oxoglutarate Oxygenase/Decarboxylase (EFE) gene can be expressed.

Fig. 2: A Schematic rappresentation of Methyl Salicylate production regulated by a photo-repressible circuit. Blue light inactivates the blue receptor cassette, resulting in a inhibition of MeSA producing cassette.

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

Why B. fruity?

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.

...Extras (link to fruit applications supplement)...

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