Team:UNITN-Trento/Project/Introduction

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, Plant Physiology 1987, 38: 155-178) However, we did not engineer B. fruity to use the complicated ethylene synthesis pathway present in plants, because of the undesirable production of hydrogen cyanide!!! (Shang Fa Yang et Al., Plant Physiol. 2001, 126(2): 742–749.) 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., Food Chemistry 2001, 76 213–218). To achieve its production we used parts submitted by the 2006 MIT iGEM team, as well as others which we built ourselves.

Do you know how plants produce ethylene? Do you know that ethylene is used commercially to ripen some fruits before they enter the market? Do you know that fruit is generally classified in two main categories? How is B. fruity activated?

We envisioned a system that is coupled to a blue light photoreceptor, the same receptor that has been used previously by other labs and iGEM teams. Our system in the OFF state (no blue light) will produce methyl salicylate and, in the absence of ethylene, will stop unwanted ripening (Figure 2), while in the ON state ( Blue light exposure) it will produce ethylene and repress methyl salicylate production, thus promoting fruit ripening (Figure 2).

Thus far we have succesfully built the device shown in Figure 1 plus many other functional genetic constructs that you can find in the Parts page.

Fig. 1: a schematic representation 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 representation of methyl salicylate production regulated by a photo-repressible circuit. Blue light blocks the blue receptor cassette inducing activity, resulting in the repression of MeSA production.

You can check our DATA page for a full description of the complete 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 in Bacillus subtilis.

In order to develop a possible commercial product it is more desirable to use a chassis able to survive without nutrients for a longer time: we thought that Bacillus subtilis could fit perfectly our purpose! It can make spores and be easily re-activated by removing the source of stress and adding, for example, water/nutrients. Moreover, B. subtilis is not a human pathogen. Although this bacterium can degrade or contaminate food, with the right precautions this chassis seems to be the best system for our project.

Follow our results to discover how we successfully ripen fruit!