Team:UNITN-Trento/Project/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 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".

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.

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.

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