Team:KU Leuven/Project

One of man’s basic needs is feeding him/herself. Finding or producing food has always been a priority of mankind, of all organisms in fact. While the world’s population expands to over 7 billion resources such as land remain limited, leaving us in a desperate struggle to increase productivity of lands used and encourage innovative agriculture. Weather, disease and pests such as aphids, can reduce crop yields by up to 75% and since weather is difficult to control most farmers try to control the damage caused by disease and pests. Many farmers use pesticides and insecticides, but society has started to question whether their ‘fresh’ food isn’t ‘tainted’ by these pesticides.

Our goal is to create a sustainable system to reduce the damage that aphids cause to our agricultural industry. The current solution usually requires huge amounts of insecticides, damaging ecosystems in several ways. First, the indiscriminate weakening of insects means that beneficial insects (pollinators, ...) are also affected. Second, insecticides affect vertebrates, including humans, damaging our health (Bjørling-Poulsen et al., 2008). Third, insecticide residues (catabolites) accumulate in each food-chain. the highest organism, be it humans or for example predatory birds, will end up with potentially high levels and the negative effects.

We aim to offer an effective and sustainable alternative for insecticides. Our modified E. coli (‘BanAphids’, meaning ‘to ban aphids’ as well as with ‘benefits’) would produce the aphid’s own alarm pheromone, E-β-farnesene, (EBF) to repel them off the plant. On top of that we want to attract aphid predators such as the ladybug via the production of methyl salicylate (MeS), a phytohormone, by our E.coli`s. This way we ensure aphids are thoroughly removed from the plant.

We have established what might be possible hurdles in introducing this system in the agricultural industry. First we have to make sure that the plant cell’s metabolism is not over burdened. Besides that we have to take into account that aphids might habituate to constitutive expression of EBF (De Vos et al., 2010, Kunert et al., 2010). Finally, we do not want to attract the aphid’s natural predators when they are not needed.

We thought of two different methods to carry out our system. One method would be to spray our BanAphids onto the plants. Keeping into account the possible hurdles we mentioned before, BanAphids will produce MeS in response to an external signal that indicates the presence of aphids, in order to reduce the burden on the plant cell’s metabolism and attract predators only when needed. This external signal is honeydew, since aphids produce high amounts of this. Honeydew is a very glucose rich substance, which is the reason why ants ‘farm’ aphids, in order to milk their honeydew.

Tet repressor under low glucose promoter

AroG, LacI construct

pCaiF is a low glucose promoter, so when aphids are present on the plant and thereby honeydew, TetR will not be transcribed. pTetR, a TetR repressible promoter, will be active in this case so that lacI and aroG* will be transcribed. aroG encodes for the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which will convert erythose-4-phosphate into 3-deoxy-arabine-heptulosonate-7-phosphate. We have mutated aroG into aroG* in order to inhibit the negative feedback mechanism of phenylalanine to increase the activity of DAHP synthase so that the chorismate concentration is increased.

Chorismate pathway

The following construct will convert the chorismate produced into salicylic acid and then into MeS. The original BioBrick from the 2006 MIT team (Bba_J45700) contained a lac promoter in front of the pchBA gene. The pchBA gene encodes an enzyme that converts chorismate into salicylic acid. Since this would interfere with our system (we use LacI), we replaced this promoter with another pTetR promoter.

MIT BioBrick 2006 for salicylic acid to MeS conversion

MeS construct to convert salicylic acid into MeS

cpram is a constitutive promoter so EBF synthase will be constitutively transcribed and EBF constitutively expressed. However, there is a lac operator present and since LacI is transcribed when honeydew is present (see above), EBF synthase transcription is inhibited in the presence of honeydew. In the absence of aphids, EBF is constitutively expressed and aphids are thus repelled. However, as mentioned before, EBF could lose its aphid repellent effect due to habituation.

EBF construct

So if certain aphids do happen to escape the EBF repellent signal, the MeS acts as a counter signal and attracts natural predators of the aphid such as ladybugs and green lacewings. Aphids will activate the MeS cycle due to the presence of honeydew.

Another way to avoid habituation of the aphids is to choose for an oscillating production of EBF, which requires a colony-wide synchronized oscillating system. We also developed this oscillating system so that the BanAphids could alternate between MeS and EBF production without the use of a direct signal, the honeydew. This way, the BanAphids would not need to be in direct contact with the aphids but could be kept separate from the plant. Our second method is therefore to keep the BanAphids in a container so that they are isolated from the environment but so that MeS and EBF can still pass. We turned to the sticker model of the Groningen 2012 iGEM team to use as a container. The sticker is composed of a plastic film, whose pores are too small for bacteria to pass but through which volatiles can still pass. The Groningen 2012 iGEM team has already investigated the biosafety of this sticker.

Sticker model