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Team:KU Leuven/Project/Glucosemodel/Design
From 2013.igem.org
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, so they can milk their honeydew.
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Tet repressor under low glucose promoter
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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 erythrose-4-phosphate and phosphoenolpyruvate into 3-deoxy-arabine-heptulosonate-7-phosphate, a precursor of chorismate. 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.
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MeS construct to convert salicylic acid into MeS
The following construct will convert the chorismate produced into salicylic acid and then into MeS. The original BioBrick from the 2006 MIT team (<a href="http://parts.igem.org/Part:BBa_J45700">Bba_J45700</a>) 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 tetR promoter.
cpram is a constitutive promoter so the EBF synthase gene will be constitutively transcribed and EBF constitutively produced. 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.
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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.
