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Team:KU Leuven/Project/Ecological/wetlab
From 2013.igem.org
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<h3>Natural Enemies</h3> | <h3>Natural Enemies</h3> | ||
<p align="justify">A description of the natural enemies we received from biobest and employed in our experiments is given <a href="https://2013.igem.org/Team:KU_Leuven/Project/aphidbiology#Model%20Organisms"> here.</a> The predatory insects, ladybugs (adult and larvae), green lacewings (larvae) and parasitic wasps (adult) were subjected to a choice experiment. Starting from a determined point on our set-up (see figures), the insects were released and given a choice of a certain MeS concentration or a control solution which was the solvent used. The insects were given 1-3 mins to make a choice and there was a 'finish line' implemented so that after the insect crossed the 'finish line', it has made its choice. We first diluted the pure MeS solution in 97% ethanol to obtain five different concentrations ranging from 10^-9 to 10^-11. We chose to use ethanol due to the fact that it evaporates easily, leaving behind the MeS. After our first day at Biobest we had very disappointing results, there was no clear reaction to be seen to either the MeS or the control solution. With advice from Biobest we tried other solvents such as hexane and paraffin oil. We chose to use paraffin oil because MeS is a volatile and paraffin oil would allow the slow release of this volatile, giving the insects more time to respond to MeS. We also tried a different set-up. The first set-up was performed under a fume hood and this might have resulted in the rapid elimination of MeS, this is why we tried the same experiment out of the fume hood but with a fan (mimicking a wind tunnel) that blows in the direction of the insects so that the MeS fumes are blown toward the insect. Under the advice from Prof. Felix Wäckers and dr. Veerle Mommaerts, we also tried to prime the insects first. Practically, priming the insects means that we let a few predator/parasitoid insects loose on a washed aphid-infested leaf. The insects that showed interest in the leaf, this means that the insect was walking around in search of aphids, were further used in the behavioural experiments. In these set of experiments we used an aphid infested leaf as a control compared to response to this leaf after introduction of a certain MeS or EBF concentration. These changes however did not demonstrate any clear behaviour form the insects other than random movement. We contemplated yet again which parameters we could change, we thought that the insects were maybe more interested in their new 'strange' environment, the set-up, than the substances but other than changing the set-up, the concentrations we used and the solvent, we didn't see many other variables that could have affected our results. <br/> | <p align="justify">A description of the natural enemies we received from biobest and employed in our experiments is given <a href="https://2013.igem.org/Team:KU_Leuven/Project/aphidbiology#Model%20Organisms"> here.</a> The predatory insects, ladybugs (adult and larvae), green lacewings (larvae) and parasitic wasps (adult) were subjected to a choice experiment. Starting from a determined point on our set-up (see figures), the insects were released and given a choice of a certain MeS concentration or a control solution which was the solvent used. The insects were given 1-3 mins to make a choice and there was a 'finish line' implemented so that after the insect crossed the 'finish line', it has made its choice. We first diluted the pure MeS solution in 97% ethanol to obtain five different concentrations ranging from 10^-9 to 10^-11. We chose to use ethanol due to the fact that it evaporates easily, leaving behind the MeS. After our first day at Biobest we had very disappointing results, there was no clear reaction to be seen to either the MeS or the control solution. With advice from Biobest we tried other solvents such as hexane and paraffin oil. We chose to use paraffin oil because MeS is a volatile and paraffin oil would allow the slow release of this volatile, giving the insects more time to respond to MeS. We also tried a different set-up. The first set-up was performed under a fume hood and this might have resulted in the rapid elimination of MeS, this is why we tried the same experiment out of the fume hood but with a fan (mimicking a wind tunnel) that blows in the direction of the insects so that the MeS fumes are blown toward the insect. Under the advice from Prof. Felix Wäckers and dr. Veerle Mommaerts, we also tried to prime the insects first. Practically, priming the insects means that we let a few predator/parasitoid insects loose on a washed aphid-infested leaf. The insects that showed interest in the leaf, this means that the insect was walking around in search of aphids, were further used in the behavioural experiments. In these set of experiments we used an aphid infested leaf as a control compared to response to this leaf after introduction of a certain MeS or EBF concentration. These changes however did not demonstrate any clear behaviour form the insects other than random movement. We contemplated yet again which parameters we could change, we thought that the insects were maybe more interested in their new 'strange' environment, the set-up, than the substances but other than changing the set-up, the concentrations we used and the solvent, we didn't see many other variables that could have affected our results. <br/> | ||
| - | After talking to Tim Belien of pcfruit, he offered us their Y-tube olfactometer and their lab, to perform some more experiments in different conditions. We compared again all the MeS concentrations with the control in a Y-tube olfactometer. The lady bug adults were first released into the Y-tube to become habituated with the set-up, after three minutes, MeS (30µl of 1/10^-9) or EBF was added to one end of the Y-tube and a control solution on filter paper on the other end. The insects were added again and their position was recorded after five min. </p> | + | After talking to Tim Belien of pcfruit, he offered us their Y-tube olfactometer and their lab, to perform some more experiments in different conditions. We compared again all the MeS concentrations with the control in a Y-tube olfactometer. The lady bug adults were first released into the Y-tube to become habituated with the set-up, after three minutes, MeS (30µl of 1/10^-9) or EBF was added to one end of the Y-tube and a control solution on filter paper on the other end. The insects were added again and their position was recorded after five min. After using the methyl salicylate concentration of 10^-10, we could see a obvious attraction of three ladybugs in a row. Afterwards we changed the side of the methyl salicylate, but we could not see the attraction anymore. We think that the Y-tube olfactometer was not clean enough and that there was still some methyl salicylate on the other tube as well. We also tried some more concentrations. When we used a methyl salicylate concentration of 10^-7, we saw repulsion of all the five ladybugs. The experiments at pcfruit gave us already some more information about the concentration of methyl salicylate that has to be produced by our genetically modified bacteria, but we certainly have to redo these experiments. Methyl salicylate concentration at 10^-10 showed an obvious attraction of three ladybugs in a row. Afterwards we changed the tube in which MeS was released, but we could not see the attraction anymore. We think that the Y-tube olfactometer was not clean enough and that there was still some methyl salicylate on the other tube as well. A methyl salicylate concentration of 10^-7, showed repulsion of all the five ladybugs. The experiments at pcfruit gave us an indication of a working MeS concentration.</p> |
</div> | </div> | ||
Revision as of 21:02, 2 October 2013
Secret garden
Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!
- A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
- For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
- We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?
Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!
E. coligy: Plants with BanAphids - insect experiments
In order to validate the BanAphids' model, we decided to investigate the effect of the two substances, E-beta-farnesene (EBF) and Methyl Salicylate (MeS) on aphids. Once we started our project, we noticed that we were not the only ones approaching this agricultural problem from this angle. Research groups like Rothamsted are currently investigating the use of EBF to repel aphids as well but in the form of GM crops. We also found that other areas of the industry were also interested in our project such as Biobest, worldwide leader in sustainable crop protection and pcfruit whose mission is to research prospects for biological fruit growing, new crop protection methods, etc. We were invited to perform our insect experiments at Biobest and pcfruit to validate the BanAphids model. We attempted to determine the working concentrations of our two substances in pure form and to examine the preference of aphids and their predators under different conditions and environments. With the data we have collected so far, we can observe that these two substances indeed do have an effect on aphids.
Insect experiments
What we wanted the insects to do!
Through many years of research, it has been found that E-beta-farnesene (EBF) is the essential compound of an aphid's alarm pheromone and that the general response to this signal is a change in gene expression that will agitate the aphids causing them to leave the plant. We tried to prove this using the synthetic compound EBF (Sigma-Aldrich, farnesene, mixture of isomers).EBF also works as an insect attractant. We also tried to prove the kairomone effect of Methyl Salicylate (Sigma-Aldrich, Methyl Salicylate ≥99%) through the use of the synthetic compound. We experimented with different concentrations of these synthetic compounds, with different solvents, with different set-ups and with different insects (Biobest).
One of the set-ups we used.
Second set-up, with wind tunnel
Natural Enemies
A description of the natural enemies we received from biobest and employed in our experiments is given here. The predatory insects, ladybugs (adult and larvae), green lacewings (larvae) and parasitic wasps (adult) were subjected to a choice experiment. Starting from a determined point on our set-up (see figures), the insects were released and given a choice of a certain MeS concentration or a control solution which was the solvent used. The insects were given 1-3 mins to make a choice and there was a 'finish line' implemented so that after the insect crossed the 'finish line', it has made its choice. We first diluted the pure MeS solution in 97% ethanol to obtain five different concentrations ranging from 10^-9 to 10^-11. We chose to use ethanol due to the fact that it evaporates easily, leaving behind the MeS. After our first day at Biobest we had very disappointing results, there was no clear reaction to be seen to either the MeS or the control solution. With advice from Biobest we tried other solvents such as hexane and paraffin oil. We chose to use paraffin oil because MeS is a volatile and paraffin oil would allow the slow release of this volatile, giving the insects more time to respond to MeS. We also tried a different set-up. The first set-up was performed under a fume hood and this might have resulted in the rapid elimination of MeS, this is why we tried the same experiment out of the fume hood but with a fan (mimicking a wind tunnel) that blows in the direction of the insects so that the MeS fumes are blown toward the insect. Under the advice from Prof. Felix Wäckers and dr. Veerle Mommaerts, we also tried to prime the insects first. Practically, priming the insects means that we let a few predator/parasitoid insects loose on a washed aphid-infested leaf. The insects that showed interest in the leaf, this means that the insect was walking around in search of aphids, were further used in the behavioural experiments. In these set of experiments we used an aphid infested leaf as a control compared to response to this leaf after introduction of a certain MeS or EBF concentration. These changes however did not demonstrate any clear behaviour form the insects other than random movement. We contemplated yet again which parameters we could change, we thought that the insects were maybe more interested in their new 'strange' environment, the set-up, than the substances but other than changing the set-up, the concentrations we used and the solvent, we didn't see many other variables that could have affected our results.
After talking to Tim Belien of pcfruit, he offered us their Y-tube olfactometer and their lab, to perform some more experiments in different conditions. We compared again all the MeS concentrations with the control in a Y-tube olfactometer. The lady bug adults were first released into the Y-tube to become habituated with the set-up, after three minutes, MeS (30µl of 1/10^-9) or EBF was added to one end of the Y-tube and a control solution on filter paper on the other end. The insects were added again and their position was recorded after five min. After using the methyl salicylate concentration of 10^-10, we could see a obvious attraction of three ladybugs in a row. Afterwards we changed the side of the methyl salicylate, but we could not see the attraction anymore. We think that the Y-tube olfactometer was not clean enough and that there was still some methyl salicylate on the other tube as well. We also tried some more concentrations. When we used a methyl salicylate concentration of 10^-7, we saw repulsion of all the five ladybugs. The experiments at pcfruit gave us already some more information about the concentration of methyl salicylate that has to be produced by our genetically modified bacteria, but we certainly have to redo these experiments. Methyl salicylate concentration at 10^-10 showed an obvious attraction of three ladybugs in a row. Afterwards we changed the tube in which MeS was released, but we could not see the attraction anymore. We think that the Y-tube olfactometer was not clean enough and that there was still some methyl salicylate on the other tube as well. A methyl salicylate concentration of 10^-7, showed repulsion of all the five ladybugs. The experiments at pcfruit gave us an indication of a working MeS concentration.
