Team:Toulouse/Results
Results
Attract
Tests on varroas
In the US patent which describes utilization of butyric acid in order to attract varroa mites, it is said that a concentration of 4 % (V/V) is used in their tests. In the final description it is specify that a butyric acid concentration more than 0.1% is efficient but previously it is assumed that an efficient amount of attractant may at the minimum be 0.00001 %.
In order to verify results of this patent, we made an attraction test on varroas. Champollion University in Albi, welcome us in their lab to do this test but there were not a lot of varroas available so we chose to make only one test in order to have a significant result. So for this test we use a 4 % butyric acid concentration as it was made in the patent.
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Figure 1: Butyric acid test pie chart and statistical test
Thanks to this test we demonstrate that a solution of 4 % in butyric acid concentration attracts varraos. But in “Cytotoxicity” part we show that this concentration is totally lethal for bacteria, so our goal is to produce at least a concentration of butyric acid of 0,00001 % because of the explanation above.
In a second time it will be interesting to do another attraction test with the right concentration we could produce.
Cloning butyrate genes
When we ordered synthesized genes we chose to have directly regulated ways, so we had ccr genes behind lacI ready for circadian circle. So we had to clone ccr with all genes necessary for butyrate production, in order to have the construction below:
Figure 2: All genes necessary to butyrate production, 5220 Kb. First arrow represents promoter, others genes, green circle RBS and red circle terminator. Purple gene comes from Streptomyces collinus, blue genes form Clostridium acetobutylicum and yellow genes form Escherichia coli.
This construction is a biobrick format so we digested it by EcoRI and PstI to confirm it is integrated into pSB1C3 as we can see in figure 2.
Figure 3: Gel electrophoresis for verification of butyrate biobrick
The first band fits 5000Kb that matches with Biobrick and the second band fits 2000Kb that matches with pSB1C3. So now we have all genes necessary to butyrate production we can test it.
Test of butyrate production
In order to test butyrate production, we cultivated ApiColi in micro-aerobic condition, then we sampled supernatant that we filtrated for NMR analysis. All protocols are well described in “Protocols” part.
We tested our genetic construction in BW25113 but we were unable to detect butyrate or a significant difference from our control for others products. So we tested to produce butyrate with a strain which is deleted for phosphate acetyltransferase, as it is explained in “Metabolic Engineering of Escherichia coli for Production of Butyric Acid” (1) and the figure below.
Figure 4: Exogenous acetate system. In red there are deleted genes. Reference (1)
We did not use same enzymes for butyrate production, as we explained in “Attract” part, but this figure could be helpful to know which genes have to be deleted for butyrate production.
Indeed, in the article they deleted three others genes to produce butyrate, but we could not deleted them because of lack of time so we tested with only pta deletion.
Figure 5: Test of butyrate production in E.coli strain deleted for pta gene. Culture in micro-aerobic condition in falcon, result after 28.5 hour culture.
Unfortunately we could not detect butyric acid on NMR analysis, but there are differences for others fermentation products. Our bacteria produced less formate and a little more actetate but the most diiference is that Ethanol is less produced. It would be possible that Acetyl-CoA is transformed in Acetoactyl-CoA thanks to enzyme we added. Then Acetyl-CoA would be less available to be transformed into ethanol. We could not detect these intermediate metabolites to confirm that hypothesis because they are intracellular. In a further experiment it would be useful to measure intracellular metabolite to see if you produced intermediate products.
In any case, our genetic construction modified the fermentative balance. Moreover, it would be very interesting to test our genetic construction with a strain deleted for all genes indicated in figure 4.
Eradicate
Tests on verroas
In order to determine which concentrations of formic acid we have to produce we tested different concentrations of formic acid on varroas as we explained in “Protocol” part.
Figure 6: Mortality of varroas as a function of time for different formic acid concentrations
Figure 7: Histogram representing mortality of varroas after 2 hours and after 7 hours
Thanks to figure 6, it is possible to see a dose-dependent between formic acid concentration and varroa mortality. So, with 10mM all varroas died before three hours but as we explain in “Protocols” part varroas stop moving for less concentrations. Figure 7 shows that even with 50µM around 30 % varroas died after 7 hours. Moreover, we would like to have a project which respects bee, so we would like to produce as little as possible formic acid. It is for this reason we set to produce at least 50µmol.L-1 during the night (7hours).
Test of formate production
For formate production we synthesized directly genes coding for pyruvate formate lyase and the activate protein so we could test formate production without cloning step, as it is explained in “Eradicate” part.
Figure 8: Substrate and products concentration for formate production in a micro-aerobic culture
Figure 9: Summary of formate production test after 3 days cultivation in micro-aerobic condition
Figure 8 shows that the only difference between ApiColi and the control is for formate production, so we plot the specific histogram for formate. Figure 9 indicates that formate production increased significantly. So we increased formate production by 10 %.
Our goal is to produce 50µM of formic acid in 7 hours that matches 77mM of formate. We produce around 25mM in 36 hours (Figure 8), namely around 5mM in 7 hours. So our production is in the same order of magnitude of our target production.
Thanks to our device considerations and all results summarized below, we show that it will be possible to reach our target production with optimization.
Device
All explanations of our tests are in "Device" part but we summarize our results here.
TPX bag
Characteristics of E.coli
Fortnight culture
Figure 9: Bacteria survival after 10 days in micro-aerobic condition. Plate 100µL after a dilution of 104
Bacteria stay alive during at least 15 days
Acids toxicity
For concentrations we want to produce, there is no toxicity problem. It seemed that bacteria are more sensitive to pH than acids so we could use a better buffered medium to be sure that there will be no problem
