Team:Toulouse/Results
Results
Content
Attract
Tests on varroas
In the US patent XXX which the concentration of butyric acid that attracts varroa mites is 4 % (V/V). In the final description it is specified that an efficient amount of attractant may at the minimum be 0.00001 %.
In order to verify the results presented in this patent, we designed an attraction test on varroas. Champollion University in Albi, welcomed us in their lab to perform 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 used a 4 % butyric acid concentration, like the one first mentioned in the patent.
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Figure 1: Butyric acid test pie chart and statistical test
This test demonstrates that a solution of 4 % butyric acid attracts varraos. However, in the “Cytotoxicity” part of the results, we also show that this 4% concentration is totally lethal for bacteria, so our goal is to produce at least a concentration of butyric acid of at least 0,00001 % (as described in the patent).
Cloning butyrate genes
We ordered synthetic genes directly as the full regulated pathway, so the ccr genes were already placed behind lacI ready for circadian circle regulation. We therefore cloned the ccr genes with all genes necessary for butyrate production, in order to have the construction below:
Figure 2: assembly of the synthetic pathway leading to butyrate production (5220 Kb). The first arrow represents the promoter, the others represent genes, the green circle is for the RBS and red circle are for terminators. Purple genes originated from Streptomyces collinus, blue genes form Clostridium acetobutylicum and yellow genes from Escherichia coli.
This construction (BBa_AREMPLIR), inserted in pSB1C3 is in the biobrick format and was then verified by an EcoRI and PstI digestion, leading to the release of the inserted fragment. We then confirm the right insertion of the fragment.
Figure 3: Gel electrophoresis of the digestion of the BBa_AREMPLIR verification of the inserted fragment encoding the pathway to produce butyrate.
The size of the first DNA fragment matches 5000 pb (butyrate construction) and the size of the second one (2000 pb) matches linearized pSB1C3.
Test of butyrate production
In order to test butyrate production, we cultivated ApiColi in micro-aerobic condition. After harvesting the supernatant of the saturated culture, we filtrated it prior NMR analysis.
We tested our genetic construction in the E. coli strain BW25113 but were unable to detect butyrate production. As a mean to verify if the pathway introduced was actually operating, we also searched for significant difference in other products present in the culture medium (acetate and ethanol). We also tested the butyrate production with a strain deleted for phosphate acetyltransferase, as is explained in “Metabolic Engineering of Escherichia coli for Production of Butyric Acid” [1] and in the figure below.
Figure 4: Exogenous acetate system. Deleted genes are coloured red. Reference [1]
Even if other enzymes for butyrate production were used, as explained in “Attract” part, this metabolic pathway figure can be helpful to understand which genes would need to be deleted for butyrate production.
Indeed, in the publication, three others genes were also deleted to produce butyrate. However, the lack of time did not allow us to perform these experiments. Only pta deletion could be achieved.
Figure 5: Test of butyrate production in E. coli strain deleted for pta gene. Culture in micro-aerobic condition in 10 mL falcon, result obtained after 28.5 hours culture.
Unfortunately we could not detect butyric acid with our NMR analysis, but we see differences for others fermentation products, a sign that something has happened in our constructed strain. Our bacteria produced less formate and a little more acetate but the biggest difference is visible for ethanol which is lower that in the control strain. It is 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 did not detect these intermediate metabolites because they are not released into the medium. In any case, our genetic construction modified the fermentative balance.
In a forthcoming experiment it would be useful to measure intracellular metabolites to see if intermediate products are actually synthesized. 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 what target quantities of formic acid need to be attained during the killing phase, we tested different concentrations of formic acid on varroas as explained in the “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
Figure 6 presents a dose-dependency of formic acid on varroa mortality. At 10mM formic acid, all varroas died before three hours but as we explain in “Protocols” part varroas also stop moving at lesser concentrations. Figure 7 shows that even with 50µM around 30 % varroas died after 7 hours. As our first goal is to respect bees, we would like to produce as little as possible of formic acid. That is why we set to produce around 50µmol.L-1 during the night (7 hours).
Test of formate production
For formate production we designed genes coding for pyruvate formate lyase and the activation protein. The assembly was cloned in pUC53 plasmid (from the gene synthesis company) so we could directly test formate production without any cloning step, as explained in “Eradicate” part.
Figure 8: Substrate and products concentration for formate production in a micro-aerobic culture (CONDITIONS)
Figure 9: Summary of formate production tests 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 plotted the specific histogram for formate. Figure 9 indicates that formate production increased significantly (10%).
As mentioned earlier, our goal is to produce 50µM of formic acid in 7 hours. To reach this quantity, we need to produce 77mM of formate. Right now the production of formate reaches 25mM in 36 hours (Figure 8), 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 the results summarized below, we show that it will be possible to reach our target production with slight optimizations of the metabolic pathway regulation.
Device: TPX Bag
Growth tests
The second step was to know if bacteria can grow inside a small bag of TPX®. Thus, the strain E. coli BW 25113 has been inoculated in a small bag and further added inside a tube and incubated at 37 °C. The monitoring of OD at 600 nm has been performed over 10 days.
Figure 1: Growth test of E. coli BW 25113 inside a small bag of TPX® (Tubes n°1 and n°2).
Both tubes contain a small bag of TPX® in which bacteria are growing (t = 17 hours, 37 °C).
Figure 2: Growth test of E. coli BW 25113 in a culture tube (Tube 3).
The culture tube, contains bacteria which are growing (t = 17 hours, 37 °C, 130 rpm) in parallel of biological sample tubes shown above. It represents the control: E. coli BW 25113 are growing under aerobic condition with agitation.
The graph below shows the monitoring of OD at 600 nm. The strain E. coli BW 25113 is growing over 10 days (Tube n°1') in the TPX® bag .
Figure 3: Growth test in TPX® bag by monitoring OD at 600 nm over 7 days (tubes n°1 and 2) over 10 days (tube n°1').
The picture below represents the strain of E. coli BW25113, previously identified as tubes n°1', n°1, n°2 and n°3 after 10 days of culture for tube 1' and 7 days for tubes 1, 2 and 3.
Figure 4: Colonies of E. coli BW 25113 on Petri dishes after an overnight incubation at 37°C. EXPLANATION
Bacteria are still alive after 10 days or 7 days (depending on the tube) while growing in TPX® bags. In all the culture tubes, the number of cells is about 4.106. Thus, the strain can survive over 10 days in the TPX® bag.
Gas diffusion tests
In order to verify if our test (described in the “Protocol” section) works, we performed a series of control reactions using butyric acid solutions in a Falcon and we quantified the gases.
We did not detect any evaporation of butyric acid by NMR and postulated that the solution we used to solubilize gases may not be sufficiently alkaline. We then performed a test with a TPX® bag containing butyric acid into a solution of sodium bicarbonate. The control is an injection of a 4% (V/V) solution of butyric acid in water.
Figure 1: NMR Spectrum of butyric acid liquid control in red and butyric acid liquid which passed through TPX bag in blue. * Blue curve is zoomed 1340 times more than red curve. Each condition was tested in two replicates.
Table 1: Concentrations of butyric acid calculated from the NMR spectrum
From these results, we can conclude that TPX® allows butyric acid
to pass outside the bag. We detect only a small quantity but
an optimization of the device could be made with a plastic containing
bigger pores in order to let more butyric acid go out.
For formic acid we were able to detect it
in gas, probably because its pka is lower than butyric acid.
Figure 2: NMR spectrum of formic acid gas control in red and formic acid gas which passed through TPX bag in blue. An internal standard present in both curves allowed us to standardize both curves. curves. Each condition was tested in two replicates.
Table 2: Concentrations of formic acid corresponding to NMR spectrum
According to these results, TPX® allows 56% of formic acid to pass outside the bag in gas phase. We show that formic acid can go through TPX plastic. Using a more porous plastic, we propose that this percentage could even further increase.
Safety tests
Based on the protocol specified in here, the bacteria’s impermeability of TPX®, has been tested by inoculating the strain
E. coli BW 25113 in M9 defined medium. To summarize, the strain (in M9 medium) has been inoculated inside the small bag of TPX®. Then, the inoculated bag was immersed in a glass measuring cylinder containing M9 medium. OD600 nm served to monitor and growth in the external medium.
Figure 1: Measuring cylinders used for the safety test of the TPX® polymer.
The first cylinder, on the right contains the small TPX® bag with E. coli BW 25113 after 27 hours of growth at 37 °C. On the right, the negative control cylinder contains a small bag of TPX® without bacteria and immersed in M9 medium after 27 hours of growth at 37 °C.
Over this time, no bacteria went out of the bag, so the sterility has been conserved over 27 hours.
Device: Biological tests
In the end, our objective is to have a bag which contains bacteria to produce alternately butyric acid
and formic acid during at least ten days in order to be practical for beekeeper.
So we faced with some biological questions as:
- Could bacteria live during ten days in micro-aerobic condition?
- Which carbon source could we have to produce continuously acids?
- Would acids be toxic for E.coli?
Characteristics of E.coli growth
In order to know better the E.coli strain we would use for our project, we made a culture in aerobic and micro-aerobic conditions. We sampled OD and supernatant as it is explained here to see what happen in it.
Micro-aerobic condition is obtained thanks to cultivation in specific falcons with holes recovered by a membrane into the plug which let pass oxygen without opening the falcon. They were incubated at 37 °C without agitation to best correspond to our real condition.
Aerobic condition is obtained with classic Erlenmeyer incubated at 37 °C with agitation.
For the medium, we use a minimal medium M9 because we want to follow acids production by NMR. And we choose a standard glucose concentration, 15mM.
Biomass, substrate and products
In order to plot biomass concentration it is necessary to convert the OD measured.
This equation was used:
$$ X=OD_{600nm}\times 0,4325 $$
Where X is the cell concentration (g.L-1)
For substrate and products concentration we plotted peak area of each molecule on NMR spectrum.
Then, we calculated concentration with this equation:
$$[A]=\frac{Area_{molecule}}{Area_TSP} \times [TSP] \times \frac{\textrm{TSP proton number}}{\textrm{A proton number}} \times DF $$
- [A] = concentration of molecule in our solution in mM
- AreaTSP = 1
- [TSP] = 1.075 mM
concentration of Trimethylsilyl propanoic acid in NMR tube, internal reference for quantification - TSP proton number = 9
- DF = Dilution Factor = 1.25
Thanks to these calculations we were able to plot biomass, substrate and products depending on time.
Figure 1: Results of aerobic culture. Culture of BW25113 in M9 medium with [glucose] = 15 mM, in Erlenmeyer at 37 °C
Figure 2: Results of micro-aerobic culture. Culture of E. coli BW25113 in M9 medium with [glucose] = 15 mM, in Falcon at 37 °C
Glucose is consumed approximately at the same rate for both conditions but it is not use for the same thing at all. In aerobic condition biomass reaches 3 g/L whereas in micro-aerobic condition there is six times less biomass. Inversely, there are far less products in aerobic conditions, and bacteria consume them when there is not glucose anymore, than in micro-aerobic condition.
For our objective to produce acids in a microporous bag, it is a really interesting results to have naturally bacteria which have slow growth and fermentation products.
We can convert formate
concentration into formic
acid to know how much more
we will have to produce to
kill varroa. Indeed, the bacteria
produce a base but it is the acid that
interests us.
The formula below
is used:
$$ pH=pKa+log \left(\frac{C_{b}}{C_{a}} \right) $$
- pH: medium used is buffered with a low concentration in acid. pH = 7.
- pKa: 3.7 for formic acid and 4.81 for butyric acid
- Cb: base concentration
- Ca: acid concentration
As it is said in the “Eradicate” part, our goal is to produce 50 µM of formic acid to kill varroa, thanks to the equation (3) we know it corresponds to 77,7 mM of formate.
At the maximum the bacteria produces 32mmol/L of formate. It is necessary to add genes involved in formate production to regulate production and multiply it by 2.4. For a perfect regulation it would be necessary to delete pfl-B in E.coli genome not to have formate production during the day.
Bacteria survival
As it is explained here we plated bacteria on Petri dish to know if they were alive or not because OD measure cannot discriminate alive bacteria from dead. This test show us that wild type bacteria can easily survive during at least 15 days. So if we bring them a carbon source during this period they should survive even better.
Figure 3: Bacteria survival results from culture test with BW25113 on M9 with 15mM of glucose during 15 days to mime real survival condition.
Choice of carbon source to produce acids during 10 days
Characteristics of Biosilta kit
En Presso B is a technology which permits to produce a lot of recombinant proteins thanks to a low substrate delivering during 24 hours. This technology is based on polymer degradation by an enzyme which permits to have the right quantity of substrate at each moment. We would like to use this technology to cultivate our cells during one or two weeks in good conditions in order to produce butyrate and formate. The medium with the polymer is solid and contained in separate bags. To know which quantity of butyrate and formate we can produce, we have to know the quantity of substrate we could obtain with the polymer so we made a kinetic test with a high enzyme concentration (50 U/L).
Figure 4: Kinetic test of enzyme which degrades polymer from Biosilta kit. [Enzyme] = 50 U/L in order to have a complete degradation of polymer.
In order to have a global idea of the rate of glucose releasing we calculate an average speed.
$$ v_{glucose1}=\frac{[glucose]}{time}=\frac{11.1}{3.97}=2.80 g.L^{-1}.h^{-1} , for [E]_{1}=50 U.L^{-1} (4) $$
With a final glucose concentration of 13 g/L for one bag of polymer, a rate of glucose releasing can be calculate in order to have glucose during 13 days.
$$ v_{glucose2}=\frac{13}{13 days}=\frac{13}{322 hours}=0.0403 g.L^{-1}.h^{-1} (5) $$
The reduction factor is calculated:
$$ RF=\frac{v_{glucose1}}{v_{glucose2}}=\frac{2.8}{0.0403}=69.44 (6)$$
So, the concentration of enzyme that we have to use is:
$$ [E]_{2}=\frac{[E]_{1}}{RF}=\frac{50}{69.44}=0.72 U.L^{-1} (7)$$
Growth culture with Biosilta kit
As we do not know any growth characteristic with Biosilta medium we tested different enzyme concentrations and not only the one which allow growth during 13 days. We made acquisition in two times because of software constraints, this is why there is a break at 5 days:
Figure 5: Bacteria growth as a function of different enzyme concentrations in Biosilta medium. Test was made in 48 wells plate with OD reader.
Except for 1.5 U/L enzyme, OD increase during 12 days so glucose releasing seems to function well. At the beginning there is exponential growth because some glucose is directly available on medium. Since 2 days until the end growth is linear, only the slope change. It is higher between 2 and 4 days than after probably because bacteria were in worse condition after few days so they are not able anymore to consume all glucose available.
Thanks to those results we know it is possible to have continuous growth during at least 12 days. The only problem is that our control, without enzyme, grows also so either another substrate is available or bacteria could able to degrade polymer that could be a problem.
In order to answer these questions we did culture in falcon in order to analyze products and to see evolution of polymer quantity. We made culture without enzyme and we test concentration of enzyme of 0.72 U/L because it this one which allows to reach the highest OD in figure 5 and it is the one we calculated above to have glucose releasing during 13 days.
Figure 6: Results of BW25113 culture on Biosilta medium without enzyme.
Figure 7: Results of BW25113 culture on Biosilta medium with [Enzyme] = 0.72 U/L.
Figure 6 shows that polymer is not degraded, so it is only enzyme of Biosilta kit which releases glucose. Enzyme concentration could be correlate to glucose rate releasing for further modelling. So bacteria find another carbon source in Biosilta medium but we were not able to determine which one. Products concentrations are nearly identical to those in M9 medium.
In figure 7, polymer area decreases, so enzyme degrades it well. At the beginning, glucose concentration is almost constant so bacteria consumed it directly when enzyme releases it. At the end glucose concentration increases a bit, bacteria either did not consume it as fast as the beginning or they consumed formate because its concentration decreases. This could be a problem for us because we look for produce more formate so we would have to think about it.
Fermentation products have high concentrations in comparison to culture in M9 with 15mM of glucose, around 20 times more for lactate, 3 times more for acetate and 2 times more for ethanol. So if we delete production ways for lactate, acetate and ethanol and degradation way of formate we would able to produce enough formate and butyrate.
Acids production modelling
With the rate of glucose calculated above, an FBA and FVA simulation were launched as explained in “Modelling” part. Some conversion between the model and the real condition are necessary and they are explain in “Modelling” part.
In order to model production in the most similar conditions to fit real experiment we chose a glucose rate of 0.0403 g.L-1.h-1 that correspond to 0.72 U/L of enzyme.
To convert formate production into formic acid concentration we use equation (3).
Figure 8: Modelling of formic acid production as a function of different growth rates for a glucose rate of 0.0403 g.L-1.h-1.
Our goal is to produce at least 50µmol/L and with this graph the maximum production could be 6µmol/L. So we have to produce nearly 10 times more formic acid. In order to reach our goal we could see which rate of glucose we needed with a reverse thought.
Figure 9 : Modelling of formic acid production as a function of glucose rate for different growth rates (in h-1).
To produce 50 µmol/L of formic acid different strategies are available. Either a low growth rate is chosen so a low glucose rate would be necessary or a high growth rate is chosen and a high glucose rate would be necessary. As bacteria have to live during at least ten days it will be better to have a continuous slow growth rate. Moreover, it will consume less glucose per hour so we would need a lower polymer concentration in our bag at the beginning. So, we chose a growth rate of 0.2h-1, and we can determine rate of glucose needed.
[Formic acid] (μmol.L-1 )=166.88 ×[Glucose] (g.L-1.h-1 ) (9) $$ [Glucose] = \frac{50}{166.88}=0.3 g.L^{-1}.h^{-1} (10) $$
Now, we will see which butyric acid concentration we could produce.
Figure 10: Modelling of butyric acid production as a function of glucose rate for different growth rates.
According to modelling results in figure 10, we would be able to produce around 100µmol/L of butyric acid that correspond to 0.0092% (V/V). As we explained in “Results” part, "attract" section, our objective is to produce at least 0.00001%, so with modelling we reach it.
Nevertheless, in order to have this right glucose rate it is necessary to calculate how much polymer is needed at the beginning and which enzyme concentration.
With the same equations as we used in “Characteristics of Biosilta kit” we can determine which quantity of glucose is needed in total during a fortnight.
$$[Glucose]=v_{glucose}\times time=0.3\times 322=96.6 g.L^{-1} (11)$$
Knowing that one Biosilta kit contains the equivalence of 13 g/L of glucose, we have to concentrate the medium 7 times. Concerning the glucose rate, the 0.3 g.L-1.h-1 value correspond to 5 U/L of enzyme. Thus, we tested different concentrations of Biosilta medium with different enzyme concentrations.
Testing different concentrations of Biosilta kit
As we do not know the exact composition of Biosilta medium, we are not able to say if there is a molecule which could be toxic at high concentrations. We could only have a global analysis on our results :
Figure 11: Bacteria growth as a function of time on Biosilta medium concentrated 6 times. Culture with BW25113 on 48 wells plate and optical reader
There is hardly any growth during three first days, bacteria probably adapt themselves to the medium. Since 3 days until the end, OD increases up to 1 for 1.5 U/L enzyme but it is still slow. Moreover, bacteria grow better with 0.72 and 1.5 U/L than with 3 or 4 U/L. It could be explained by an excess of glucose that inhibits bacteria growth. Indeed, enzyme could release too much glucose that bacteria would not consume as fast, then glucose accumulated itself in medium. We tested with a less concentrated medium in order to see if latency period could be reduce.
Figure 12: Bacteria growth as a function of time on Biosilta medium concentrated 4 times. Culture with BW25113 on 48 wells plate and optical reader
With a 4 times concentrated medium, there is no latency period anymore but enzyme concentration seem not to affect bacteria growth. Bacteria probably consume all free glucose in medium and then enzyme does not have enough time to degrade polymer. A longer period test would be necessary to know if bacteria were able to consume glucose as fast as enzyme released it. Maybe by testing a twice concentrated medium, we would be able to answer it.
Figure 13: Bacteria growth as a function of time on Biosilta medium concentrated twice. Culture with BW25113 on 48 wells plate and optical reader
Decline of curve for 0.72U/L was not expected because in “normal” Biosilta medium, figure 5, bacteria grew during 12 days. We cannot explain this result, but it shows that it is complicated to work with a medium with an unknown composition.
Curves for 3 and 4 U/L enzyme or very similar so it seems that bacteria are not able to consume all glucose released by enzyme. As we only measured OD we do not know if bacteria would assimilate glucose for another way that growth metabolism.
Thanks to figure 11, 12 and 13 we know that it would not be possible to have enough polymer in our medium. As a solution we think to use a dialysis system: in one side there will be bacteria and in the other side there will be the polymer with enzyme. Membrane which separates them will allow only small molecules to pass like glucose. Thanks to this system our device will have enough substrate for two weeks.
Regarding the rate of glucose assimilation we could do additional tests where we would measure glucose in medium to determine maximum rate of assimilation. An optimization of this assimilation could be essential.
Testing acids toxicity
Effects of medium
In order to optimize resistance of BW25113 to different acids concentrations we tested two medium: LB and M9 with 15mM of glucose.
Figure 14: Optic density in function of time for different formic acid concentrations and two medium. LB medium is represented with green curves and M9 medium with blue curves. Each condition is tested in three replicates so standard deviation is represented in orange.
Figure 15: Optic density in function of time for different butyric acid concentrations and two medium. LB medium is represented with green curves and M9 medium with blue curves. Each condition is tested in three replicates so standard deviation is represented in orange.
In M9 medium, growth is slower at the beginning in both figures but OD max is almost the same for both medium.
For formic acid, the only significant difference is for 10mM with a slower growth in LB than in M9. For butyric acid the difference is stronger because in LB bacteria do not grow anymore with 109mM whereas in M9 there is growth.
In fact, M9 is buffered and not LB so we measured pH in both medium with different acids concentrations in order to see if there were a correlation.
Figure 16: pH in function of concentration in mM for formic acid and butyric acid. LB medium is represented with green curves and M9 medium with blue curves. pH was measured with pH paper because only order of magnitude interested us. Each condition was tested three times and give us the exactly the same results.
It is clear that in M9 medium pH stay at pH 7 for higher acids concentrations than LB medium. Moreover, thanks to previous figures, it is possible to see that bacteria do not grow anymore when pH is around 5. This results show that bacteria are sensitive to acid pH, but they may resist to higher acids concentrations if the medium was better buffered. We will now see if it would be interesting or not to buffer better our medium.
Formic acid toxicity
Figure 17: Toxicity test of formic acid, OD of BW on M9 15mM glucose.
Figure 17 shows a dose/response relationship between formic acid concentration and bacteria growth. We would like to produce at least 50µmol/L of formic acid in order to kill varroas and bacteria growth normally up to 1mM. So we should not have toxicity problem during the treatment.
Butyric acid toxicity
Figure 18: Toxicity test of butyric acid, OD of BW on M9 15mM glucose.
The higher is butyric concentration, the less is bacteria growth, as our previous results with formic acid. However we have an intermediate result with 109mM of butyric acid. We do not have a specific butyric acid concentration to produce and modelling show us that we could produce around 15mM with all optimizations so we would not have any butyric acid toxicity during our treatment.
Nota Bene: For our conclusions about acids toxicity, we consider that acids evaporate during day for formic acid and night for butyric acid, so there would not be a lot of acid accumulation.
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