Difference between revisions of "Team:Toulouse/Results"

In the US patent US 8647615 B1, the concentration of butyric acid that attracts varroa mites is 4 % (V/V), but the final description specified the concentration is efficient from 0.00001 %.

In order to verify the results presented in this patent and to make sure we are able to perform such experiment, we designed an attraction test for varroas. Champollion University in Albi welcomed us in their lab to get access to varroas. At the time of this test, there were not a lot of varroas available, so we add to make only one test in order to hope a significant result. Hence, we used the mentioned 4 % butyric acid concentration, like the one first mentioned in the patent.

This test demonstrated 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 lethal for bacteria. We therefore decided to aim for a minimal concentration of 0.00001 % butyric acid, even if further experimentation will have to demonstrate the efficiency of this lower threeshold.

In a nutshell:

butyrate is an effective attractant for the varroas. Its synthetic pathway has been implemented in E. coli. Butyrate production from this pathway remains to be demonstrated prior to testing the strain with varroas.

In a nutshell:

formate is effective to kill varroas from as low as 50 µmol.L^-1. Overexpressing its biosynthetic genes allow obtaining a 10 % increase in production. Optimizing this production will have to be achieved before testing the strain capacity to kill varroas.

We investigated if the bacteria can grow inside a small bag of TPX®. Thus, the strain E. coli BW 25113 has been inoculated in a small bag that was sealed. The bag was placed inside a tube and incubated at 37 °C. Growth was visualy assessed in the bags after 17 hours at 37°C (figure 9), or with a culture in tube with agitation as a control over the same period (figure 11). This showed that the cells were able to proliferate in the plastic bag.

Figure 10: Growth test of E. coli BW 25113 inside a small bag of TPX®.
(t = 17 hours, 37 °C).

Figure 11: Control of figure 10 with a growth test of E. coli BW 25113 in a culture tube.
The culture tube contains bacteria growing in parallel of the biological sample tubes shown above (t = 17 hours, 37 °C, 130 rpm).

Figure 12: Growth test in TPX® bag by monitoring OD at 600 nm over 7 days to 10 days.

We then checked the viability of the cells after 10 days of growth in the TPX® bag (tube 1, 1' and 2) or in the control tube (tube 3), by spreading diluted volume of the cultures on petri plates. Similar numbers of colonies were obtained, indicating that the cells survival is the same in both culture conditions.

Figure 13: Colonies of E. coli BW 25113 on Petri dishes after an overnight incubation at 37°C to check survivability.

Figure 14: 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 15: Concentrations of butyric acid calculated from the NMR spectrum

Figure 16: NMR spectrum of formic acid gas control in red and formic acid gas which passed through TPX bag in blue.

Table 2: Concentrations of formic acid corresponding to NMR spectrum

Accordingly to these results, TPX® allows 56% of formic acid to pass outside the bag in the gas phase. Therefore, formic acid permeate through the TPX plastic. Using a more porous plastic as proposed for the butyrate, we suppose this percentage to further increase.

The bacteria impermeability of the TPX® was tested through inoculation of the strain E. coli BW 25113 in M9 defined medium complemented with glucose inside the bag. Then, the inoculated bag was immersed in a glass measuring cylinder containing M9 medium with glucose. OD600 nm served to monitor a putative growth in the external medium.

Figure 17: Measuring cylinders used for the safety test of the TPX® polymer.
The cylinder on the left contains the TPX® bag with E. coli BW 25113 immersed in M9 medium after 27 hours of growth at 37 °C. On the right, the negative control cylinder contains a bag of TPX® without bacteria after 27 hours of growth at 37 °C.

In a nutshell:

The TPX® material successfully contains growing bacteria while allowing the diffusion of formate and, in a lesser extend, of butyrate. These tests will profit to all Igem teams looking for a solution to contain their strains .

Our final objective is to prepare a bag containing bacteria producing alternatively butyric acid (during the day) and formic acid (during the night) and for a period of time of at least ten days (so that beekeepers don't have to change them every morning...).

So we face some biological questions:

In order to characterize the E. coli strain growth in the conditions we plan to use in our device, we used a culture in aerobic and micro-aerobic conditions, measured the growth rate (OD) and analysed the supernatant to measure the concentration of fermentation products.

Micro-aerobic condition is obtained by cultivation in specific Falcon tubes with holes and covered with a membrane that let the oxygen pass through. They were incubated at 37 °C without agitation (mimicking the aerobic and lack of agitation condition present in our device).

We used a minimal M9 medium to identify formic or butyric acid production by NMR.

$$ X=OD_{600nm}\times 0,4325 $$

Where X is the cell concentration (g.L^-1)

$$[A]=\frac{Area_{molecule}}{Area_TSP} \times [TSP] \times \frac{\textrm{TSP proton number}}{\textrm{A proton number}} \times DF $$

Glucose is consumed approximately at the same rate for both conditions but is converted differently depending on the condition. In aerobic condition the biomass reaches 3 g/L whereas in micro-aerobic condition there is six times less biomass. On the contrary, there are far less products in aerobic conditions.

Our results are in line with our objective to produce acids in a microporous bag because we demonstrate that with microaerobic conditions, bacteria grow slowly and produce fermentation products.

To convert the concentration of formate into formic acid we use the famous Henderson Hasselbalch equation:

$$ pH=pKa+log \left(\frac{C_{b}}{C_{a}} \right) $$

As mentioned in the Eradicate> part, our goal is to produce 50 µM of formic acid to kill varroa, and this corresponds to 77,7 mM of formate.

In our microaerobic conditions, the natural E. coli strain produces 32 mM of formate. It is therefore necessary to further improve the metabolic production by adding genes involved in formate production in order to increase it by a factor of 240%. For a perfect regulation it would be necessary to delete the chromosomic version of pflB in E. coli genome to avoid formate production during the day.

As explained here calculation of the survival of bacteria was perfomed via colony counting after plating on solid agar medium. Wild-type bacteria can easily survive at least 15 days in aerobic or microaerobic conditions without a carbon source. We hypothesize that in the presence of a carbon source, we may be able to extend even further their survival time period.

Figure 20: Bacteria survival test performed with the BW25113 strain on M9 with 15 mM of glucose during 15 days.

En Presso B is a technology which enables production of a lot of recombinant proteins owing to a slow carbon source delivery during 24 hours. This technology is based on polymer degradation by an depolymerisation enzyme leading to a perfect control of the quantity of substrate released in the medium for a given period of time. We wanted to use this technology to cultivate our cells during one or two weeks in conditions allowing the production of butyrate and formate. The medium with the polymer was solid and contained in separate bags. In order to quantify the production of butyrate and formate in these conditions, we first performed a test with a high concentration of the enzyme (50 U/L) and measured the kinetics of the polymer degradation.

Figure 21: Kinetics of Biosilta polymer degradation measured with glucose release.

$$ v_{glucose1}=\frac{[glucose]}{time}=\frac{11.1}{3.97}=2.80 g.L^{-1}.h^{-1} , for [E]_{1}=50 U.L^{-1} (4) $$

$$ v_{glucose2}=\frac{13}{13 days}=\frac{13}{322 hours}=0.0403 g.L^{-1}.h^{-1} (5) $$

$$ RF=\frac{v_{glucose1}}{v_{glucose2}}=\frac{2.8}{0.0403}=69.44 (6)$$

$$ [E]_{2}=\frac{[E]_{1}}{RF}=\frac{50}{69.44}=0.72 U.L^{-1} (7)$$

As we do not know any growth characteristics of bacteria in the Biosilta medium we tested different enzyme concentrations and not only the one calculated precedently. Acquisition of the growth stopped due to a problem with the plate reading system where the bacteria where growing. Hence, there is a break at 5 days.

Except for the 1.5 U/L enzyme concentration, there is an increase of OD in all conditions during 12 days. This meanes that glucose releasing functions as expected. At the beginning there is an exponential growth because some glucose is directly available in the medium. This phase is therefore not the one to analyse. After 2 days and until the end, the growth rate changes gradually and becomes constant, at the highest between 2 and 4 days and decreasing thereafter.

We therefore know that it is possible to have continuous growth during at least 12 days. However, in our control without enzyme, there is also some bacterial growth so either another substrate is available or bacteria can readily degrade the polymer. More tests will be needed to verify if and how E. coli can use this polymer.

In order to answer these questions we performed some cultures in Falcon tubes in order to analyse the products and the evolution of the polymer quantity. Culture without enzyme and with a concentration of enzyme of 0.72 U/L were performed.

Figure 23 (right panel) shows that the polymer is not degraded by E. coli which is probably capable of finding another carbon source within the Biosilta medium. The concentration of products (left panel) is clearly different in the two conditions: with the enzyme, more fermentation products are formed.



Fermentation products have high concentrations in comparison to the cultures in performed in M9 (15 mM glucose), around 20 times more for lactate, 3 times more for acetate and 2 times more for ethanol. We hypothesize that by deleting the various production pathways for lactate, acetate and ethanol and the degradation pathway for formate we should be able to produce enough formate.

With the rate of glucose release calculated above, an FBA and FVA simulation were launched as explained in Modeling part. Some conversion between the model and the real condition are necessary and they are also explained there.

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 used equation (3).

Our goal was to produce at least 50 µmol/L. The graph clearly shows a maximum production of 6 µmol/L. To reach our goal we then have to change the growth rate.

To produce 50 µmol/L of formic acid different strategies are possible. If we choose a low growth rate then a low glucose rate is be necessary and vice-versa. As bacteria have to live during at least ten days it was better to have a continuous slow growth rate. Moreover, it would consume less glucose per hour so we would need a lower polymer concentration in our bag at the beginning. Thus, we chose a growth rate of 0.2h^-1, and we could determinate the glucose release value needed.

[Formic acid] (μmol.L^-1 )=166.88 ×[Glucose] (g.L^-1.h^-1 ) (9) $$ [Glucose] = \frac{50}{166.88}=0.3 g.L^{-1}.h^{-1} (10) $$

According to the modelling results described in figure 10, we have to produce around 100µmol/L of butyric acid that corresponds to 0.0092% (V/V). As explained in the "Results" part, "attract" section, our objective was to produce at least 0.00001%, so the modelling indicates that we can theoretically reach it.

Nevertheless, in order to have this right glucose rate it was 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 could 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 had 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.

As we did 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 :

There was hardly any growth during three first days, bacteria probably adapted themselves to the medium. During 3 days until the end, OD increased up to 1 for 1.5 U/L enzyme but it was still slow. Moreover, bacteria grew 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 this fast, then glucose accumulated itself in medium. We tested with a less concentrated medium in order to see if latency period could be reduce.

With a 4 times concentrated medium, there was no latency period anymore but enzyme concentration did not seem to affect bacteria growth. Bacteria probably consumed all free glucose in medium and then enzyme did not have enough time to degradate the polymer. A longer period test would have been necessary to know if bacteria were able to consume glucose as fast as the enzyme released it. Maybe by testing a twice concentrated medium, we would have been able to answer it.

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.

In order to optimize resistance of BW25113 to different acids concentrations we tested two medium: LB and M9 with 15mM of glucose.

In M9 medium, growth was slower at the beginning in both figures but OD max was almost the same for both medium.

For formic acid, the only significant difference was for 10mM with a slower growth in LB than in M9. For butyric acid the difference was stronger because in LB bacteria did not grow anymore with 109mM, whereas in M9 there was growth.

In fact, M9 was buffered and not LB so we measured pH in both medium with different acids concentrations in order to see if there were a correlation.

It is clear that in M9 medium pH stayed at pH 7 for higher acids concentrations than LB medium. Moreover, thanks to previous figures, it is possible to see that bacteria did not grow anymore when pH was around 5. This results show that bacteria are sensitive to acid pH, but they may have resisted to higher acids concentrations if the medium were better buffered. We will now see if it would be interesting or not to have buffered better our medium.

Figure 34 shows a dose/response relationship between formic acid concentration and bacteria growth. We wanted to produce at least 50µmol/L of formic acid in order to kill varroas and bacteria growth normally up to 1mM. So, there should not have been toxicity problems during the treatment.

The higher butyric concentration was, the less was bacteria growth, as our previous results with formic acid. However we had an intermediate result with 109mM of butyric acid. We did not have a specific butyric acid concentration to produce and modeling showed us that we could produce around 15mM with all optimizations, so we would not have any butyric acid toxicity during our treatment.

Note: 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 in the medium.