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 varroas. However, in the Cytotoxicity part of the results, we also showed 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 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 enables obtention of a 10 % increase in production. Optimizing this production will have to be achieved before testing the strain capacity to kill varroas.

We want to express the butyrate synthetic pathway by day and the formate synthetic pathway by night. As a first step to develop our regulation module, we replaced the butyrate pathway by the RFP reporter gene and the formate pathway by GFP reporter gene. Doing so, it should be easy to assess the functioning of the light sensor and the NOT logic gate since the colonies should be red by day and green by night.
To do so, we synthesized the pOMPC-cI gene (new biobrick BBa_K1587006)Through subcloning the GFP reporter gene (BBa_E0240) was placed under control of this promoter. Likewise, the RFP reporter gene (BBa_K081014) was cloned behind the PL-lacI synthetic gene. Both constructions were placed together on the same plasmid but unfortunately sequencing revealed that there was some incoherence in the RFP sequence, preventing further testing.

Figure : NOT logic gate test construction

For our project we need to implement a light sensor system in our strain. To that end, we needed to clone the three genes (cph8, hoI, pcyA) of this system behind a strong promoter. We received the strain containing the three genes on different plasmids as courtesy from Dr Clark Lagarias, Christopher Voigt, and Nico Claassens [REF]. These genes were amplified by PCR and subcloned in pSB1C3 (at this step, a RBS was added to sequence during the PCR). The new biobricks for hoI and pcYA were sent to the registry under the accession number BBa_K1587000 and BBa_K1587002, respectively. The cph8 biobrick was not obtained but we managed to build a biobrick (BBa_K1587008) with the cph8 encoding sequence under the control of a strong promoter originated from the biobrick (BBa_J23119).

Figure : Targeted light sensor construction

Figure : cph8 biobrick

We did not succeed in associating the three genes together behind a strong promoter in pSB1C3 using the In Fusion cloning technique before the iGEM deadline. Consequently, we were not able to test our light sensor system soon enough for this project.

In a nutshell:

About 70% of the clonings for the light sensor and the NOT logic gate has been done, but the last cloning steps remain to be finished before the tests.

We investigated if the bacteria could 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

According to these results, TPX® lets 56% of formic acid to pass outside the bag in the gas phase. Therefore, formic acid permeats through the TPX plastic. Using a more porous plastic as proposed for the butyrate, we supposed 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 was 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 faced 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 under aerobic and micro-aerobic conditions, measured the growth rate (OD) and analyzed the supernatant to measure the concentration of fermentation products.

Micro-aerobic conditions were obtained with culture 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).

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

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 was consumed approximately at the same rate for both conditions but was converted differently depending on the condition. Under aerobic conditions the biomass reached 3 g/L whereas under micro-aerobic conditions there was six times less biomass. On the contrary, there were far less products in aerobic conditions.

Our results were in line with our objective to produce acids in a microporous bag because we demonstrated that with microaerobic conditions, bacteria grew slowly and produced fermentation products.

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

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

Under our microaerobic conditions, the wild type E. coli strain produced 32 mM of formate. It was 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 have been 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 hypothesized that in 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.

In a nutshell:

E. coli can produce various fermentation products when cultured in microaerobiosis and can survive up to two weeks in normal culture conditions. This validates the possible use of long lasting cultures in our device.

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 previously. 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 was an increase of OD in all conditions during 12 days. This means that glucose releasing worked as expected. At the beginning there is an exponential growth because some glucose is directly available in the medium. This phase was 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 knew that it was possible to have continuous growth during at least 12 days. However, in our control without enzyme, there was also some bacterial growth so either another substrate was available or bacteria could realy 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 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 hypothesized 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 glucose release production, it was necessary to calculate how much polymer and enzyme concentration are needed.

With the same equations as we used in Characteristics of Biosilta kit we determined which total quantity of glucose is needed for 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 should therefore concentrate the medium 7 times. To obtain a glucose release of 0.3 g.L^-1.h^-1 value, we nee 5 U/L of enzyme. Thus, we tested different concentrations of Biosilta medium with different enzyme concentrations.

We do not have the exact composition of Biosilta medium and cannot ascertain innocuity of higher titers of the polymer on growth. The best approach was to actually test it directly.

From these three figures it is evident that we cannot reach the growth level that is expected and that concentrated Biosilta is actually detrimental to growth.

In a nutshell:

Biosilta medium can be used in many various conditions that can actually exactly control the glucose release rate and hence the growth of bacteria. However ,our target quantity of formic acid cannot be attained with normal medium concentrations and it is not possible to use higher doses of polymer in order to promote the growth to acceptable values (growth inhibition).

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

In M9 medium, the growth is slower at the beginning of the growth curve, however the maximum value of OD reached is almost the same for both medium.

There is a visible difference between the two culture media. Growth in LB is clearly less tolerant to either formic or butyric acid. As M9 (and not LB) is a buffered culture medium we measured the pH of both media with different acids concentrations.

It is clear that in M9 medium the pH remains at pH 7 at high acid concentrations but not in the LB medium. Moreover we now know that bacteria do not grow when the pH is around 5. We will now see if buffering our medium has a positive action on growth.