Difference between revisions of "Team:Toulouse/Design"

TrApiColi has been designed in order to take into account ethical reflection, safety and ease of use for beekeepers. The use of genetically modified organisms in a field and more, associated with edible products underlies both law enforcement and applying of regulations and public perception account about it. Thus, as TrApiColi is composed of a small bag made of TPX® where engineered bacteria are confined, we tested the impermeability of bacteria through the bag.

In order to know if our test that we described in “Protocol” test functions, we made a control with acids solutions in a falcon and we sample gas in balance.

For butyric acid we did not detect gas butyric acid by NMR, the solution we used to solubilize gas should be not basic enough. So we made a test with a TPX® bag containing butyric acid into a solution of sodium bicarbonate. As a control we sampled directly a solution of 4% (V/V) of butyric acid.

Figure 1: NMR Spectrum of butyric acid liquid control in red and butyric acid liquid which passed through TPX bag in blue. Red curve is zoomed 1340 times more than blue curve. Each condition was tested in two replicates.

Table 1: Concentrations of butyric acid corresponding to NMR spectrum

Thanks to these results, TPX® overlook butyric acid outside the bag. We detect only a small quantity but an optimization of test could be made or a plastic with bigger porous could be use.

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. At the left top internal standard shows that it is the same scale for both curves. Each condition was tested in two replicates.

Table 2: Concentrations of formic acid corresponding to NMR spectrum

According to these results, TPX® overlook 56 % of formic acid outside the bag in gas phase. We show that formic acid can go through TPX plastic. And with a better test, as we proposed above this percentage could increase.

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:

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.

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.

In order to plot biomass concentration it is necessary to convert the OD measured.
This equation was used:

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

For substrate and products concentration we plotted peak area of each molecule on NMR spectrum.
Then, we calculated concentration with this equation:

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

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:

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

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.

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.