Team:Toulouse/Experiments

iGEM Toulouse 2015

Device

Device: biological tests

At 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
Area_TSP = 1
[TSP] = 1.075mM
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 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 6 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
C_b: base concentration
C_a: acid concentration

As it is said in the “Eradicate” part, our goal is to produce 50µM of formic acid to kill varroa, thanks to the equation (3) we know it corresponds to 77,7mM 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.