Team:UNIPV-Pavia/Methods Materials/Sensing ethanol concentration: our do-it-yourself kit

From 2009.igem.org

Protocols

Sensing ethanol concentration: our do-it-yourself kit

In order to sense the presence of ethanol in the culture broth, we initially used a commercial enzymic kit, which gave us disappointing results, as not even the calibration resulted linear and sometimes the enzyme failed the reaction.

Then, inspired by the threat of being caught drunk by the police at the wheel, we looked into whether it was possible to set up a similar testing system to the one used by police officers for breath tests.

We discovered that there are two different ethanol sensitive devices: the first type is based on measuring the variation of potential on a charged semiconductor, but we immediately discarded this as it was not possible for us to reproduce a similar device in the short term.

The second system looked much more achievable. Actually, it is nothing more than a colorimetric assay, where a redox reaction between a dichromate ion (Cr₂O_7^2-) and ethanol reduces the chrome’s charge from +6 to +3 and turns the solution from orange to blue in a linear relationship with the amount of ethanol dissolved into the medium.

The components for this reaction are: - Potassium dichromate (K₂Cr₂O₇) in variable concentrations, as explained later. - H₂SO₄ 5M as solvent - Ethanol Whereas the stoichiometry of this reaction is:

CH₃CH₂OH + K₂Cr₂O₇ + H₂SO₄ → CH₃COOH + Cr₂(SO₄)₃ + K₂SO₄ + 11 H₂O

The dichromate concentration limits the range of confidence in measurement: the more dichromate ions are present, the more the system will sense higher ethanol concentrations.

Thus, we performed a primary experiment with different dichromate concentration, as explained in Hyun-Beom; 2008.

The three concentrations we tested were 5%, 8% and 10% w/v, while standard curves were performed with different concentrations of ethanol first in water, then in LB medium, as follows: 0%; 0.5%; 1.5%; 2.5%; 3.5%; 4.5%. Measurement has been performed with a Tecan™ instrument, checking absorbance 595nm every 5 minutes for more than two hours.

As expected, dichromate 5% lost linearity in a very short range of concentrations: between 0% and 0.5% of ethanol, while dichromate 8% lost linearity at about 1.5% - 2.5% of ethanol. In contrast, dichromate 10% (equivalent to 340 mM) maintained linearity above ethanol 3.5%, when the curve started to flatten, reaching its saturation plateau. The theoretical yield of ethanol should settle at about 4.5%, so we decided to use a solution of 10% dichromate, with the possibility of increasing its concentration if wider range measures were required.

Similar results have been obtained when the standard curve was performed dissolving ethanol into LB medium as well, even if the absorbance values were higher than those measured in distilled water. This is caused by normal higher optical density of LB medium than that of water, but we figured out that it could still create some background noise.

For this purpose, we decided to perform further experiments to find out at what time we would have the best measurement, changing number and frequency of measurements, and we found that the reaction is surprisingly quick, reaching saturation in less than 15 minutes from the beginning of measurement. We concluded that best measurements could be taken within 10 minutes from the beginning of reaction.

Finally, we considered whether glucose could interfere with measurement, because of its reducing potential. For this purpose, we performed two other experiments, the first exchanging glucose with ethanol in the standard curve; the second performing a standard curve in which samples were composed of different concentrations of ethanol and LB medium, with 10% glucose added (which is our medium selected for long time fermentation). Unfortunately, results were not encouraging: glucose is sensed by this system almost as well as ethanol, and a measurement with 10% glucose in the medium produces too much noise for it to be considered accurate.

At this point, we faced three different possibilities: the first was to look for a gas chromatograph, which is the fastest and most reliable instrument for this kind of measurement. The second was distilling the supernatant, instead of measuring it straightaway, in order to separate everything that was dissolved into the medium (salts, proteins and cell particles and, above all, sugars) and extracting just water and ethanol. A distillation at 78°C could separate ethanol from water, but we considered it satisfactory to separate water and ethanol from all the rest. Actually, once some samples had been distilled, we considered that it was quite an imprecise method for all the possible loss that could occur, so we discarded it.

Finally, we considered following the protocol explained by Hyun-Beom; 2008. They used tri-n-butyl phosphate, which is a non-polar solvent, to extract ethanol in an aqueous solution.

PHASE SEPARATION PROTOCOL

1) Centrifuge samples 6000rpm for 15 minutes, in order to remove cell particles, than move the supernatant into a new tube.

2) Move 1ml of the supernatant previously collected into a 2ml tube, than add 1ml tributyl-n-phosphate (TBP).

3) Keep shaking into an incubator 150rmp for 15min. You should see a clear phase separation, where the lower phase has the classical color of LB medium, while the higher is transparent.

4) Move 250ul of the higher phase into a new 1,5ml tube, then add 250ul dichromate reagent.

5) Keep shaking into an incubator 150rmp for 15min.

6) Move 100ul of the lower phase into a 96-well plate.

7) Measure absorbance at 600nm wavelength.

We use to prepare a standard curve made of 6 to 8 spots. This system has been used and tested several times in the late phase of the competition, but we didn’t have enough time to set up the best experimental procedure, even if the one we just described, gave us pretty satisfying results.