Difference between revisions of "Team:Pasteur Paris/Demonstrate"

We demonstrated the effective degradation of PAHs using anthracene as a representative molecule. It is possible to measure its concentration since anthracene absorbs at 254 nm, and therefore the kinetics of the reaction can be followed as well. We prepared our enzyme mix composed of E1(E1_1, E1_2), E2, E3, E4 by expression in E. coli (see below) (See Biobrick parts section for correspondence of enzyme names)

Figure 1 : schema of anthracene

Anthracene was obtained in solution in 80% glycerol/water v/v at 0.1 mg/ml (Generous gift Chimie ParisTech).

The five open reading frames coding for the four enzymes of the degradation pathway were independently expressed in BL21De3 cells. Cell pellets stored at -80°c, were thawed in lysis buffer (Tris-Cl 50 mM, pH 7.4, NaCl 100 mM, Glycerol 5% v/v) and sonicated. After centrifugation, supernatants were kept on ice for the enzymatic assay. (See Protein expression protocol).

Buffers for enzymatic reaction:

The Reaction mixes:

For the Controls :

The enzymes volumes are substituted with extracts of cells transformed with pET32a only, and induced for the same length of time.

An absorbance blank was performed using the enzyme mix in reaction buffer at 254 nm. This prevented the detection of DNA in the enzyme solution from the cell extracts. Anthracene was then added and a time point taken. Several other time points were taken after 3, 6, 12 and 24 minutes. The reaction was performed three times and the mean value of absorbance plotted against time to follow the kinetics. Error bars are shown as the standard deviation of the three measurements.

Figure 2.A. Degradation of Anthracene by the E1, E2, E3, and E4 cocktail of enzymes. Absorbance of anthracene was followed at 254 nm over time. Three independent measurements were made and the average plotted. The SD for each point is plotted as error bar.

Figure 2.B. Control reaction for the degradation of anthracene with the enzyme cocktail E1, E2, E3, E4. Time plot of the pET32a transformed BL21De3 cells extract with anthracene.

On the Figure 2.A.: we can clearly see the concentration of anthracene decreasing in a constant and monotonous way over time. So, there is a degradation of the anthracene during the first 12 min and then the concentration stabilises itself. In the second graph Figure 2.B., we expected to see only a constant value. As it can be seen, except for point at time of 3 min, the concentration stays constant which means that there is no degradation without the catalysis.

We can conclude that our cocktail of four enzymes degrades effectively anthracene, a member of the PAH family, and therefore possibly other poly-aromatic compounds such as the benzo[a]pyrene.

Based on the ideation session, interviews with professionals, questionnaires to the public, and modelling of air flow, amount of enzymes and biopolymer to support it that can be possible etc .. we designed and Air filter system to the scale that can be used in the household indoors. The device is equipped with a front cover air filter for large particles or fibres. It has a vinyl crafted surface for a prototype of Graetzel solar cells for its power supply. It also has holder for the bio-filter and charcoal filter units. It has a back cover to allow for air exit. And finally, it has a stand a strap system to either stand on a desk or set against a wall.

Figure 3. The Aether system for air purification and degradation of poly aromatic hydrocarbons.