Part I :Science
Proof of the efficiency of the degradation of Anthracene a Poly Aromatic hydrocarbon.
1. The molecule targeted: Anthracene
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 anthraceneAnthracene was obtained in solution in 80% glycerol/water v/v at 0.1 mg/ml (Generous gift from Chimie ParisTech).
2. Preparation of the enzymes
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).
3. Solutions tested :
Buffers for enzymatic reaction:
|Composition of the buffers for :||Reaction volumes (μl): A||The blank volumes (μl): B|
|Solution of trisphenylphosphine (5M)||13.2 μL (0.050M)||13.2 μL (0.050M)|
|Solution of Anthracene||660 μL||0 μL|
|Solution of NaCl (1M)||66 μL (0.050M)||66 μL (0.050M)|
|Water||0 μL||660 μL|
The Reaction mixes:
|Reaction buffer||67.2 μL of A||67.2 μL of B|
|Enzyme||10 μL for each enzymes (50 μL when there are the five)||10 μL for each enzymes (50 μL when there are the five)|
|Water||Complementary to 120 μL as a total volume (which means 2.8 μL when there are the five enzymes)||Complementary to 120 μL as a total volume (which means 2.8 μL when there are the five enzymes)|
For the Controls :
The enzymes volumes are substituted with extracts of cells transformed with pET32a only, and induced for the same length of time.
4. Reactions kinetics
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.
On the Figure 5.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 5.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.