Team:Aix-Marseille/Results
Production of Laccase E.coli
Figure A: Schematic representation of “01-35-02”
Laccase T.thermophilus from iGEM parts
Then we tried to add to this BioBrick a promoter and a His-Tag.
Unfortunately we managed to add only the promoter.
Figure A: Schematic representation of “01-35-02”Then we inserted our BioBrick into E.coli strain (BL21) to express it. We induced it by addition of IPTG into the cell culture. We made a Western-Blot using a primary antibody against the His-tag and an anti-mouse secondary antibody conjugate with HRP (horseradish peroxidase). The expected size of the protein “01-35-02” is 53 kDa.
Laccase T.thermophilus from IDT
From this optimised laccase, we added a promoter and a His-Tag.
This new BioBrick is named “01-30-02” with an expected size of about 1500 pb
Figure A : Schematic representation of “01-30-02”
Enzymatic activity
Question 1: Can the laccase oxidize the cytochrome C?
To answer this question, we used absorbance properties of the cytochrome C (FIG.1). Indeed, the reduced cytochrome C (curve in red) absorbs at 550 nm whereas the oxidized cytochrome C (curve in green) doesn’t absorb. By spectrophotometry, we analyzed the change in oxidation state. When the cytochrome C is alone, we don’t observe oxidation (FIG.2, blue curve). When we add the laccase, the cytochrome C oxidizes as we can see a decreased absorbance at 550 nm (FIG.2, red curve). We can see the effect of the laccase on the cytochrome C.
Conclusion 1: The laccase can oxidize the cytochrome C.
Question 2: Can the cytochrome C and the laccase oxidize chewing-gum polymer?
The aim of our project is to show that the chewing gum can be degraded by the laccase and the cytochrome C in presence of the light.
Using the X compound, the light and the laccase, the styrene that is a close chewing gum polymer can be oxidized (FIG.3 & FIG.4). The X compound is a rare metal so we don’t want to use it.
Hypothesis: Could the cytochrome C substitute the X compound to oxidize the styrene?
To test our hypothesis, we used an oxygraph. This engine allows us to measure dioxygen concentration consumed during the reoxidation of the X compound or the cytochrome C by the laccase.
The laccase coupled with the X compound can oxidize the styrene (FIG.4). Replacing the X compound by the cytochrome C, we don’t observe a decreased dioxygen concentration and then the polymer degradation (FIG.4).
Enzymatic activity
Question 1: Can the laccase oxidize the cytochrome C?
To answer this question, we used absorbance properties of the cytochrome C (FIG.1). Indeed, the reduced cytochrome C (curve in red) absorbs at 550 nm whereas the oxidized cytochrome C (curve in green) doesn’t absorb. By spectrophotometry, we analyzed the change in oxidation state. When the cytochrome C is alone, we don’t observe oxidation (FIG.2, blue curve). When we add the laccase, the cytochrome C oxidizes as we can see a decreased absorbance at 550 nm (FIG.2, red curve). We can see the effect of the laccase on the cytochrome C.
Conclusion 1: The laccase can oxidize the cytochrome C.
Question 2: Can the cytochrome C and the laccase oxidize chewing-gum polymer?
The aim of our project is to show that the chewing gum can be degraded by the laccase and the cytochrome C in presence of the light.
Using the X compound, the light and the laccase, the styrene that is a close chewing gum polymer can be oxidized (FIG.3 & FIG.4). The X compound is a rare metal so we don’t want to use it.
Hypothesis: Could the cytochrome C substitute the X compound to oxidize the styrene?
To test our hypothesis, we used an oxygraph. This engine allows us to measure dioxygen concentration consumed during the reoxidation of the X compound or the cytochrome C by the laccase.
The laccase coupled with the X compound can oxidize the styrene (FIG.4). Replacing the X compound by the cytochrome C, we don’t observe a decreased dioxygen concentration and then the polymer degradation (FIG.4).
