Team:Aix-Marseille/Results
Production of Laccase E.coli
To obtain our Laccase E.coli, we used the BioBrick Bba_K863006 and we removed its stop codon. Then we added to this BioBrick a promoter and a His-Tag.
Thanks to digestion, ligation and transformation, we managed to get our BioBrick named “01-35-02” with a size of about 1700 pb:
Figure A: Schematic representation of “01-35-02”
Figure A: Schematic representation of “01-35-02”
Figure B: Digestion from “01-35-02” miniprep to check the size of the insert
The band corresponds to the expected size for the insert, which is about 1700 pb.”
Figure C: Western Blot of the expression of “01-35-02” into E. coli
The expected size of the protein is about 53 kDa.
Laccase T.thermophilus from iGEM parts
To get our Laccase T. thermophilus, we used the BioBrick Bba_K863011 and we removed its stop codon.
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.
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.
Figure E: Digestion from “01-36” miniprep to check the size of the insert
The band corresponds to the expected size for the insert, which is about 1500 pb.”
Laccase T.thermophilus from IDT
We ordered from IDT a laccase optimized for an expression into E.coli.
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”
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”
Figure B: Digestion from “01-30-02” miniprep to check the size of the insert
The band corresponds to the expected size for the insert, which is about 1500 pb.
Enzymatic activity
All tests were performed using laccases and cytochromes C obtained by ISM2 (Institut des Sciences Moléculaires de Marseille)and LISM (Laboratoire d’Ingénierie des Systèmes Macromoléculaires)).
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.
Figure 1 : Absorbance propertiez of cytochrome C
Figure 2: Oxydation of cytochrom C by laccase
onclusion 1: The laccase can oxidize the cytochrome C.
