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:
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 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
• 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.
• 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.
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.
• 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.
TEXTE
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