Difference between revisions of "Team:Aix-Marseille/Results"
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| + | <h2 class="title wow Hinge"><span style="color:#8E3B8C">Laccase <i>T.thermophilus</i> from IDT</h2></span> | ||
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| + | We ordered from IDT a laccase optimized for an expression into <i>E.coli</i>.<br /> From this optimised laccase, we added a promoter and a His-Tag.<br /> This new BioBrick is named “01-30-02” with an expected size of about 1500 pb <br /><br /> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/a/a8/Figure_A2.png" width="500" height="179" space="0"> | ||
| + | <span>Figure A : Schematic representation of “01-30-02”</span><br /><br /> | ||
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| + | <img src="https://static.igem.org/mediawiki/2015/5/58/Figure_B2.png" space200> <br /> | ||
| + | <span>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.</span><br /><br /> | ||
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Revision as of 06:55, 17 September 2015
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.
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.
