Difference between revisions of "Team:Aix-Marseille/Results"
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| − | <h2 class="title wow Hinge"><span style="color:#8E3B8C">Production of Laccase E.coli</h2></span> | + | <h2 class="title wow Hinge"><span style="color:#8E3B8C">Production of Laccase <i>E.coli</i></h2></span> |
<div class="space30"></div> | <div class="space30"></div> | ||
<p class="space20"><div align="justify"> | <p class="space20"><div align="justify"> | ||
| − | 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. | + | To obtain our Laccase <i>E.coli</i>, 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: <br /><br /> | Thanks to digestion, ligation and transformation, we managed to get our BioBrick named “01-35-02” with a size of about 1700 pb: <br /><br /> | ||
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<img src= https://static.igem.org/mediawiki/2015/c/c1/Figure_C.png class="img-responsive" width="250" height="156"> | <img src= https://static.igem.org/mediawiki/2015/c/c1/Figure_C.png class="img-responsive" width="250" height="156"> | ||
| − | <span>Figure C: Western Blot of the expression of “01-35-02” into E. coli | + | <span>Figure C: Western Blot of the expression of “01-35-02” into <i>E. coli</i> |
The expected size of the protein is about 53 kDa. </span> | The expected size of the protein is about 53 kDa. </span> | ||
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<span>Figure A: Schematic representation of “01-35-02”</span><br /><br /> | <span>Figure A: Schematic representation of “01-35-02”</span><br /><br /> | ||
| − | 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. <br /> | + | Then we inserted our BioBrick into <i>E.coli</i> 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. <br /> |
</div></p> | </div></p> | ||
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<p class="space20"><div align="justify"> | <p class="space20"><div align="justify"> | ||
| − | We ordered from IDT a laccase optimized for an expression into E. coli.<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 /> | + | 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"> | <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 /> | <span>Figure A : Schematic representation of “01-30-02”</span><br /><br /> | ||
Revision as of 19:27, 16 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.
