Difference between revisions of "Team:Aix-Marseille/Results"

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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)). </p>
 
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)). </p>
  
<p><span style="color:#FF0000">Question 1: Can the laccase oxidize the cytochrome C?</p>
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<p><span style="color:#FF0000"><span style ="font-family:Courier New">Question 1: Can the laccase oxidize the cytochrome C?</p>
 
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.  
 
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. </p></div>
 
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. </p></div>
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     <div class="space30"></div> <p class="space20"><div align="justify"><span style ="font-family:Courier New">
 
     <div class="space30"></div> <p class="space20"><div align="justify"><span style ="font-family:Courier New">
     <p><span style="color:#FF0000">Question 3: Do our enzymes have the same enzymatic activity?</p>
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     <p><span style="color:#FF0000"><span style ="font-family:Courier New">Question 3: Do our enzymes have the same enzymatic activity?</p>
 
<p>We tried to clone several laccases from T. thermophilus, E. coli, B. subtilis and Laccase 15 (from a uncultured bacterium) and several cytochromes C from S. oneidensis and Synechosystis sp.</p>
 
<p>We tried to clone several laccases from T. thermophilus, E. coli, B. subtilis and Laccase 15 (from a uncultured bacterium) and several cytochromes C from S. oneidensis and Synechosystis sp.</p>
 
<p>We cloned with success two laccases (T. thermophilus and E. coli) and one cytochrome C (S. oneidensis). We tried to produce these proteins into the E.coli BL21 strain.</p>
 
<p>We cloned with success two laccases (T. thermophilus and E. coli) and one cytochrome C (S. oneidensis). We tried to produce these proteins into the E.coli BL21 strain.</p>

Revision as of 12:50, 17 September 2015

Chew fight

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 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.

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”


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



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).


Figure 3: Putative schema of reaction with the laccase and the cytochrom C

Conclusion 2: The laccase coupled with the cytochrome C can’t oxidize the styrene.


Figure 4: Oxydation of cytochrom C by laccase



Question 3: Do our enzymes have the same enzymatic activity?

We tried to clone several laccases from T. thermophilus, E. coli, B. subtilis and Laccase 15 (from a uncultured bacterium) and several cytochromes C from S. oneidensis and Synechosystis sp.

We cloned with success two laccases (T. thermophilus and E. coli) and one cytochrome C (S. oneidensis). We tried to produce these proteins into the E.coli BL21 strain.

We purified the E.coli laccase and performed enzymatic tests to determine if it has an activity. To do this, we used the ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)), which is oxidized by laccases. This compound is colored in green when it is oxidized.


Unfortunately, we don’t observe activity with purified E. coli laccase. We know that copper binds laccases. So we incubate our laccase with copper and with no success.

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Chew figth project, for the iGEM competition. See you soon in Boston !