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

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    <div align="center"><h2 class="title wow bounce in up"><span style="color:#8E3B8C"><span style="font-family:Armalite Rifle"><h2 class="title wow Hinge">Enzymatic activity</h2></span></div>
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    <p class="space20"><div align="justify"><span style ="font-family:Courier New">
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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>
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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.
 +
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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<img src="http://i.imgur.com/lhuqROK.png"  width="500" height="350" space="0">
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<span><p class="space20"><div align="justify"><span style ="font-family:Courier New">Figure 1 : Absorbance properties of cytochrome C</span><br /><br />
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                          <div class="success-work project">
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                        <div class="success-work-desc">
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                            <img src="http://i.imgur.com/xZ4I4FX.png" width="500" height="350"  space200> <br />
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<span><p class="space20"><div align="justify"><span style ="font-family:Courier New">Figure 2: Oxydation of cytochrom C by laccase</p></div></span><br /> 
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                      <p><span style="color:#FF0000"><span style ="font-family:Courier New">Conclusion 1: The laccase can oxidize the cytochrome C.</p>
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<p><span style="color:#FF0000"><span style ="font-family:Courier New">Question 2: Can the cytochrome C and the laccase oxidize chewing-gum polymer?</p>
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<p><span style ="font-family:Courier New">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.</p>
 +
<p><span style ="font-family:Courier New">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.</p>
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</p>
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<span style ="font-family:Courier New"><h4>Hypothesis:</h4> <p><span style ="font-family:Courier New">Could the cytochrome C substitute the X compound to oxidize the styrene?</p>
 +
<p><span style ="font-family:Courier New">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.</p>
 +
<p><span style ="font-family:Courier New">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).</p>
 +
</div><br />
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<img src="http://i.imgur.com/nqiJuod.png"  width="500" height="350" space="0">
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<span><p class="space20"><div align="justify"><span style ="font-family:Courier New">Figure 3: Putative schema of reaction with the laccase and the cytochrom C</span>
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</p>
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        </div>
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<p class="space20"><div align="justify"><span style ="font-family:Courier New"><span style="color:#FF0000">Conclusion 2: The laccase coupled with the cytochrome C can’t oxidize the styrene.</p>
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</div>
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    <div class="col-md-6 left">
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                            <img src="http://i.imgur.com/L9jlTEB.png" width="500" height="350"  space200> <br />
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<span><p class="space20"><div align="justify"><span style ="font-family:Courier New">Figure 4: Oxydation of cytochrom C by laccase</p></div></span><br /><br />
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<section style="padding:30px 0px 50px;" class="arrow_box" id="team">
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    <div class="container">
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    <div class="row">
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    <div class="col-md-6 left">
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    <div class="space30"></div> <p class="space20"><div align="justify"><span style ="font-family:Courier New">
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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><span style ="font-family:Courier New">We tried to clone several laccases from <i>T.thermophilus</i>, <i>E.coli</i>, <i>B. subtilis</i> and Laccase 15 (from a uncultured bacterium) and several cytochromes C from <i>S. oneidensis</i> and <i>Synechosystis sp.</i></p>
 +
<p><span style ="font-family:Courier New">We cloned with success two laccases (<i>T. thermophilus</i> and <i>E.coli</i>) and one cytochrome C (<i>S.oneidensis</i>). We tried to produce these proteins into the <i>E.coli</i> BL21 strain.</p>
 +
<p><span style ="font-family:Courier New">We purified the <i>E.coli</i> 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.</p><br/>
 +
 +
<p><span style ="font-family:Courier New">Unfortunately, we don’t observe activity with purified <i>E.coli</i> laccase. We know that copper binds laccases. So we incubate our laccase with copper and with no success.</p>
 +
        </div>
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 +
                            <img src="http://i.imgur.com/diY7clP.png" width="500" height="350"  space200> <br />
 +
<span><p class="space20"><div align="justify"><span style ="font-family:Courier New">Figure 5: Mersure of ABTS oxydation by the laccase from <i>E.coli</i> we purified</p></div></span><br />
 +
<p class="space20"><div align="justify"><span style ="font-family:Courier New"><span style="color:#FF0000">Conclusion 3: With or without copper, the purified E.coli laccase can’t oxidize the styrene.</p></div>
 +
 +
<h4>Discussion:</h4>
 +
<p class="space20"><div align="justify"><span style ="font-family:Courier New">We can make some hypothesis concerning these results.</p><br/>
 +
1) We produced the <i>E. coli</i> laccase in aerobic condition. However, the <i>E.coli</i> laccase could have a bad folding in these conditions and then needs to be produced in anaerobic condition. <br/><br/>
 +
2) Usually, a laccase shows a blue color given by copper presence. Our purified <i>E.coli</i> laccase is not blue. Maybe the copper is absent. Ni-NTA column is known to remove metal from protein. We can hypothesis that the use of the Ni-NTA is not appropriate. <br/><br/>
 +
In perspective, we would like to perform the production of our <i>E.coli</i> laccase in anaerobic condition. Using another purification column such as an anion-exchange column or size exclusion, we hope to conserve copper center and enzymatic activity.</p>
 +
 +
 +
  </div>
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    </section>
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<section style="padding:30px 0px 50px;" class="arrow_box" id="team">
 
    
 
    
 
<div class="container">
 
<div class="container">

Revision as of 17:24, 17 September 2015

Chew fight

Results

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 properties 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.


Figure 5: Mersure of ABTS oxydation by the laccase from E.coli we purified


Conclusion 3: With or without copper, the purified E.coli laccase can’t oxidize the styrene.

Discussion:

We can make some hypothesis concerning these results.


1) We produced the E. coli laccase in aerobic condition. However, the E.coli laccase could have a bad folding in these conditions and then needs to be produced in anaerobic condition.

2) Usually, a laccase shows a blue color given by copper presence. Our purified E.coli laccase is not blue. Maybe the copper is absent. Ni-NTA column is known to remove metal from protein. We can hypothesis that the use of the Ni-NTA is not appropriate.

In perspective, we would like to perform the production of our E.coli laccase in anaerobic condition. Using another purification column such as an anion-exchange column or size exclusion, we hope to conserve copper center and enzymatic activity.

1-What is cytochrome C?

The cytochrome complex, or cyt c is a small hemeprotein found in association with the inner membrane of the mitochondrion. It is interesting because unlike other cytochromes, cytochrome c is highly soluble (100g/L). It is capable of undergoing oxidation and reduction. It transfers electrons in the electron transport chain (between complex III and IV), where it is indispensable.

More than its biological functions, cytochrome c can catalyse several reactions such as hydroxylation and aromatic oxidation, and shows peroxidase activity by oxidation of various electron donors such as 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS).

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.

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.”


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 properties 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.


Figure 5: Mersure of ABTS oxydation by the laccase from E.coli we purified


Conclusion 3: With or without copper, the purified E.coli laccase can’t oxidize the styrene.

Discussion:

We can make some hypothesis concerning these results.


1) We produced the E. coli laccase in aerobic condition. However, the E.coli laccase could have a bad folding in these conditions and then needs to be produced in anaerobic condition.

2) Usually, a laccase shows a blue color given by copper presence. Our purified E.coli laccase is not blue. Maybe the copper is absent. Ni-NTA column is known to remove metal from protein. We can hypothesis that the use of the Ni-NTA is not appropriate.

In perspective, we would like to perform the production of our E.coli laccase in anaerobic condition. Using another purification column such as an anion-exchange column or size exclusion, we hope to conserve copper center and enzymatic activity.

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