Difference between revisions of "Team:TJUSLS China/Proof"

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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Medals">gold medal criterion for proof of concept</a>. </p>
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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        <ul>
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            <li><a href="#part1">Mutation</a></li>
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            <li><a href="#part2">Surface display in E.coli</a></li>
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            <li><a href="#part3">Surface display in Pichia Pastoris</a></li>
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            <li><a href="#part4">Co-display in Pichia Pastoris</a></li>
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    Proof of Concept
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        <h3 id="part1">Mutation:</h3>
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        <img src="https://static.igem.org/mediawiki/igem.org/2/22/ProofTJU1.jpg">
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        <div class="sec-wenzi-content">Figure 1. The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein.</div>
  
 +
        <img src="https://static.igem.org/mediawiki/igem.org/5/53/ProofTJU2.jpg">
 +
        <div class="sec-wenzi-content">Figure 2. PETase’s self-degrading condition in different temperature. We take the quantity of PETase in the day the experiment begines as 100%. We can now see when stored in low temperature, the protein was degraded slowly, but in room temperature, the protein degrades rapidly. The Quantity of PETase left was measured by protein gel and analysised by a computer program called GEL-PRO.</div>
  
 +
        <h3 id="part2">Surface display in E.coli</h3>
 +
        <div class="sec-wenzi-list">Surface display in E.coli by using INP</div>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/6/64/ProofTJU3.jpg">
 +
        <div class="sec-wenzi-content">Figure 3  Relative enzyme activity of  engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃.</div>
  
 +
        <img src="https://static.igem.org/mediawiki/igem.org/3/3c/ProofTJU4.jpg">
 +
        <div class="sec-wenzi-content">Figure 4  Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 25℃ with different amount of bacteria.</div>
  
 +
        <img src="https://static.igem.org/mediawiki/igem.org/e/e0/ProofTJU5.jpg">
 +
        <div class="sec-wenzi-content">Figure 5  Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced with 0.1mM IPTG for 24h.</div>
  
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<div class="column full_size">
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        <img src="https://static.igem.org/mediawiki/igem.org/8/82/ProofTJU6.jpg">
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        <div class="sec-wenzi-content">Figure 6  Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃ with 0.1mM IPTG for 1h, 4h, 8h, 12h, 16h and 20h.</div>
  
 +
        <div class="sec-wenzi-list">Surface display in E.coli by using LPP-OmpA</div>
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        <img src="https://static.igem.org/mediawiki/igem.org/6/66/ProofTJU8.jpg">
 +
        <div class="sec-wenzi-content">Figure 7  Relative enzyme activity of  engineering bacteria E.coli(BL21)/pET22b(+)LAP when induced at 16℃.</div>
  
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<p>
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        <div class="sec-wenzi-list">Surface display in E.coli by using BrkA</div>
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iGEM teams are great at making things work! We value teams not only doing an incredible job with theoretical models and experiments, but also in taking the first steps to make their project real.  
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        <img src="https://static.igem.org/mediawiki/igem.org/6/60/ProofTJU9.jpg">
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</p>
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        <div class="sec-wenzi-content">Figure 8 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)Brk when induced at 16℃ and 25 ℃ with 0.02mM IPTG.And the last two were induced with 0.09mM IPTG.</div>
  
 +
        <div class="sec-wenzi-list">Surface display in E.coli by using AIDA</div>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/d/d5/ProofTJU10.jpg">
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        <div class="sec-wenzi-content">Figure 9. Reletive enzyme activity of engineering  bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃</div>
  
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<h4> What should we do for our proof of concept? </h4>
 
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<p>
 
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You can assemble a device from BioBricks and show it works. You could build some equipment if you're competing for the hardware award. You can create a working model of your software for the software award. Please note that this not an exhaustive list of activities you can do to fulfill the gold medal criterion. As always, your aim is to impress the judges!
 
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</p>
 
  
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        <h3 id="part3">Surface display in Pichia Pastoris</h3>
  
 +
        <img src="https://static.igem.org/mediawiki/igem.org/0/0f/ProofTJU11.jpg">
  
 +
        <img src="https://static.igem.org/mediawiki/igem.org/e/e5/ProofTJU12.jpg">
 +
        <div class="sec-wenzi-content">Figure 10: The activity of P. pastoris PETase-GCW21. a&b used the first group of yeast; c&d used the second of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.</div>
  
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</div>
 
  
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        <img src="https://static.igem.org/mediawiki/igem.org/4/48/ProofTJU13.jpg">
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        <img src="https://static.igem.org/mediawiki/igem.org/2/2d/ProofTJU14.jpg">
 +
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        <div class="sec-wenzi-content">Figure 11: The activity of P. pastoris PETase-GCW51. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.</div>
 +
 +
        <img src="https://static.igem.org/mediawiki/igem.org/d/d7/ProofTJU15.jpg">
 +
        <img src="https://static.igem.org/mediawiki/igem.org/0/0c/ProofTJU16.jpg">
 +
        <div class="sec-wenzi-content">Figure 12: The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.</div>
 +
 +
        <h3 id="part4">Co-display in Pichia Pastoris</h3>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/6/67/ProofTJU17.jpg">
 +
        <div class="sec-wenzi-content">Figure 13: The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 co-display transformants in different hours  and amount of yeast.</div>
 +
 +
        <img src="https://static.igem.org/mediawiki/igem.org/d/d5/ProofTJU18.jpg">
 +
        <div class="sec-wenzi-content">Figure 14: The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 co-display transformants in different hours  and amount of yeast.</div>
 +
 +
        <img src="https://static.igem.org/mediawiki/igem.org/b/b9/ProofTJU19.jpg">
 +
        <div class="sec-wenzi-content">Figure 15: The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformants in different hours  and amount of yeast.</div>
 +
 +
        <img src="https://static.igem.org/mediawiki/igem.org/a/a0/ProofTJU20.jpg">
 +
        <div class="sec-wenzi-content">Figure 16: The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformants in different hours  and amount of yeast.</div>
 +
 +
        <img src="https://static.igem.org/mediawiki/igem.org/e/ea/ProofTJU21.jpg">
 +
        <div class="sec-wenzi-content">Figure 17: The activity of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformant in best condition</div>
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Latest revision as of 11:42, 19 October 2016

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Proof of Concept

Mutation:

Figure 1. The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein.
Figure 2. PETase’s self-degrading condition in different temperature. We take the quantity of PETase in the day the experiment begines as 100%. We can now see when stored in low temperature, the protein was degraded slowly, but in room temperature, the protein degrades rapidly. The Quantity of PETase left was measured by protein gel and analysised by a computer program called GEL-PRO.

Surface display in E.coli

Surface display in E.coli by using INP
Figure 3 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃.
Figure 4 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 25℃ with different amount of bacteria.
Figure 5 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced with 0.1mM IPTG for 24h.
Figure 6 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃ with 0.1mM IPTG for 1h, 4h, 8h, 12h, 16h and 20h.
Surface display in E.coli by using LPP-OmpA
Figure 7 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)LAP when induced at 16℃.
Surface display in E.coli by using BrkA
Figure 8 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)Brk when induced at 16℃ and 25 ℃ with 0.02mM IPTG.And the last two were induced with 0.09mM IPTG.
Surface display in E.coli by using AIDA
Figure 9. Reletive enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃

Surface display in Pichia Pastoris

Figure 10: The activity of P. pastoris PETase-GCW21. a&b used the first group of yeast; c&d used the second of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.
Figure 11: The activity of P. pastoris PETase-GCW51. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.
Figure 12: The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.

Co-display in Pichia Pastoris

Figure 13: The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 14: The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 15: The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 16: The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 17: The activity of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformant in best condition

Copyright 2016 TJUSLS