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

 
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                 </script>
 
                 </script>
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                 <li onmouseover="getHover(0)"><a href="https://2016.igem.org/Team:TJUSLS_China/home">TJUSLS</a></li>
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                 <li onmouseover="getHover(0)"><a href="https://2016.igem.org/Team:TJUSLS_China">TJUSLS</a></li>
 
                 <li onmouseover="getHover(1)" class=""><a href="" class="root">project</a>
 
                 <li onmouseover="getHover(1)" class=""><a href="" class="root">project</a>
 
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                     <ul>
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         <img class="sec-menu-close" src="https://static.igem.org/mediawiki/2016/b/b5/T--TJUSLS_China--sec-menu-close.png"/>
 
         <ul>
 
         <ul>
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             <li><a href="#part1">Stucture</a></li>
+
             <li><a href="#part1">Mutation</a></li>
 
             <li><a href="#part2">Surface display in E.coli</a></li>
 
             <li><a href="#part2">Surface display in E.coli</a></li>
−
 
+
            <li><a href="#part3">Surface display in pichia pastoris、fusing secretion of hydrophobin and PETase and their co - display
 +
</a></li>
 
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<div class="second-content">
 
<div class="second-content">
 
     <div class="sec-list-wenzi">
 
     <div class="sec-list-wenzi">
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        <h3 id="part1">Stucture:</h3>
+
 
−
         <div class="sec-wenzi-content">One of the vital results of our team this year is managing to analyze the high resolution of the PETase protein structure!</div>
+
         <div class="sec-wenzi-content"><b>One of the vital results of our team this year is managing to analyze the high resolution of the PETase protein structure.To verify if the surface display is successful, we also do western blot, immunofluorescence assay and using HPLC to detecte enzyme activity.(<a href="https://2016.igem.org/Team:TJUSLS_China/Proof">click here</a> to see the HPLC results)</b></div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/c/c9/Tjuresults2.jpg" width="600" height="300">
+
        <h3 id="part1">Mutation:</h3>
 +
         <img src="https://static.igem.org/mediawiki/igem.org/c/c9/Tjuresults2.jpg">
 
         <div class="sec-wenzi-content">Figure1 The result of Overlap PCR of different conditions.</div>
 
         <div class="sec-wenzi-content">Figure1 The result of Overlap PCR of different conditions.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/d/d9/Tjuresults3.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/d/d9/Tjuresults3.jpg">
 
         <div class="sec-wenzi-content">Figure2. The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG.</div>
 
         <div class="sec-wenzi-content">Figure2. The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/6/63/Tjuresults4.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/6/63/Tjuresults4.jpg">
 
         <div class="sec-wenzi-content">Figure3. The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.</div>
 
         <div class="sec-wenzi-content">Figure3. The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.</div>
  
 
         <h3 id="part2">Surface display in E.coli</h3>
 
         <h3 id="part2">Surface display in E.coli</h3>
 +
        <div class="sec-wenzi-list">INP</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/3/35/Tjuresults5.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/3/35/Tjuresults5.jpg">
 
         <div class="sec-wenzi-content">Figure 1.  Overlap PCR results of anchoring motif and passenger protein(NP). (a)The first round of overlap PCR results. Lane 1 is for INPN. Lane 2 is for PETase. M is marker. (b) The third round of overlap PCR results. Lane 1 is the overlap product of INPN and PETase. M is the lane of DNA marker.</div>
 
         <div class="sec-wenzi-content">Figure 1.  Overlap PCR results of anchoring motif and passenger protein(NP). (a)The first round of overlap PCR results. Lane 1 is for INPN. Lane 2 is for PETase. M is marker. (b) The third round of overlap PCR results. Lane 1 is the overlap product of INPN and PETase. M is the lane of DNA marker.</div>
  
−
        <img src="https://static.igem.org/mediawiki/igem.org/e/e0/ProofTJU5.jpg" height="300" width="600">
 
−
        <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>
 
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/d/d4/Tjuresults6.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/d/d4/Tjuresults6.jpg">
 
         <div class="sec-wenzi-content">Figure 2  Enzyme digestion validation. Lane 1 is for double enzyme(NdeI & XhoI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 2 is for single enzyme(NdeI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 3 is for single enzyme(XhoI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 4 is for recombinant plasmid pET22b (+)-INPN-PETase. . Lane 5 is for double enzyme(NdeI & XhoI) digestion of pET22b(+). Lane 6 is for single enzyme(NdeI) digestion of the pET22b(+). Lane 7 is for single enzyme(XhoI) digestion of pET22b(+). Lane 8 is for pET22b (+).  M is DNA marker.</div>
 
         <div class="sec-wenzi-content">Figure 2  Enzyme digestion validation. Lane 1 is for double enzyme(NdeI & XhoI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 2 is for single enzyme(NdeI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 3 is for single enzyme(XhoI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 4 is for recombinant plasmid pET22b (+)-INPN-PETase. . Lane 5 is for double enzyme(NdeI & XhoI) digestion of pET22b(+). Lane 6 is for single enzyme(NdeI) digestion of the pET22b(+). Lane 7 is for single enzyme(XhoI) digestion of pET22b(+). Lane 8 is for pET22b (+).  M is DNA marker.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/9/9f/Tjuresults7.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/9/9f/Tjuresults7.jpg">
 
         <div class="sec-wenzi-content">Figure 3  Standardization of anchoring protein INPN and fusion protein INPN-PETase. (a) The PCR results of anchoring motif INPN and the fusion protein INPN-PETase. Lane 1 is for the N-terminal of ice nucleation protein. Lane 2 is for the whole length of INPN-PETase. M is DNA marker.(b) The double enzyme(EcoRI&SpeI) digestion validation of the recombination standardization plasmid. Lane 1 is for pSBIC3-INPN. Lane 2 is for pSBIC3-INPN-PETase. M is DNA marker.</div>
 
         <div class="sec-wenzi-content">Figure 3  Standardization of anchoring protein INPN and fusion protein INPN-PETase. (a) The PCR results of anchoring motif INPN and the fusion protein INPN-PETase. Lane 1 is for the N-terminal of ice nucleation protein. Lane 2 is for the whole length of INPN-PETase. M is DNA marker.(b) The double enzyme(EcoRI&SpeI) digestion validation of the recombination standardization plasmid. Lane 1 is for pSBIC3-INPN. Lane 2 is for pSBIC3-INPN-PETase. M is DNA marker.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults8.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults8.jpg">
 
         <div class="sec-wenzi-content">Figure 4  Standardization of the anchoring motif INPNC and fusion protein INPNC-PETase. (a) The PCR results of anchoring motif INPN and the fusion protein INPN-PETase. Lane 1 is for the N-terminal of ice nucleation protein. Lane 2 is for the whole length of INPN-PETase. M is DNA marker.(b) The double enzyme(EcoRI&SpeI) digestion validation of the recombination standardization plasmid. Lane 1 is for pSBIC3-INPN. Lane 2 is for pSBIC3-INPN-PETase. M is DNA marker.</div>
 
         <div class="sec-wenzi-content">Figure 4  Standardization of the anchoring motif INPNC and fusion protein INPNC-PETase. (a) The PCR results of anchoring motif INPN and the fusion protein INPN-PETase. Lane 1 is for the N-terminal of ice nucleation protein. Lane 2 is for the whole length of INPN-PETase. M is DNA marker.(b) The double enzyme(EcoRI&SpeI) digestion validation of the recombination standardization plasmid. Lane 1 is for pSBIC3-INPN. Lane 2 is for pSBIC3-INPN-PETase. M is DNA marker.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/3/30/Tjuresults9.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/3/30/Tjuresults9.jpg">
 
         <div class="sec-wenzi-content">Figure5  NP induced by 37℃</div>
 
         <div class="sec-wenzi-content">Figure5  NP induced by 37℃</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/b/b4/Tjuresults10.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/b/b4/Tjuresults10.jpg">
 
         <div class="sec-wenzi-content">Figure6 NP induced by 16℃</div>
 
         <div class="sec-wenzi-content">Figure6 NP induced by 16℃</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/4/48/Tjuresults11.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/4/48/Tjuresults11.jpg">
 
         <div class="sec-wenzi-content">Figure7 NP induced by 25℃</div>
 
         <div class="sec-wenzi-content">Figure7 NP induced by 25℃</div>
  
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/9/93/Tjuresults12.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/9/93/Tjuresults12.jpg">
 
         <div class="sec-wenzi-content">Figure8  NP induced by different concentration of IPTG</div>
 
         <div class="sec-wenzi-content">Figure8  NP induced by different concentration of IPTG</div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/4/45/Tjuresults13.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/4/45/Tjuresults13.jpg">
 
         <div class="sec-wenzi-content">Figure9  NP induced by different concentration of IPTG</div>
 
         <div class="sec-wenzi-content">Figure9  NP induced by different concentration of IPTG</div>
  
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/d/d8/Tjuresults14.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/d/d8/Tjuresults14.jpg">
 
         <div class="sec-wenzi-content">Figure10 NP induced 24h by different concentration of IPTG at 16℃ and 25℃</div>
 
         <div class="sec-wenzi-content">Figure10 NP induced 24h by different concentration of IPTG at 16℃ and 25℃</div>
  
−
         <h3>LPP-OmpA</h3>
+
         <div class="sec-wenzi-list">LPP-OmpA</sec-wenzi-list></div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/b/bd/Tjuresults15.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/b/bd/Tjuresults15.jpg">
 
         <div class="sec-wenzi-content">Figure 1. The first round of overlap PCR results of anchoring motif and passenger protein(Lpp-OmpA). (a) Lane A and B are for  Lpp-OmpA. M1 is marker. (b) Lane C and D are for PETase. M2 is the lane of DNA marker.</div>
 
         <div class="sec-wenzi-content">Figure 1. The first round of overlap PCR results of anchoring motif and passenger protein(Lpp-OmpA). (a) Lane A and B are for  Lpp-OmpA. M1 is marker. (b) Lane C and D are for PETase. M2 is the lane of DNA marker.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/6/69/Tjuresults16.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/6/69/Tjuresults16.jpg">
 
         <div class="sec-wenzi-content">Figure 2.The third round of overlap PCR results. Lane A is the overlap product of Lpp-OmpA and PETase. M is the lane of DNA marker.</div>
 
         <div class="sec-wenzi-content">Figure 2.The third round of overlap PCR results. Lane A is the overlap product of Lpp-OmpA and PETase. M is the lane of DNA marker.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/4/4e/Tjuresults17.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/4/4e/Tjuresults17.jpg">
 
         <div class="sec-wenzi-content">Figure 3  Enzyme digestion validation. Lane A is for double enzyme(NdeI & XhoI) digestion of the recombinant plasmid pET22b(+)-Lpp-OmpA-PETase. M is DNA marker.</div>
 
         <div class="sec-wenzi-content">Figure 3  Enzyme digestion validation. Lane A is for double enzyme(NdeI & XhoI) digestion of the recombinant plasmid pET22b(+)-Lpp-OmpA-PETase. M is DNA marker.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/6/63/Tjuresults41.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/6/63/Tjuresults41.jpg">
 
         <div class="sec-wenzi-content">Figure4  Lpp-OmpA induced by 37℃.</div>
 
         <div class="sec-wenzi-content">Figure4  Lpp-OmpA induced by 37℃.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/9/94/Tjuresults42.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/9/94/Tjuresults42.jpg">
 
         <div class="sec-wenzi-content">Figure5  Lpp-OmpA induced by 25℃.</div>
 
         <div class="sec-wenzi-content">Figure5  Lpp-OmpA induced by 25℃.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/f/f8/Tjuresults43.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/f/f8/Tjuresults43.jpg">
 
         <div class="sec-wenzi-content">Figure6  Lpp-OmpA induced by 16℃. </div>
 
         <div class="sec-wenzi-content">Figure6  Lpp-OmpA induced by 16℃. </div>
  
  
 
         <div class="sec-wenzi-list">BrkA</div>
 
         <div class="sec-wenzi-list">BrkA</div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/6/69/Tjuresults18.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/6/69/Tjuresults18.jpg">
 
         <div class="sec-wenzi-content">Figure 1.M is DNA marker.Lane 1 and Lane 2 are the results of  B1 signal, Lane 3 and 4 are the results of B2 Petase.PCR results Petase.  M is DNA marker. Lane1-4 are overlap PCR results of  B1 and B2.</div>
 
         <div class="sec-wenzi-content">Figure 1.M is DNA marker.Lane 1 and Lane 2 are the results of  B1 signal, Lane 3 and 4 are the results of B2 Petase.PCR results Petase.  M is DNA marker. Lane1-4 are overlap PCR results of  B1 and B2.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/b/b8/Tjuresults19.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/b/b8/Tjuresults19.jpg">
 
         <div class="sec-wenzi-content">Figure 2.C. M is DNA marker.Lane 1 and Lane 2 are the results of B3, Lane 3 and 4 are the results of B4.D. M is DNA marker. Lane1-5 are  oeverlap PCR results of B3 and B4. </div>
 
         <div class="sec-wenzi-content">Figure 2.C. M is DNA marker.Lane 1 and Lane 2 are the results of B3, Lane 3 and 4 are the results of B4.D. M is DNA marker. Lane1-5 are  oeverlap PCR results of B3 and B4. </div>
  
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/2/20/Tjuresults20.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/2/20/Tjuresults20.jpg">
 
         <div class="sec-wenzi-content">Figure 3.E. M is DNA marker.Lane 1 and Lane 2 are the results of full length.F. M is DNA marker. Lane 1 is plasmid digestion.G.M is marker. Lane 1 and 2 are digestion verification.H.M is marker. Lane 1 is PCR verification.</div>
 
         <div class="sec-wenzi-content">Figure 3.E. M is DNA marker.Lane 1 and Lane 2 are the results of full length.F. M is DNA marker. Lane 1 is plasmid digestion.G.M is marker. Lane 1 and 2 are digestion verification.H.M is marker. Lane 1 is PCR verification.</div>
  
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/9/94/Tjuresults21.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/9/94/Tjuresults21.jpg">
 
         <div class="sec-wenzi-content">Figure 4.M is marker. Lane 1 is PCR result of Standard β-barrel domain, anchor sequence.J. M is marker. Lane 1 is Enzyme digestion result of Standard β-barrel domain, anchor sequence.</div>
 
         <div class="sec-wenzi-content">Figure 4.M is marker. Lane 1 is PCR result of Standard β-barrel domain, anchor sequence.J. M is marker. Lane 1 is Enzyme digestion result of Standard β-barrel domain, anchor sequence.</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/d/da/Tjuresults22.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/d/da/Tjuresults22.jpg">
 
         <div class="sec-wenzi-content">Figure 5.K.M is marker. Lane 1 is the pcr result of Standard full length.L.M is marker. Lane 1 is Enzyme digestion result of Standard full length.</div>
 
         <div class="sec-wenzi-content">Figure 5.K.M is marker. Lane 1 is the pcr result of Standard full length.L.M is marker. Lane 1 is Enzyme digestion result of Standard full length.</div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/1/14/Tjuresults23.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/1/14/Tjuresults23.jpg">
 
         <div class="sec-wenzi-content">Figure6. Pre-expression in different inducing condition</div>
 
         <div class="sec-wenzi-content">Figure6. Pre-expression in different inducing condition</div>
  
 
         <div class="sec-wenzi-list">AIDA</div>
 
         <div class="sec-wenzi-list">AIDA</div>
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/5/53/Tjuresults44.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/5/53/Tjuresults44.jpg">
 
         <div class="sec-wenzi-content">Figure 1. PCR amplification of the signal peptide. </div>
 
         <div class="sec-wenzi-content">Figure 1. PCR amplification of the signal peptide. </div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/d/d2/Tjuresults45.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/d/d2/Tjuresults45.jpg">
 
         <div class="sec-wenzi-content">Figure 2. PCR amplification of the PETase. </div>
 
         <div class="sec-wenzi-content">Figure 2. PCR amplification of the PETase. </div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/b/bf/Tjuresults46.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/b/bf/Tjuresults46.jpg">
 
         <div class="sec-wenzi-content">Figure 3. Overlap PCR amplification of the signal peptide and the  PETase. </div>
 
         <div class="sec-wenzi-content">Figure 3. Overlap PCR amplification of the signal peptide and the  PETase. </div>
  
  
−
         <img src="https://static.igem.org/mediawiki/igem.org/e/ec/Tjuresults47.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/e/ec/Tjuresults47.jpg">
 
         <div class="sec-wenzi-content">Figure 4. PCR amplification of the two parts of anchoring protein called ap protein. The name of the two parts called pre ap and suf ap.</div>
 
         <div class="sec-wenzi-content">Figure 4. PCR amplification of the two parts of anchoring protein called ap protein. The name of the two parts called pre ap and suf ap.</div>
−
         <img src="https://static.igem.org/mediawiki/igem.org/3/32/Tjuresults48.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/3/32/Tjuresults48.jpg">
 
         <div class="sec-wenzi-content">Figure 5. Overlap PCR amplification of the pre ap and suf ap. </div>
 
         <div class="sec-wenzi-content">Figure 5. Overlap PCR amplification of the pre ap and suf ap. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/1/12/Tjuresults49.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/1/12/Tjuresults49.jpg">
 
         <div class="sec-wenzi-content">Figure 6. Overlap PCR amplification of the signal peptide, PETase and ap protein. </div>
 
         <div class="sec-wenzi-content">Figure 6. Overlap PCR amplification of the signal peptide, PETase and ap protein. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/8/89/Tjuresults50.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/8/89/Tjuresults50.jpg">
 
         <div class="sec-wenzi-content">Figure 7. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein. </div>
 
         <div class="sec-wenzi-content">Figure 7. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/f/f9/Tjuresults51.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/f/f9/Tjuresults51.jpg">
 
         <div class="sec-wenzi-content">Figure 8. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein. </div>
 
         <div class="sec-wenzi-content">Figure 8. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein. </div>
  
  
  
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         <img src="https://static.igem.org/mediawiki/igem.org/9/9d/Tjuresults52.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/9/9d/Tjuresults52.jpg">
 
         <div class="sec-wenzi-content">Figure 9. Enzyme digestion test of the whole sequence of signal peptide,PETase and ap protein. </div>
 
         <div class="sec-wenzi-content">Figure 9. Enzyme digestion test of the whole sequence of signal peptide,PETase and ap protein. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults53.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults53.jpg">
 
         <div class="sec-wenzi-content">Figure 10. This is the pre-expression using E.coli BL21 at 16 ℃. </div>
 
         <div class="sec-wenzi-content">Figure 10. This is the pre-expression using E.coli BL21 at 16 ℃. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/e/ed/Tjuresults54.jpg">
 
         <div class="sec-wenzi-content">Figure 11. This is the pre-expression using E.coli BL21 at 25 ℃. </div>
 
         <div class="sec-wenzi-content">Figure 11. This is the pre-expression using E.coli BL21 at 25 ℃. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/0/04/Tjuresults55.jpg">
 
         <div class="sec-wenzi-content">Figure 12. This is the pre-expression using E.coli BL21 at 37 ℃. </div>
 
         <div class="sec-wenzi-content">Figure 12. This is the pre-expression using E.coli BL21 at 37 ℃. </div>
  
  
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         <div class="sec-wenzi-list">Surface display in pichia pastoris、fusing secretion of hydrophobin and PETase and their co - display</div>
+
         <h3 id="part3">Surface display in pichia pastoris、fusing secretion of hydrophobin and PETase and their co - display</h3>
  
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         <div class="sec-wenzi-content">Figure 1. PCR amplification of the anchoring protein sequence GCW21, GCW51 and GCW61 .A. PCR results of the genome of GS115.  M is DNA marker.Lane 1 is the results for the genome of yeast GS115; B. PCR results for anchoring proteins.  M is DNA marker. Lane 1 , lane 2 and lane 3 are the results for GCW21, GCW51 and GCW61 respectively. </div>
 
         <div class="sec-wenzi-content">Figure 1. PCR amplification of the anchoring protein sequence GCW21, GCW51 and GCW61 .A. PCR results of the genome of GS115.  M is DNA marker.Lane 1 is the results for the genome of yeast GS115; B. PCR results for anchoring proteins.  M is DNA marker. Lane 1 , lane 2 and lane 3 are the results for GCW21, GCW51 and GCW61 respectively. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/8/8f/Tjuresults25.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/8/8f/Tjuresults25.jpg">
 
         <div class="sec-wenzi-content">Figure 2 . Overlap PCR  results of anchoring proteins and PETase .A. The first round overlap PCR results.  M is DNA marker, Lane 1 is for GCW21, lane 2 is for GCW 51, lane 3 is for GCW61, lane 4 is for PETase.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for GCW21-PETase, lane 2 is for GCW 51-PETase, lane 3 is for GCW61-PETase. </div>
 
         <div class="sec-wenzi-content">Figure 2 . Overlap PCR  results of anchoring proteins and PETase .A. The first round overlap PCR results.  M is DNA marker, Lane 1 is for GCW21, lane 2 is for GCW 51, lane 3 is for GCW61, lane 4 is for PETase.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for GCW21-PETase, lane 2 is for GCW 51-PETase, lane 3 is for GCW61-PETase. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/8/8b/Tjuresults26.jpg">
 
         <div class="sec-wenzi-content">Figure 3 . Overlap PCR  results of anchoring proteins and HFB1/HGF1. The first round overlap PCR results.  M is DNA marker, Lane 1 is for HFB1, lane 2 is forHGF1.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for GCW61-HFB1, lane 2 is for GCW61-HGF1.</div>
 
         <div class="sec-wenzi-content">Figure 3 . Overlap PCR  results of anchoring proteins and HFB1/HGF1. The first round overlap PCR results.  M is DNA marker, Lane 1 is for HFB1, lane 2 is forHGF1.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for GCW61-HFB1, lane 2 is for GCW61-HGF1.</div>
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         <img src="https://static.igem.org/mediawiki/igem.org/9/9b/Tjuresults27.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/9/9b/Tjuresults27.jpg">
 
         <div class="sec-wenzi-content">Figure 4 . Overlap PCR  results of HFB1 and PETase .A. The first round overlap PCR results.  M is DNA marker, Lane 1 is for PETase, lane 2 is for HFB1.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for HFB1 - PETase.</div>
 
         <div class="sec-wenzi-content">Figure 4 . Overlap PCR  results of HFB1 and PETase .A. The first round overlap PCR results.  M is DNA marker, Lane 1 is for PETase, lane 2 is for HFB1.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for HFB1 - PETase.</div>
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         <img src="https://static.igem.org/mediawiki/igem.org/7/7c/Tjuresults28.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/7/7c/Tjuresults28.jpg">
 
         <div class="sec-wenzi-content">Figure 5 . Overlap PCR  results of HGF1 and PETase .A. The first round overlap PCR results.  M is DNA marker, Lane 1 is for HGF1 , lane 2 is for PETase.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for HGF1 - PETase.</div>
 
         <div class="sec-wenzi-content">Figure 5 . Overlap PCR  results of HGF1 and PETase .A. The first round overlap PCR results.  M is DNA marker, Lane 1 is for HGF1 , lane 2 is for PETase.  B. The third round overlap PCR results. M is DNA marker, Lane 1 is for HGF1 - PETase.</div>
  
  
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         <img src="https://static.igem.org/mediawiki/igem.org/9/9c/Tjuresults29.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/9/9c/Tjuresults29.jpg">
 
         <div class="sec-wenzi-content">Figure 6 . PCR and dual-enzyme digestion check results of  ppiczaA-HFB1/HGF1-GCW61 .  A.The results of PCR check.M is DNA Marker.Lane 1 is for PCR check  result of ppiczaA-HFB1-GCW61.Lane 2 is for PCR check result of ppiczaA-HGF1-GCW61.    B.The results of dual-enzyme digestion check.M is DNA Marker.Lane 1 is for dual-enzyme digestion check result of ppiczaA-HFB1-GCW61.Lane 2 is for dual-enzyme digestion check result of ppiczaA-HGF1-GCW61. </div>
 
         <div class="sec-wenzi-content">Figure 6 . PCR and dual-enzyme digestion check results of  ppiczaA-HFB1/HGF1-GCW61 .  A.The results of PCR check.M is DNA Marker.Lane 1 is for PCR check  result of ppiczaA-HFB1-GCW61.Lane 2 is for PCR check result of ppiczaA-HGF1-GCW61.    B.The results of dual-enzyme digestion check.M is DNA Marker.Lane 1 is for dual-enzyme digestion check result of ppiczaA-HFB1-GCW61.Lane 2 is for dual-enzyme digestion check result of ppiczaA-HGF1-GCW61. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/8/8d/Tjuresults30.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/8/8d/Tjuresults30.jpg">
 
         <div class="sec-wenzi-content">Figure 7 . PCR and dual-enzyme digestion check results of ppic9-PETase-GCW21/51/61 .  A.The results of dual-enzyme digestion check.M is DNA Marker.Lane 1 is for dual-enzyme digestion check result of ppic9-PETase-GCW21.Lane 2 is for dual-enzyme digestion check result of ppic9-PETase-GCW51. Lane 3 is for dual-enzyme digestion check result of ppic9-PETase-GCW61.    B.The results of PCR check.M is DNA Marker.Lane 1 is for PCR check result of ppic9-PETase-GCW21.Lane 2 is for PCR check result of ppic9-PETase-GCW51. Lane 3 is for PCR check result of ppic9-PETase-GCW61. </div>
 
         <div class="sec-wenzi-content">Figure 7 . PCR and dual-enzyme digestion check results of ppic9-PETase-GCW21/51/61 .  A.The results of dual-enzyme digestion check.M is DNA Marker.Lane 1 is for dual-enzyme digestion check result of ppic9-PETase-GCW21.Lane 2 is for dual-enzyme digestion check result of ppic9-PETase-GCW51. Lane 3 is for dual-enzyme digestion check result of ppic9-PETase-GCW61.    B.The results of PCR check.M is DNA Marker.Lane 1 is for PCR check result of ppic9-PETase-GCW21.Lane 2 is for PCR check result of ppic9-PETase-GCW51. Lane 3 is for PCR check result of ppic9-PETase-GCW61. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/d/d1/Tjuresults31.jpg">
 
         <div class="sec-wenzi-content">Figure 8 . The results of  linearization.  A .The linearization result of vectors ppic9-PETase-GCW21.M is DNA marker ,lane 1 is for ppic9-PETase-GCW21.  B .The linearization of vectors ppic9-PETase-GCW51.M is DNA marker ,lane 1 is for ppic9-PETase-GCW51.    C .The linearization of vectors ppic9-PETase-GCW61. M is DNA marker ,lane 1 is for ppic9-PETase-GCW61.  D .The linearization of vectors ppiczaA-HGF1-GCW61.M is DNA marker ,lane 1 is for ppiczaA-HGF1-GCW61.  E .The linearization of vectors ppiczaA-HFB1-GCW61.M is DNA marker ,lane 1 is for  ppiczaA-HFB1-GCW61. </div>
 
         <div class="sec-wenzi-content">Figure 8 . The results of  linearization.  A .The linearization result of vectors ppic9-PETase-GCW21.M is DNA marker ,lane 1 is for ppic9-PETase-GCW21.  B .The linearization of vectors ppic9-PETase-GCW51.M is DNA marker ,lane 1 is for ppic9-PETase-GCW51.    C .The linearization of vectors ppic9-PETase-GCW61. M is DNA marker ,lane 1 is for ppic9-PETase-GCW61.  D .The linearization of vectors ppiczaA-HGF1-GCW61.M is DNA marker ,lane 1 is for ppiczaA-HGF1-GCW61.  E .The linearization of vectors ppiczaA-HFB1-GCW61.M is DNA marker ,lane 1 is for  ppiczaA-HFB1-GCW61. </div>
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         <img src="https://static.igem.org/mediawiki/igem.org/a/a9/Tjuresults32.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/a/a9/Tjuresults32.jpg">
 
         <div class="sec-wenzi-content">Figure 9 . PCR check results of ppic9-PETase-GCW21 transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW21 transformants.M is DNA marker ,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 transformants.  B. The desired gene  primer pcr check results of ppic9-PETase-GCW21 transformants.M is DNA marker ,lane 1 to 15 are for desired gene  primer pcr check results of ppic9-PETase-GCW21 transformants.</div>
 
         <div class="sec-wenzi-content">Figure 9 . PCR check results of ppic9-PETase-GCW21 transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW21 transformants.M is DNA marker ,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 transformants.  B. The desired gene  primer pcr check results of ppic9-PETase-GCW21 transformants.M is DNA marker ,lane 1 to 15 are for desired gene  primer pcr check results of ppic9-PETase-GCW21 transformants.</div>
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         <img src="https://static.igem.org/mediawiki/igem.org/1/15/Tjuresults33.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/1/15/Tjuresults33.jpg">
 
         <div class="sec-wenzi-content">Figure 10 . PCR check results of ppic9-PETase-GCW51 transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW51 transformants.M is DNA marker ,lane 1 to 9 are for aox primer pcr check results of ppic9-PETase-GCW51 transformants.  B. The desired gene  primer pcr check results of ppic9-PETase-GCW51 transformants.M is DNA marker ,lane 1 to 9 are for desired gene  primer pcr check results of ppic9-PETase-GCW51 transformants.</div>
 
         <div class="sec-wenzi-content">Figure 10 . PCR check results of ppic9-PETase-GCW51 transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW51 transformants.M is DNA marker ,lane 1 to 9 are for aox primer pcr check results of ppic9-PETase-GCW51 transformants.  B. The desired gene  primer pcr check results of ppic9-PETase-GCW51 transformants.M is DNA marker ,lane 1 to 9 are for desired gene  primer pcr check results of ppic9-PETase-GCW51 transformants.</div>
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         <img src="https://static.igem.org/mediawiki/igem.org/a/a6/Tjuresults34.jpg">
 
         <div class="sec-wenzi-content">Figure 11. PCR check results of ppic9-PETase-GCW61 transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW61 transformants.M is DNA marker ,lane 1 to 8 are for aox primer pcr check results of ppic9-PETase-GCW61 transformants.  B. The desired gene  primer pcr check results of ppic9-PETase-GCW61 transformants.M is DNA marker ,lane 1 to 8 are for desired gene  primer pcr check results of ppic9-PETase-GCW61 transformants./div>
 
         <div class="sec-wenzi-content">Figure 11. PCR check results of ppic9-PETase-GCW61 transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW61 transformants.M is DNA marker ,lane 1 to 8 are for aox primer pcr check results of ppic9-PETase-GCW61 transformants.  B. The desired gene  primer pcr check results of ppic9-PETase-GCW61 transformants.M is DNA marker ,lane 1 to 8 are for desired gene  primer pcr check results of ppic9-PETase-GCW61 transformants./div>
  
  
  
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             <img src="https://static.igem.org/mediawiki/igem.org/e/e7/Tjuresults35.jpg">
 
             <div class="sec-wenzi-content">Figure 12 . PCR check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HFB1-GCW61,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.  B&C.  The desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HFB1-GCW61,lane 1 to 8 are  for desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.</div>
 
             <div class="sec-wenzi-content">Figure 12 . PCR check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HFB1-GCW61,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.  B&C.  The desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HFB1-GCW61,lane 1 to 8 are  for desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.</div>
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             <img src="https://static.igem.org/mediawiki/igem.org/3/3f/Tjuresults36.jpg">
 
             <div class="sec-wenzi-content">Figure 13 . PCR check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.    A&B. The aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9-PETase-GCW51,C3 is  ppiczaA-HFB1-GCW61 ,C4 is ppiczaA,C5 is ppic9. lane 1 to 20 are for aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.    C.  The desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9-PETase-GCW51,C3 is  ppiczaA-HFB1-GCW61 ,C4 is ppiczaA,C5 is ppic9.lane 1 to 8 are  for desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.</div>
 
             <div class="sec-wenzi-content">Figure 13 . PCR check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.    A&B. The aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9-PETase-GCW51,C3 is  ppiczaA-HFB1-GCW61 ,C4 is ppiczaA,C5 is ppic9. lane 1 to 20 are for aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.    C.  The desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9-PETase-GCW51,C3 is  ppiczaA-HFB1-GCW61 ,C4 is ppiczaA,C5 is ppic9.lane 1 to 8 are  for desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.</div>
  
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             <img src="https://static.igem.org/mediawiki/igem.org/4/48/Tjuresults37.jpg">
 
             <div class="sec-wenzi-content">Figure 14 . PCR check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.    A. The aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppiczaA,C3 is  ppic9 ,C4 is ppic9-PETase-GCW61,C5 is ppiczaA-HFB1-GCW61. lane 1 to 11 are for aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.    B.  The desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9 ,C3 is  ppiczaA ,C4 is ppic9-PETase-GCW61,C5 is ppiczaA-HFB1-GCW61. lane 1 to 11 are  for desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.</div>
 
             <div class="sec-wenzi-content">Figure 14 . PCR check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.    A. The aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppiczaA,C3 is  ppic9 ,C4 is ppic9-PETase-GCW61,C5 is ppiczaA-HFB1-GCW61. lane 1 to 11 are for aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.    B.  The desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9 ,C3 is  ppiczaA ,C4 is ppic9-PETase-GCW61,C5 is ppiczaA-HFB1-GCW61. lane 1 to 11 are  for desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.</div>
  
  
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             <img src="https://static.igem.org/mediawiki/igem.org/d/d1/Tjuresults38.jpg">
 
             <div class="sec-wenzi-content">Figure 15 . PCR check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.  B&C.  The desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9,C3 is  ppiczaA ,C4 is ppic9-PETase-GCW21,C5 is ppiczaA-HGF1-GCW61,lane 1 to 15 are  for desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.</div>
 
             <div class="sec-wenzi-content">Figure 15 . PCR check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.  B&C.  The desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9,C3 is  ppiczaA ,C4 is ppic9-PETase-GCW21,C5 is ppiczaA-HGF1-GCW61,lane 1 to 15 are  for desired gene  primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.</div>
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             <img src="https://static.igem.org/mediawiki/igem.org/a/ae/Tjuresults39.jpg">
 
             <div class="sec-wenzi-content">Figure 16 . PCR check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.  A&B. The aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,lane 1 to 17 are for aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.  C&D.  The desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,lane 1 to 17 are  for desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.</div>
 
             <div class="sec-wenzi-content">Figure 16 . PCR check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.  A&B. The aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,lane 1 to 17 are for aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.  C&D.  The desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,lane 1 to 17 are  for desired gene  primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.</div>
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             <img src="https://static.igem.org/mediawiki/igem.org/0/0a/Tjuresults40.jpg" height="300" width="600">
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             <img src="https://static.igem.org/mediawiki/igem.org/0/0a/Tjuresults40.jpg">
 
             <div class="sec-wenzi-content">Figure 17 . PCR check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 12 are for aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.  B.  The desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 12 are  for desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.
 
             <div class="sec-wenzi-content">Figure 17 . PCR check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.  A. The aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 12 are for aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.  B.  The desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 12 are  for desired gene  primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.
  

Latest revision as of 00:35, 20 October 2016

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Results
One of the vital results of our team this year is managing to analyze the high resolution of the PETase protein structure.To verify if the surface display is successful, we also do western blot, immunofluorescence assay and using HPLC to detecte enzyme activity.(click here to see the HPLC results)

Mutation:

Figure1 The result of Overlap PCR of different conditions.
Figure2. The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG.
Figure3. The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.

Surface display in E.coli

INP
Figure 1. Overlap PCR results of anchoring motif and passenger protein(NP). (a)The first round of overlap PCR results. Lane 1 is for INPN. Lane 2 is for PETase. M is marker. (b) The third round of overlap PCR results. Lane 1 is the overlap product of INPN and PETase. M is the lane of DNA marker.
Figure 2 Enzyme digestion validation. Lane 1 is for double enzyme(NdeI & XhoI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 2 is for single enzyme(NdeI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 3 is for single enzyme(XhoI) digestion of the recombinant plasmid pET22b(+)-INPN-PETase. Lane 4 is for recombinant plasmid pET22b (+)-INPN-PETase. . Lane 5 is for double enzyme(NdeI & XhoI) digestion of pET22b(+). Lane 6 is for single enzyme(NdeI) digestion of the pET22b(+). Lane 7 is for single enzyme(XhoI) digestion of pET22b(+). Lane 8 is for pET22b (+). M is DNA marker.
Figure 3 Standardization of anchoring protein INPN and fusion protein INPN-PETase. (a) The PCR results of anchoring motif INPN and the fusion protein INPN-PETase. Lane 1 is for the N-terminal of ice nucleation protein. Lane 2 is for the whole length of INPN-PETase. M is DNA marker.(b) The double enzyme(EcoRI&SpeI) digestion validation of the recombination standardization plasmid. Lane 1 is for pSBIC3-INPN. Lane 2 is for pSBIC3-INPN-PETase. M is DNA marker.
Figure 4 Standardization of the anchoring motif INPNC and fusion protein INPNC-PETase. (a) The PCR results of anchoring motif INPN and the fusion protein INPN-PETase. Lane 1 is for the N-terminal of ice nucleation protein. Lane 2 is for the whole length of INPN-PETase. M is DNA marker.(b) The double enzyme(EcoRI&SpeI) digestion validation of the recombination standardization plasmid. Lane 1 is for pSBIC3-INPN. Lane 2 is for pSBIC3-INPN-PETase. M is DNA marker.
Figure5 NP induced by 37℃
Figure6 NP induced by 16℃
Figure7 NP induced by 25℃
Figure8 NP induced by different concentration of IPTG
Figure9 NP induced by different concentration of IPTG
Figure10 NP induced 24h by different concentration of IPTG at 16℃ and 25℃
LPP-OmpA
Figure 1. The first round of overlap PCR results of anchoring motif and passenger protein(Lpp-OmpA). (a) Lane A and B are for Lpp-OmpA. M1 is marker. (b) Lane C and D are for PETase. M2 is the lane of DNA marker.
Figure 2.The third round of overlap PCR results. Lane A is the overlap product of Lpp-OmpA and PETase. M is the lane of DNA marker.
Figure 3 Enzyme digestion validation. Lane A is for double enzyme(NdeI & XhoI) digestion of the recombinant plasmid pET22b(+)-Lpp-OmpA-PETase. M is DNA marker.
Figure4 Lpp-OmpA induced by 37℃.
Figure5 Lpp-OmpA induced by 25℃.
Figure6 Lpp-OmpA induced by 16℃.
BrkA
Figure 1.M is DNA marker.Lane 1 and Lane 2 are the results of B1 signal, Lane 3 and 4 are the results of B2 Petase.PCR results Petase. M is DNA marker. Lane1-4 are overlap PCR results of B1 and B2.
Figure 2.C. M is DNA marker.Lane 1 and Lane 2 are the results of B3, Lane 3 and 4 are the results of B4.D. M is DNA marker. Lane1-5 are oeverlap PCR results of B3 and B4.
Figure 3.E. M is DNA marker.Lane 1 and Lane 2 are the results of full length.F. M is DNA marker. Lane 1 is plasmid digestion.G.M is marker. Lane 1 and 2 are digestion verification.H.M is marker. Lane 1 is PCR verification.
Figure 4.M is marker. Lane 1 is PCR result of Standard β-barrel domain, anchor sequence.J. M is marker. Lane 1 is Enzyme digestion result of Standard β-barrel domain, anchor sequence.
Figure 5.K.M is marker. Lane 1 is the pcr result of Standard full length.L.M is marker. Lane 1 is Enzyme digestion result of Standard full length.
Figure6. Pre-expression in different inducing condition
AIDA
Figure 1. PCR amplification of the signal peptide.
Figure 2. PCR amplification of the PETase.
Figure 3. Overlap PCR amplification of the signal peptide and the PETase.
Figure 4. PCR amplification of the two parts of anchoring protein called ap protein. The name of the two parts called pre ap and suf ap.
Figure 5. Overlap PCR amplification of the pre ap and suf ap.
Figure 6. Overlap PCR amplification of the signal peptide, PETase and ap protein.
Figure 7. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein.
Figure 8. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein.
Figure 9. Enzyme digestion test of the whole sequence of signal peptide,PETase and ap protein.
Figure 10. This is the pre-expression using E.coli BL21 at 16 ℃.
Figure 11. This is the pre-expression using E.coli BL21 at 25 ℃.
Figure 12. This is the pre-expression using E.coli BL21 at 37 ℃.

Surface display in pichia pastoris、fusing secretion of hydrophobin and PETase and their co - display

Figure 1. PCR amplification of the anchoring protein sequence GCW21, GCW51 and GCW61 .A. PCR results of the genome of GS115. M is DNA marker.Lane 1 is the results for the genome of yeast GS115; B. PCR results for anchoring proteins. M is DNA marker. Lane 1 , lane 2 and lane 3 are the results for GCW21, GCW51 and GCW61 respectively.
Figure 2 . Overlap PCR results of anchoring proteins and PETase .A. The first round overlap PCR results. M is DNA marker, Lane 1 is for GCW21, lane 2 is for GCW 51, lane 3 is for GCW61, lane 4 is for PETase. B. The third round overlap PCR results. M is DNA marker, Lane 1 is for GCW21-PETase, lane 2 is for GCW 51-PETase, lane 3 is for GCW61-PETase.
Figure 3 . Overlap PCR results of anchoring proteins and HFB1/HGF1. The first round overlap PCR results. M is DNA marker, Lane 1 is for HFB1, lane 2 is forHGF1. B. The third round overlap PCR results. M is DNA marker, Lane 1 is for GCW61-HFB1, lane 2 is for GCW61-HGF1.
Figure 4 . Overlap PCR results of HFB1 and PETase .A. The first round overlap PCR results. M is DNA marker, Lane 1 is for PETase, lane 2 is for HFB1. B. The third round overlap PCR results. M is DNA marker, Lane 1 is for HFB1 - PETase.
Figure 5 . Overlap PCR results of HGF1 and PETase .A. The first round overlap PCR results. M is DNA marker, Lane 1 is for HGF1 , lane 2 is for PETase. B. The third round overlap PCR results. M is DNA marker, Lane 1 is for HGF1 - PETase.
Figure 6 . PCR and dual-enzyme digestion check results of ppiczaA-HFB1/HGF1-GCW61 . A.The results of PCR check.M is DNA Marker.Lane 1 is for PCR check result of ppiczaA-HFB1-GCW61.Lane 2 is for PCR check result of ppiczaA-HGF1-GCW61. B.The results of dual-enzyme digestion check.M is DNA Marker.Lane 1 is for dual-enzyme digestion check result of ppiczaA-HFB1-GCW61.Lane 2 is for dual-enzyme digestion check result of ppiczaA-HGF1-GCW61.
Figure 7 . PCR and dual-enzyme digestion check results of ppic9-PETase-GCW21/51/61 . A.The results of dual-enzyme digestion check.M is DNA Marker.Lane 1 is for dual-enzyme digestion check result of ppic9-PETase-GCW21.Lane 2 is for dual-enzyme digestion check result of ppic9-PETase-GCW51. Lane 3 is for dual-enzyme digestion check result of ppic9-PETase-GCW61. B.The results of PCR check.M is DNA Marker.Lane 1 is for PCR check result of ppic9-PETase-GCW21.Lane 2 is for PCR check result of ppic9-PETase-GCW51. Lane 3 is for PCR check result of ppic9-PETase-GCW61.
Figure 8 . The results of linearization. A .The linearization result of vectors ppic9-PETase-GCW21.M is DNA marker ,lane 1 is for ppic9-PETase-GCW21. B .The linearization of vectors ppic9-PETase-GCW51.M is DNA marker ,lane 1 is for ppic9-PETase-GCW51. C .The linearization of vectors ppic9-PETase-GCW61. M is DNA marker ,lane 1 is for ppic9-PETase-GCW61. D .The linearization of vectors ppiczaA-HGF1-GCW61.M is DNA marker ,lane 1 is for ppiczaA-HGF1-GCW61. E .The linearization of vectors ppiczaA-HFB1-GCW61.M is DNA marker ,lane 1 is for ppiczaA-HFB1-GCW61.
Figure 9 . PCR check results of ppic9-PETase-GCW21 transformants. A. The aox primer pcr check results of ppic9-PETase-GCW21 transformants.M is DNA marker ,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 transformants. B. The desired gene primer pcr check results of ppic9-PETase-GCW21 transformants.M is DNA marker ,lane 1 to 15 are for desired gene primer pcr check results of ppic9-PETase-GCW21 transformants.
Figure 10 . PCR check results of ppic9-PETase-GCW51 transformants. A. The aox primer pcr check results of ppic9-PETase-GCW51 transformants.M is DNA marker ,lane 1 to 9 are for aox primer pcr check results of ppic9-PETase-GCW51 transformants. B. The desired gene primer pcr check results of ppic9-PETase-GCW51 transformants.M is DNA marker ,lane 1 to 9 are for desired gene primer pcr check results of ppic9-PETase-GCW51 transformants.
Figure 11. PCR check results of ppic9-PETase-GCW61 transformants. A. The aox primer pcr check results of ppic9-PETase-GCW61 transformants.M is DNA marker ,lane 1 to 8 are for aox primer pcr check results of ppic9-PETase-GCW61 transformants. B. The desired gene primer pcr check results of ppic9-PETase-GCW61 transformants.M is DNA marker ,lane 1 to 8 are for desired gene primer pcr check results of ppic9-PETase-GCW61 transformants./div>
Figure 12 . PCR check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants. A. The aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HFB1-GCW61,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants. B&C. The desired gene primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HFB1-GCW61,lane 1 to 8 are for desired gene primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HFB1-GCW61 codisplay transformants.
Figure 13 . PCR check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants. A&B. The aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9-PETase-GCW51,C3 is ppiczaA-HFB1-GCW61 ,C4 is ppiczaA,C5 is ppic9. lane 1 to 20 are for aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants. C. The desired gene primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9-PETase-GCW51,C3 is ppiczaA-HFB1-GCW61 ,C4 is ppiczaA,C5 is ppic9.lane 1 to 8 are for desired gene primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 codisplay transformants.
Figure 14 . PCR check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants. A. The aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppiczaA,C3 is ppic9 ,C4 is ppic9-PETase-GCW61,C5 is ppiczaA-HFB1-GCW61. lane 1 to 11 are for aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants. B. The desired gene primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9 ,C3 is ppiczaA ,C4 is ppic9-PETase-GCW61,C5 is ppiczaA-HFB1-GCW61. lane 1 to 11 are for desired gene primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HFB1-GCW61 codisplay transformants.
Figure 15 . PCR check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants. A. The aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 15 are for aox primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants. B&C. The desired gene primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C1 is GS115,C2 is ppic9,C3 is ppiczaA ,C4 is ppic9-PETase-GCW21,C5 is ppiczaA-HGF1-GCW61,lane 1 to 15 are for desired gene primer pcr check results of ppic9-PETase-GCW21 & ppiczaA-HGF1-GCW61 codisplay transformants.
Figure 16 . PCR check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants. A&B. The aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,lane 1 to 17 are for aox primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants. C&D. The desired gene primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,lane 1 to 17 are for desired gene primer pcr check results of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 codisplay transformants.
Figure 17 . PCR check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants. A. The aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 12 are for aox primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants. B. The desired gene primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.M is DNA marker ,C is ppiczaA-HGF1-GCW61,lane 1 to 12 are for desired gene primer pcr check results of ppic9-PETase-GCW61 & ppiczaA-HGF1-GCW61 codisplay transformants.

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