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

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         <ul>
 
         <ul>
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             <li><a href="#part1">Project Design</a></li>
+
             <li><a href="#part1">Stucture</a></li>
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             <li><a href="#part2">Lab Work</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">Shipment</a></li>
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         </ul>
 
         </ul>
 
     </div>
 
     </div>
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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">Mutation:</h3>
+
         <h3 id="part1">Stucture:</h3>
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         <img src="https://static.igem.org/mediawiki/igem.org/2/22/ProofTJU1.jpg" width="600" height="300">
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        <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">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>
+
        Figure3. The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.
 +
         <img src="https://static.igem.org/mediawiki/igem.org/c/c9/Tjuresults2.jpg" width="600" height="300">
 +
         <div class="sec-wenzi-content">Figure1 The result of Overlap PCR of different conditions.</div>
  
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         <img src="https://static.igem.org/mediawiki/igem.org/5/53/ProofTJU2.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/d/d9/Tjuresults3.jpg" height="300" width="600">
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         <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>
+
         <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-list">Surface display in E.coli by using INP</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/6/64/ProofTJU3.jpg" height="300" width="600">
+
         <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">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" height="300" width="600">
+
        <h3 id="part3">Surface display in E.coli</h3>
−
         <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/3/35/Tjuresults5.jpg" height="300" width="600">
 +
         <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">
 
         <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>
 
         <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/8/82/ProofTJU6.jpg" height="300" width="600">
+
         <img src="https://static.igem.org/mediawiki/igem.org/d/d4/Tjuresults6.jpg" height="300" width="600">
−
         <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-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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <div class="sec-wenzi-content">Figure10 NP induced 24h by different concentration of IPTG at 16℃ and 25℃</div>
 +
 
 +
        <div class="sec-wenzi-list">LPP-OmpA</div>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/b/bd/Tjuresults15.jpg" height="300" width="600">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <div class="sec-wenzi-content">Figure6  Lpp-OmpA induced by 16℃. </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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <div class="sec-wenzi-content">Figure6. Pre-expression in different inducing condition</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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <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">
 +
        <div class="sec-wenzi-content">Figure 5. Overlap PCR amplification of the pre ap and suf ap. </div>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/1/12/Tjuresults49.jpg" height="300" width="600">
 +
        <div class="sec-wenzi-content">Figure 6. Overlap PCR amplification of the signal peptide, PETase and ap protein. </div>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/8/89/Tjuresults50.jpg" height="300" width="600">
 +
        <div class="sec-wenzi-content">Figure 7. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein. </div>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/f/f9/Tjuresults51.jpg" height="300" width="600">
 +
        <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-list">Surface display in E.coli by using LPP-OmpA</div>
 
−
        <img src="https://static.igem.org/mediawiki/igem.org/6/66/ProofTJU8.jpg" height="300" width="600">
 
−
        <div class="sec-wenzi-content">Figure 7  Relative enzyme activity of  engineering bacteria E.coli(BL21)/pET22b(+)LAP when induced at 16℃.</div>
 
  
−
        <div class="sec-wenzi-list">Surface display in E.coli by using BrkA</div>
 
−
        <img src="https://static.igem.org/mediawiki/igem.org/6/60/ProofTJU9.jpg" height="300" width="600">
 
−
        <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/9/9d/Tjuresults52.jpg" height="300" width="600">
−
         <img src="https://static.igem.org/mediawiki/igem.org/d/d5/ProofTJU10.jpg" height="300" width="600">
+
         <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. Reletive enzyme activity of engineering  bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃</div>
+
        <img src="https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults53.jpg" height="300" width="600">
 +
        <div class="sec-wenzi-content">Figure 10. This is the pre-expression using E.coli BL21 at 16 ℃. </div>
 +
         <img src="https://static.igem.org/mediawiki/igem.org/e/ed/Tjuresults54.jpg" height="300" width="600">
 +
         <div class="sec-wenzi-content">Figure 11. This is the pre-expression using E.coli BL21 at 25 ℃. </div>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/0/04/Tjuresults55.jpg" height="300" width="600">
 +
        <div class="sec-wenzi-content">Figure 12. This is the pre-expression using E.coli BL21 at 37 ℃. </div>
  
−
        <div class="sec-wenzi-list">Surface display in Pichia Pastoris</div>
 
−
        <img src="https://static.igem.org/mediawiki/igem.org/0/0f/ProofTJU11.jpg" height="300" width="600">
 
  
−
        <img src="https://static.igem.org/mediawiki/igem.org/e/e5/ProofTJU12.jpg" height="300" width="600">
+
         <div class="sec-wenzi-list">Surface display in pichia pastoris、fusing secretion of hydrophobin and PETase and their co - display</div>
−
         <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>
+
  
 +
        <img src="https://static.igem.org/mediawiki/igem.org/f/f0/Tjuresults24.jpg" height="300" width="600">
 +
        <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/8/8f/Tjuresults25.jpg" height="300" width="600">
 +
        <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/8/8b/Tjuresults26.jpg" height="300" width="600">
 +
        <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/9/9b/Tjuresults27.jpg" height="300" width="600">
 +
        <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/7/7c/Tjuresults28.jpg" height="300" width="600">
 +
        <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>
  
−
        <img src="https://static.igem.org/mediawiki/igem.org/4/48/ProofTJU13.jpg" height="300" width="600">
 
−
        <img src="https://static.igem.org/mediawiki/igem.org/2/2d/ProofTJU14.jpg" height="300" width="600">
 
  
−
         <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/9/9c/Tjuresults29.jpg" height="300" width="600">
 +
         <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/8/8d/Tjuresults30.jpg" height="300" width="600">
 +
        <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/d/d1/Tjuresults31.jpg" height="300" width="600">
 +
        <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/a/a9/Tjuresults32.jpg" height="300" width="600">
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        <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>
 +
        <img src="https://static.igem.org/mediawiki/igem.org/1/15/Tjuresults33.jpg" height="300" width="600">
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        <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" height="300" width="600">
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        <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/d/d7/ProofTJU15.jpg" height="300" width="600">
 
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        <img src="https://static.igem.org/mediawiki/igem.org/0/0c/ProofTJU16.jpg" height="300" width="600">
 
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        <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>
 
  
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        <div class="sec-wenzi-list">Co-display in Pichia Pastoris</div>
 
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        <img src="https://static.igem.org/mediawiki/igem.org/6/67/ProofTJU17.jpg" height="300" width="600">
 
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        <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>
 
  
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         <img src="https://static.igem.org/mediawiki/igem.org/d/d5/ProofTJU18.jpg" height="300" width="600">
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         <img src="https://static.igem.org/mediawiki/igem.org/e/e7/Tjuresults35.jpg" height="300" width="600">
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         <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>
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         <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" height="300" width="600">
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            <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/b/b9/ProofTJU19.jpg" height="300" width="600">
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            <img src="https://static.igem.org/mediawiki/igem.org/4/48/Tjuresults37.jpg" height="300" width="600">
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        <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>
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            <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/a/a0/ProofTJU20.jpg" height="300" width="600">
 
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        <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>
 
  
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        <img src="https://static.igem.org/mediawiki/igem.org/e/ea/ProofTJU21.jpg" height="300" width="600">
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            <img src="https://static.igem.org/mediawiki/igem.org/d/d1/Tjuresults38.jpg" height="300" width="600">
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        <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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            <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" height="300" width="600">
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            <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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            <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.
  
  
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            </div>
  
  

Revision as of 14:42, 18 October 2016

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Results

Stucture:

One of the vital results of our team this year is managing to analyze the high resolution of the PETase protein structure!
Figure3. The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.
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

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 5 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced with 0.1mM IPTG for 24h.
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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