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Revision as of 15:58, 18 October 2016
Results
![](https://static.igem.org/mediawiki/2016/8/86/T--TJUSLS_China--second-logo.png)
Stucture:
One of the vital results of our team this year is managing to analyze the high resolution of the PETase protein structure!
![](https://static.igem.org/mediawiki/igem.org/c/c9/Tjuresults2.jpg)
Figure1 The result of Overlap PCR of different conditions.
![](https://static.igem.org/mediawiki/igem.org/d/d9/Tjuresults3.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/6/63/Tjuresults4.jpg)
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
![](https://static.igem.org/mediawiki/igem.org/3/35/Tjuresults5.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/e/e0/ProofTJU5.jpg)
Figure 5 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced with 0.1mM IPTG for 24h.
![](https://static.igem.org/mediawiki/igem.org/d/d4/Tjuresults6.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/9/9f/Tjuresults7.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults8.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/3/30/Tjuresults9.jpg)
Figure5 NP induced by 37℃
![](https://static.igem.org/mediawiki/igem.org/b/b4/Tjuresults10.jpg)
Figure6 NP induced by 16℃
![](https://static.igem.org/mediawiki/igem.org/4/48/Tjuresults11.jpg)
Figure7 NP induced by 25℃
![](https://static.igem.org/mediawiki/igem.org/9/93/Tjuresults12.jpg)
Figure8 NP induced by different concentration of IPTG
![](https://static.igem.org/mediawiki/igem.org/4/45/Tjuresults13.jpg)
Figure9 NP induced by different concentration of IPTG
![](https://static.igem.org/mediawiki/igem.org/d/d8/Tjuresults14.jpg)
Figure10 NP induced 24h by different concentration of IPTG at 16℃ and 25℃
LPP-OmpA
![](https://static.igem.org/mediawiki/igem.org/b/bd/Tjuresults15.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/6/69/Tjuresults16.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/4/4e/Tjuresults17.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/6/63/Tjuresults41.jpg)
Figure4 Lpp-OmpA induced by 37℃.
![](https://static.igem.org/mediawiki/igem.org/9/94/Tjuresults42.jpg)
Figure5 Lpp-OmpA induced by 25℃.
![](https://static.igem.org/mediawiki/igem.org/f/f8/Tjuresults43.jpg)
Figure6 Lpp-OmpA induced by 16℃.
BrkA
![](https://static.igem.org/mediawiki/igem.org/6/69/Tjuresults18.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/b/b8/Tjuresults19.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/2/20/Tjuresults20.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/9/94/Tjuresults21.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/d/da/Tjuresults22.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/1/14/Tjuresults23.jpg)
Figure6. Pre-expression in different inducing condition
AIDA
![](https://static.igem.org/mediawiki/igem.org/5/53/Tjuresults44.jpg)
Figure 1. PCR amplification of the signal peptide.
![](https://static.igem.org/mediawiki/igem.org/d/d2/Tjuresults45.jpg)
Figure 2. PCR amplification of the PETase.
![](https://static.igem.org/mediawiki/igem.org/b/bf/Tjuresults46.jpg)
Figure 3. Overlap PCR amplification of the signal peptide and the PETase.
![](https://static.igem.org/mediawiki/igem.org/e/ec/Tjuresults47.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/3/32/Tjuresults48.jpg)
Figure 5. Overlap PCR amplification of the pre ap and suf ap.
![](https://static.igem.org/mediawiki/igem.org/1/12/Tjuresults49.jpg)
Figure 6. Overlap PCR amplification of the signal peptide, PETase and ap protein.
![](https://static.igem.org/mediawiki/igem.org/8/89/Tjuresults50.jpg)
Figure 7. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein.
![](https://static.igem.org/mediawiki/igem.org/f/f9/Tjuresults51.jpg)
Figure 8. PCR amplification test of the whole sequence of signal peptide,PETase and ap protein.
![](https://static.igem.org/mediawiki/igem.org/9/9d/Tjuresults52.jpg)
Figure 9. Enzyme digestion test of the whole sequence of signal peptide,PETase and ap protein.
![](https://static.igem.org/mediawiki/igem.org/f/f6/Tjuresults53.jpg)
Figure 10. This is the pre-expression using E.coli BL21 at 16 ℃.
![](https://static.igem.org/mediawiki/igem.org/e/ed/Tjuresults54.jpg)
Figure 11. This is the pre-expression using E.coli BL21 at 25 ℃.
![](https://static.igem.org/mediawiki/igem.org/0/04/Tjuresults55.jpg)
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
![](https://static.igem.org/mediawiki/igem.org/f/f0/Tjuresults24.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/8/8f/Tjuresults25.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/8/8b/Tjuresults26.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/9/9b/Tjuresults27.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/7/7c/Tjuresults28.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/9/9c/Tjuresults29.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/8/8d/Tjuresults30.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/d/d1/Tjuresults31.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/a/a9/Tjuresults32.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/1/15/Tjuresults33.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/a/a6/Tjuresults34.jpg)
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>
![](https://static.igem.org/mediawiki/igem.org/e/e7/Tjuresults35.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/3/3f/Tjuresults36.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/4/48/Tjuresults37.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/d/d1/Tjuresults38.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/a/ae/Tjuresults39.jpg)
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.
![](https://static.igem.org/mediawiki/igem.org/0/0a/Tjuresults40.jpg)
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.
![](https://static.igem.org/mediawiki/2016/6/62/T--TJUSLS_China--up.jpg)