Team:TJUSLS China/Results

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• Mutation
• Surface display in E.coli
• Surface display in pichia pastoris、fusing secretion of hydrophobin and PETase and their co - display

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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