Team:TJUSLS China/Proof
Proof of Concept
Mutation:
Figure 1. The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein.
Figure 2. PETase’s self-degrading condition in different temperature. We take the quantity of PETase in the day the experiment begines as 100%. We can now see when stored in low temperature, the protein was degraded slowly, but in room temperature, the protein degrades rapidly. The Quantity of PETase left was measured by protein gel and analysised by a computer program called GEL-PRO.
Surface display in E.coli
Surface display in E.coli by using INP
Figure 3 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃.
Figure 4 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 25℃ with different amount of bacteria.
Figure 5 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced with 0.1mM IPTG for 24h.
Figure 6 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃ with 0.1mM IPTG for 1h, 4h, 8h, 12h, 16h and 20h.
Surface display in E.coli by using LPP-OmpA
Figure 7 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)LAP when induced at 16℃.
Surface display in E.coli by using BrkA
Figure 8 Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)Brk when induced at 16℃ and 25 ℃ with 0.02mM IPTG.And the last two were induced with 0.09mM IPTG.
Surface display in E.coli by using AIDA
Figure 9. Reletive enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃
Surface display in Pichia Pastoris
Figure 10: The activity of P. pastoris PETase-GCW21. a&b used the first group of yeast; c&d used the second of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.
Figure 11: The activity of P. pastoris PETase-GCW51. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.
Figure 12: The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.
Co-display in Pichia Pastoris
Figure 13: The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 14: The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-HFB1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 15: The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 16: The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformants in different hours and amount of yeast.
Figure 17: The activity of ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-HGF1-GCW61 co-display transformant in best condition
