1. Prove our vectors successfully constructed
2. Prove three enzymes successfully produced
3. Prove enzymes are functional
Prove our vectors successfully constructed
In LIGGREEN synthesis, monolignols react mainly with two enzymes, peroxidase and laccase. We decided to synthesize these enzymes through synthetic biology. In our experiments, we use P. pastoris as our host. Our goal is to transform the insert into P. pastoris. We did westernblot to prove the yeast transform was successful and the enzymes were produced.
Figure 1：Plasmid digest( EcoRI, AgeI) after miniprep (pGAPZ A-SalI_His4, pGAPZ A-SalI)
Figure 2：Plasmid digest( EcoRI, AgeI) after miniprep (Px16, Px18, Lac1)
Figure 3：PCR result of E.coli colony (pGAPZ A_Px16, pGAPZ A_Px18, pGAPZ A_Lac1)
According to gel electrophoresis, we have confirm that our cloning is successful.
Prove three enzymes successfully produced
We use western blot to analyze our protein expression. To detect Lac1, Px16, Px18, we select mouse anti-FLAG tag, mouse anti-HA tag, mouse anti-V5 tag, respectively as our primary antibody. As our secondary antibody, we use goat anti-mouse HRP. We to detect secondary antibody, we apply standard enhanced chemilumescent (ECL) substrates for detecting horseradish peroxidase (HRP) enzyme activity.
Figure 4：Western blot (lac1 protein extraction from pellet) of high copy strain, middle copy strain, high copy strain at 96hr
Figure 5：Western blot (px18 protein extraction from pellet) of high copy strain
Figure 6：Western blot (px18 protein extraction from soup) of high copy strain
Figure 7：Western blot (px16 protein extraction from pellet) of low copy strain
Prove enzymes are functional
Coniferyl alcohol will react with our enzymes, Px16, Px18 and Lac1. We want to prove that coniferyl alcohol will polymerize into an oligomer. We use UV-visible for measurements. Through UV-visible, we can prove the difference in absorbance wavelength between monomer and oligomer.
Figure 8：the difference between monomer and monomer + Lac1
Figure 9：the difference between monomer and monomer + Px16 and Px18
Figure 10：the difference between monomer and monomer + Lac1 + Px16 and Px18
According to UV-visible, when the wavelength reaches some specific positions, the oligomer will shift to the left more than the monomer. Therefore, we found that only the peroxidase case did not produce spectral absorption. We have shown that our enzymes can polymerize the monomer into the oligomer.