PROJECT ACHIEVEMENTS

1. Prove our vectors successfully constructed
2. Prove three enzymes successfully produced
3. Prove enzymes are functional
4. Establishment of material production line

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

This is our original expectation. We want to produce the required enzymes, Px16, Px18 and Lac1, through P. pastoris. We have completed the prove of our part. Then, we have completed the improvement of engineering yeast.

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.

Western blot：

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.

UV-visible :

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.