PROJECT ACHIEVEMENTS

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

Prove our vectors successfully be 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 strain. 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 be 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.

This is the schematic structure of our gene design. you can see, GAP promoter ,which is constitutive type of promoter, then Kozak sequence, alpha factor ,which is secretion signal for directing secreted expression of the recombinant protein, After that, is our target gene, HA Tag for western blot protein detection, and His tag for protein purification Lastly, AOXI terminator is common one in P. pastoris.
Up to now, you may already found out we put only one target gene in this backbone And your right it is important part of our gene design that is our target genes are separated into three different inserts rather than put it in one.

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.