Team:SDSZ China/Experi-PPO
Team:SDSZ China
Polyphenol Oxidase
Introduction
Polyphenol Oxidase (PPO), also named catechol oxidase, is an enzyme found widely in plant (potato, litchi, peach, tobacco, etc.) tissues. PPO is naturally attached to thylakoid membrane, and as the plant cell breaks, the enzyme is activated, catalyzing polyphenol oxidization reactions. In corresponding reactions, the emittance of luminescence comes from the oxidation of luciferin, and most of the times the reaction system includes ATP and Mg2+.
Tea polyphenol contains Epigallocatechin (EGC), Epicatechin (EC), Epigallocatechin gallate (EGCG) and Epicatechin gallate (ECG). Under the catalyzation of PPO, the catechol would oxidize according to the following reactions:
Epigallocatechin + Epicatechin→theaflavins；
Epigallocatechin gallate + Epicatechin→theaflavins -3- gallate；
Epicatechin gallate + Epigallocatechin→theaflavins -3’- gallate；
Epigallocatechin gallate + Epicatechin gallate→theaflavin digallate
As a result, the light-yellow tea polyphenol will turn into red theaflavins.
Design
In order to reproduce and simplify the existing bluephage detection system, we decided to use PPO instead of β-glucuronidase enzyme in bluphage. First, PPO is from plant tissue, meaning that we don’t need to knock out channel proteins in E. coli. Secondly, the reactant, tea polyphenol, is easy to acquire. Third, the final product, theaflavins, has anti-hyperlipidemia, anti-oxidization, anti-aging and anti-cancer effects. Besides pollutant detection, our PPO-producing strain can potentially play a role in theaflavin industrial production.
After reviewing several literatures, we chose potato PPO because it is relatively effective in oxidizing catechol and well-studied. We acquired the gene sequence through NCBI blast, and thanks to Twist, we acquired the synthesized sequence for free.
We decided to adopt pET expression system because its strong T7 bacteriophage promoter make it suitable for expressing the plant protein. Also, the expression can be regulated by IPTG induction, a widely used induction method. With the help of this efficient vector, our protein can be highly expressed.
Experiments
1. Polymerase Chain Reaction
First, we designed primer that contains endonuclease cutting sites, and used PCR to amplify PPO DNA.
To prevent mutation during PCR process, we adopted high fidelity taq DNA polymerase, and designed the PCR procedure according to the manual of the enzyme and the Tm of primers:
2. Endonuclease digestion
We used Nde1 and Bgl2 to digest PPO DNA and pET30a vector, and ran agarose gel to separate the successfully digested DNA and other DNA fragments. We then collected and purified the gel to acquire pure digested DNA.
3. Ligation
T4 DNA ligase was used for ligating PPO fragment to pET30a vector. As our experiment failed again and again, we began to doubt the efficiency of the T4 DNA ligase in our lab. At this time, Ms. Shang, a doctor student in Dr. Dou’s lab in Beijing Normal University kindly provided us with new T4 ligase. We heartily appreciate their generosity.
4. Transformation
We used heat shock protocols to transform the plasmid into Dh5a for selection. Before 42℃ water bath, the competent cell was treated with two different methods: 30-minutes ice bath and 5-minutes ice bath plus 5-minutes liquid nitrogen bath. Then the transformation mix was spread on plates containing kanamycin and incubated overnight. The result showed that ice bath is more effective than nitrogen bath.
5. Colony PCR
To select for the correct plasmid, we used colony PCR. We used PPO-F and PPO-R again as primers. But there were no correct responses. After several repetitions, we speculated that the digestion and ligation method may not be so effective. Therefore, we turned to in-fusion and Gibson assembly.
6. In-Fusion
Compared to digestion and ligation, in-fusion is more efficient, requiring only 15 minutes reaction. We designed primers containing 20 bp homogeneous sequence and used PCR to add the sequence to PPO DNA, and linearized pET30a vector with endonuclease EcoRV. The recombinant enzyme would then combine PPO sequence to the plasmid through homologous recombination. The in-fusion mix was also a gift from Ms. Shang in BNU. Thanks again!
After transformation and colony PCR, we finally derived strains with plasmid that contains PPO.
7. Plasmid Extraction
After overnight incubation in liquid LB broth, we extracted plasmid from the strain, and briefly verified the plasmid using agarose gel.
Then we sent the plasmid for DNA sequencing. Because PPO sequence is 1770 bp long, the signal might be too weak at the middle of our sequence if we simply use the universal T7 promoter and terminator primers, we designed sequencing primers.
Rose
Introduction
2-Phenylethanol (2-PE) is an aromatic alcohol that has rose-like scent that exists widely in plant essential oil. The industrial production of 2-PE has many approaches, including chemical synthesis, plant extraction and microbial fermentation. The last approach is safer compared to chemical synthesis, for it doesn’t require benzene and styrene as reactants; and more economic compared to plant extraction, for it is more efficient. But the industrial pathway was through yeast, and the process is relatively complicated, involving numerous enzymes.
Thanks to 2018 iGEM team FJNU China, we learned about a simplified approach that could synthesize 2-PE in E. coli. The synthetic pathway is shown below. Under the catalyzation of TyrB, Aro10 and PAR, L-phenylalanine is turned into 2-PE.
*This reaction pathway comes from 2018 iGEM team FJNU China
Design
Even after successfully incorporating PPO into BL21 strain, we still cannot certify that the PPO is expressed in every strain we use in detection. If PPO is not expressed, there won’t be color change even with bacteriophages.
Luckily, our communication with 2018 iGEM team FJNU China opens a new window for us. Their project was about producing 2-PE in E. coli so as to make the industrial production of this aromatic substance more efficient. Then we thought about incorporating 2-PE to our expression system. Through adding the 2-PE related genes to our plasmid as a fragrant reporter, when smelling the scent, we can make sure that the proteins are expressed. In this way, we can prevent false negative response due to lack of protein expression.
We decided to acquire the rose parts from FJNU China, and add the parts to pET30a-PPO and pET30a-luciferase using endonuclease digestion and ligation.
-Possible Design