Team:SDSZ China/Des
Team:SDSZ China
Luci
Luciferase is the joint name for all enzymes in nature that induce bioluminescence. One of the most typical types is firefly luciferase from North American firefly (Photinus Pyralis). 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+.
Reaction process:
—Luciferase + ATP—> luciferyl adenylate + PPi
—Luciferyl adenylate + O2—> Oxyluciferin + AMP+ light
Considering that bioluminescence would be better detected and observed in seriously polluted water than simply chromogenic reactions, our team decided to construct plasmids with firefly luciferase sequence that can be induced in E.coli. cells to express luciferase proteins for further detection of phages in fecal-polluted water samples.
As the cultured solution of strain BL21 of E.coli. cells is induced by IPTG and protein expression is confirmed by SDS-PAGE, different concentrations of bacteriophage are added to the same batch of bacteria solution.
-Bacteriophages of different concentrations attack E.coli.cell membranes to allow for the release of the expressed proteins inside the cells.
-Proteins of firefly luciferase leak into the background culture to interact with the substrates (Mg2+, ATP, D-luciferin).
-Enzymatic reaction occurs and bioluminescence is observed.
PPO
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.
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.
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.
PSP SYSTEM
Psp system stands for phage shock protein system, including a set of intracellular and membrane proteins that are active in perturbation of cell membranes.
We have considered applying Psp system to our design, inserting the sequence of luciferase or PPO on the downstream sequence activated by the binding of PspF protein. Under this circumstance, the expression of the protein is directly related to the attack of bacteriophages that perturbs cell membrane, and may possibly reduce false positive responses due to unexpected cell lysis other than caused by phage attack.
Rose
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.
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.