Description

Overview

m-Phenylenediamine not only has health hazards to human being like allergy, but also has environmental hazards such as water pollution. Instead of using traditional degradation methods, we resorted to microbial biodegradation, the breakdown of organic matter by microorganisms.
We aimed to applicate three approaches:
1) Adaptive evolution of E. coli (MG1655);
2) Construction of Laccase Expressing E. coli, and adaptive evolution of it;
3) Adaptive evolution of bacteria from aniline-polluted sludge. (The strain was isolated by Jiangsu Nanzi Environmental Protection Technology co. LTD)

Adaptive Lab Evolution (ALE)

Adaptive evolution with E. coli MG1655 (gradual increase of m-phenylenediamine, reduction of carbon and nitrogen sources).

The adaptive evolution, also known as directed evolution, laboratory evolution, or domestication, is currently a well-recognized strain improvement technique that enables scientists to effectively alter strains’ certain phenotypes or physiological characteristics of strains (such as bacterial growth rate, substrate consumption rate, tolerance to extreme temperature and pH, different organic solvents, etc.) in a relatively short period of time. In addition, it will not affect other excellent traits except the target phenotype. The most commonly used adaptive evolution method in laboratories is the microorganisms, which are continuously sub-cultured under specific conditions (giving selection pressure) the phenotypic or physiological properties adapted to specific conditions are obtained through continuous enrichment of the spontaneous mutations of the strain. In the process of microbial evolution, the presence of selective pressure can ensure that the random mutation of the strains is targeted and eliminated while the genotypes compatible with the environment are preserved, especially in the artificial breeding process, by manually applying the directed selection pressure to make the microorganisms along the desired The evolution of the direction to obtain the target trait.

Construction of Laccase Expressing E. coli

Laccase is a member of the poly copper oxidation family. It is able to catalyze and oxidize m-phenylenediamine. It is widely used in many industries, including food manufactories, paper mills, textile mills, paints factories, and organic chemical factories. Our team constructed a plasmid containing laccase gene fragments and transformed it into Escherichia coli MG1655 strain (and other strains) to make exogenous DNA highly expressed in E. coli competent cells. In the following experiments, we completed the transformation of the recombinant plasmid of the reaction product and verified its inhibitory effect and degradation on m-phenylenediamine (MPD).
(1) Primer design of the target gene fragment and vector fragment;
(2) construct a standard plasmid; transforming the plasmid into E. coli;
(3) cultivate E. coli, add a certain concentration of the solution of m-phenylenediamine into E.coli; and continuously add m-phenylenediamine, which competes with other carbon and nitrogen sources; collecting samples for each dosing;
(4) analyze the concentration of m-phenylenediamine by high performance liquid chromatography to observe whether adaptive evolution occurs;
(5) conduct whole genome sequencing and analyze key genes;
Get the E.coli acquiring the ability to degrade the compound obtaining strains that efficiently degrade m-phenylenediamine (or tert-butanol) to protect the environment.

Adaptive evolution of Bacteria from aniline-polluted sludge

The idea is to use the bacteria in activated sludge to degrade the m- phenylenediamine. (The strain was isolated by Jiangsu Nanzi Environmental Protection Technology co. LTD江苏南资环保科技有限公司). The technician from Jiangsu Nanzi Environmental Protection Technology informed us that the unknown strain from aniline-polluted sludge is able to degrade m-phenylenediamine analogue, p-toluidine and m-toluidine. Since that, the strain from aniline-polluted sludge have potential to gain the ability to degrade m-phenylenediamine by adaptive evolution.

Signal peptide

A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short (5-30 amino acids long) peptide presenting at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. If we find the protein(s) which is(are) able to degrade m-phenylenediamine, we will use the GFP to test if the cell can secrete the protein(s) regarding to the degradation to improve the efficiency of degradation.

Here is signal sequences list [1]:

Reference

1. Choi J H, Lee S Y. Secretory and extracellular production of recombinant proteins using Escherichia coli[J]. Applied microbiology and biotechnology, 2004, 64(5): 625-635.