Team:Saint Rose School B/Description

Problem

Despite that the process in cellulose industries have modernized, these continue to be one of the most polluting industries in the world, since the main paper bleaching process is the one that causes pollution and where the objective is to oxidize the lignin present in the pulp of cellulose, because its structure presents chromophore groups responsible for the staining of non bleached pulps. That is why was created a diversity of bleaching process, emphasized in the Conventional process with the most pollution rate, in this -process- elemental chlorine was used, a component used in the World War I, this process is very common nowadays and its first objective is to oxidize the lignin. While the process less contaminant is the Solvopulping, it is a very explosive process, because it used alcohol, due to this it has become a very infrequent process and very few people apply this.

There are other paper bleaching techniques, for example: Totally Chlorine Free (TCF), Elemental Chlorine Free (ECF), Thermo Mechanical Pulp (TMP), Chemi-ThermoMechanical Pulp (CTMP), etc.

The consequences of these paper bleaching processes are as many as for example physical deformities or reproductive deterioration on fish, including liver disorders, skin and gills damage; hormonal changes and schools behavior disorders in humans. Blood test in fish had shown composition and population structure damage, and last, disruption of cell functions; all this happened when wastewater and contaminants are released to the sea, rivers and lakes.

Humans are also affected, since the different pollutants can cause immune, reproductive and nervous system disorders. Also, carcinogenic and mutagenic compounds have been identified . While in the fetus phase this can damage the endocrine system, they can enter cells organisms and block, imitate or alter the actions of the hormones, and have negative effects on the development of neurological, reproductive, behavioral and immune systems. Dioxins are generally found in the atmosphere attached to dust particles (flying ash). Due to its low solubility in water and its great ability to adhere to soil particles, their mobility is extremely low and accumulated in the soil. It can also contaminate throughout the food chain.

The idea of change in the components for the paper bleaching process without polluting have been test in different situations, however none of these have been successful, since they still generate waste contaminants. The question is: Is it possible to bleach the paper without causing damage to the environment we live in?

Solution

Based on the information of past inquiries, potential enzymes for the degradation of lignin have been identified inside of a white rot fungus. Through approaches based on biotechnology we have proposed the development of synthetic yeast, with the objective of solving the problem of lignin degradation, since it is one of the main inhibitors in biofuel production and one of the main problems in paper industries.

To solve this we will used a yeast called Saccharomyces cerevisiae, which we have modified for it to act as a lignin degradation organism when it gets to contact the cellulose pulp,to achieve this we will used a Cellobiose response regulator and its promoter Cellobiohydrolase I. In our project, we will try to use two different genes obtained from Phanerochaete chrysosporium: lignin peroxidase and laccase. Lignin peroxidase is one of the most important enzymes on lignin degradation inside cell walls; it differences itself from other peroxidases because of their high redox potential which allows to directly oxidize non-phenolic aromatic compounds; and Laccase, belonging to the blue copper oxidase group, catalyzes the oxidation of organic or inorganic substrate. Some fungus and plants that are an important part of lignin degradation and toxic phenol elimination derived from this process.


How to: We achieve this process by biotechnology, where we introduce our biology modeling in rot yeast samples such as S. Cerevisiae since it is easy to work with and its time to duplication is around 90 minutes. To make this possible we need two enzymes: Lignin Peroxidase and Laccase, this enzymes belong to the White-rot fungi with manganese peroxidase , but this is not required in our project.

Design

Our model works with two plasmids, the first has a constitutive promoter, who is gonna activate the ClbR and the LuxI, the last one converts SAM into AHL who bound to LuxR activates the promoter PLux and this is going to activate a cyan fluorescent protein to verify that is transcribing. In the second plasmid is going to express Lignin Peroxidase the gene of interest and a colour to prove the transcription, this active under the control of the ClbR when bound to cellulose (who is going to get in the yeast), they are going to active the Cbh1. At the same time in the first plasmid is going to express the LuxR after the laccase, these activated from Cbh1 too.

Parts

Lignin Peroxidase (LiP): is an enzyme that is the initiator and one of the main enzymes responsible for the breakdown of lignin inside the cell walls of plant cells. Some organisms such as the Reticulitermes flavipes, or the eastern subterranean termite uses LiP to digest woody debris with higher efficiency.This enzyme has the ability to oxidize aromatic compounds in lignin.

Laccase: catalyzes the oxidation of a substrate, organic or inorganic, in some fungi and plants are an important part of the degradation of lignin.

Laccase is a copper-containing enzyme obtained from the gut rumen of Reticulitermes flavipes (the eastern subterranean termite), where its evolutionary uniqueness in its ability to efficiently degrade the lignin of woody debris when working in concert with other lignocellulases. Laccase functions via the catalyzation of one-electron substrate oxidations with a concurrent four-electron reduction of molecular oxygen to water. Unlike the peroxidases, they only require oxygen for activity, and are not dependant on peroxide

Cellobiose response regulator: Controls the expression of cellulase-related genes, ClbR have the control over the function of the complete bacteria. Cellobiose bound to ClbR and active Cbh1.
Cellobiohydrolase I: Cbh1 promoter has been considered the strongest promoter in Trichoderma reesei, and is generally used to construct high-efficient expression vectors to yield homologous and heterologous proteins.
LuxR: In complex with HSL, LuxR binds to the Lux promoter.
LuxI: Enzyme is a synthase that converts SAM into a small molecule called an acyl-homoserine lactone (AHL).
PLux: The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with SAM
RFP: Red fluorescent protein from Discosoma striata (coral).
CFP: This part was created by DNA 2.0 as part of their IP-Free series of fluorescent and chromogenic proteins. It is available to use under the BioBrick Public Agreement.