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, between these emphasized the Conventional, process with the most rate of pollution, in this -process- it used the elemental chlorine, a component before used in the World War I, this process is the most know it today and the first with the objective of oxidize the lignin too. While the process less contaminant is the Solvopulping, although is a process so explosive, because it is used de alcohol, due to this is become to a process non frequently used and very few know it too. Like these paper bleaching exist others too, like: Totally Chlorine Free (TCF), Elemental Chlorine Free (ECF), ThermoMechanical Pulp (TMP), Chemi-ThermoMechanical Pulp (CTMP), etc.

The consequences of these paper bleaching process are as many as the physical deformities of the fish or the reproductive deterioration, such as liver disorders, damage to the skin and gills; hormonal changes and changes in the schools behavior; in the blood composition and population structure of fish and lastly, disruption of the cell functions; all this happens 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 can damage the endocrine system: they can enter cells 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 accumulate in the soil. Its hydrophobicity allows its along the food chain. The idea of change in the components for the bleaching of the paper without polluting have been tried in different situations, however none of these have been successful, since they still generate waste contaminants. The question is: It is possible to bleach the paper without causing damage to the environment we live in?

Solution

Based on the information of past researches 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 to solve 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 give a solution we will used a yeast called Saccharomyces cerevisiae, which we will modify for it to act as a lignin degradation organism when it gets to contact the cellulose pulp, for achieve this we will used a Cellobiose response regulator and his 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 substrata. In some fungus and plants that are an important part of lignin degradation and toxic phenol elimination derived from this process.
How to: We make this by biotechnology, where we introduce our biology modeling in rot yeast S. Cerevisiae, because is easily to work and it’s time to duplication is 90 minutes. To do this possible we need two enzymes: Lignin Peroxidase and Laccase, this enzymes belong to the White-rot fungi with manganese peroxidase , but this not is required to our project.

Design

Our modeling it´s working with two plasmid, the first have a constitutive promoter, who is gonna active the ClbR and the LuxI, the last one converts SAM into AHL who bound to LuxR active 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: catalyze 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.