Cellulose Synthase (Cesa) Project

Cellulose is the most abundant polysaccharide on Earth and is incredibly valuable for multiple uses including paper, cellophane, and biofuel. Although cellulose in everywhere, we are still limited by the amount of production by the plants and constantly use more. The goal of our experiment is to introduce a secondary cell wall cellulose synthase complex into the primary cell wall to ultimately increase the production of cellulose in plants. Our hope is that if we use a primary cell wall promoter followed by secondary CesA’s (Cellulose Synthases) in Arabidopsis thaliana, we can produce more cellulose and create stronger plants.

Introduction

Cellulose is the most abundant polysaccharide on Earth and is incredibly valuable for multiple uses including paper, cellophane, and biofuel. Although cellulose in everywhere, we are still limited by the amount of production by the plants and constantly use more. The goal of our experiment is to introduce a secondary cell wall cellulose synthase complex into the primary cell wall to ultimately increase the production of cellulose in plants. Our hope is that if we use a primary cell wall promoter followed by secondary CesA’s (Cellulose Synthases) in Arabidopsis thaliana, we can produce more cellulose and create stronger plants.

Background

Cellulose is produced in both the primary and secondary plant cell walls. Research has shown that secondary cellulose synthase (CesA) complexes produce greater quantities of cellulose than those located in the primary cell wall. (1) Additionally, research indicates the possibility of a hybrid plant cell wall through the ability to exchange promoters for cellulose synthase genes. An intricate protein complex in nature, research of cellulose synthase is continually ongoing. Due to the limited amount of definitive information known about the complex, the synthesis of a hybrid plant cell wall would provide verification of standing hypotheses and valuable novel information.

(1) http://mplant.oxfordjournals.org/content/4/2/199.full

Method

To assemble our construct, we isolated the CesA 1 Promoter from Arabidopsis thaliana by extracting the genomic DNA and using PCR amplification. The CesA 4, 7, and 8 genes were obtained from cDNA via the Arabidopsis Biological Resource Center and tissue samples. PCR amplification from vector pDMC107 enabled the isolation of GFP and NOS terminator. Ultimately, the construct was tied together via Gibson assembly synthesising all tags and markers from the primers used.

Further Study

Due to limitations resulting from the lack of previous synthetic biology application within plants, we were unable to obtain any results. We have successfully isolated the desired CesA 1 promoter, CesA genes 7 and 8 and the Nos terminator parts. However, we were not able to isolate the CesA 4 gene needed for assembly of the plasmid. Thus successful construct assembly of these parts is still needed for bacterial and plant transformation. Following promising transformation, fluorescence testing and characterization must be done.

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