Team:Stanford-Brown/Cellulose

SB iGEM 2015

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Abstract

We used the acetic acid bacterium Gluconacetobacter hansenii to produce bacterial cellulose. Because of its fibrous, tough, water-insoluble properties [1] we used bacterial cellulose as a substrate for biOrigami. After making the cellulose, we refine it into a flat, paper-like sheet using a DIY paper-making protocol. One unique aspect of cellulose is the existence of a class of proteins known as cellulose binding domains (CBDs) that can attach to a cellulose sheet. We design a universal CBD (uCBD) that allows for the attachment of any protein onto a cellulose sheet. The system involves fusing a CBD to a form of streptavidin; fusing the desired protein to a biotin acceptor; and adding biotin to the cell culture, causing the protein of interest to link to biotin, which then links to streptavidin and then to the cellulose sheet. Using our uCBD device, we can extend our self-folding system to serve additional functions, such a sensor for the detection of inorganic molecules, or binding enzymes to catalyze reactions on the surface of the sheet. We have contributed the BioBricks of our uCBD system to the registry. See our biobricks below:

See our BioBricks

Introduction

We use G. hansenii to produce cellulose as an alternative substrate for our biOrigami. Combine with cellulose binding domain and spore coat protein, cotZ, we can attach spores onto the cellulose surface for self-folding.

Cellulose is a polysaccharide made up of the monomer, B-D glucose, linked by (1->4) glycosidic bond [1]. It is an important structural component of the plant cell walls and has been used in various textile applications such as paper production [2]. Cellulose is also produced by a Gram-negative bacterium, Gluconacetobacter hansenii, which secretes highly-crystalline cellulose [3]. Because of the few requirements needed to produce bacterial cellulose, G. hansenii is seen as a model organism for the study of cellulose synthesis in plants [3]. We grew a large culture of G. hansenii in growth medium to produce large batch of bacterial cellulose. After retrieving the cellulose from the medium, we used a paper-making protocol to turn them into cellulose sheets (see protocol below). The cellulose would be an important substrate for our biOrigami when combined with the spores to make bioHYDRAS (link to bioHYDRA page). We can use CBDs to attach the spores to the cellulose sheet. This can be accomplished by using a DNA linker connecting the CBD sequence to a protein expressed on the spore coat, Cotz. Since Cotz localizes to the spore coat, the hybrid Cotz-CBD will allow the spores to attach to the sheet. Furthermore, we can expand on the unique properties of this CBD to allow attachment of any protein onto a cellulose surface. We call our CBD system uCBD to note its universal protein attachment abilities.
The system is inspired by the previous 2014 Stanford-Brown-Spelman iGEM team (link to page); however, it has several improvements. We used the cellulosomal-scaffolding protein A (cipA) of Clostridium thermocellum as our CBD. We chose cipA because of its high cellulose binding affinity (tested by the Imperial 2014 IGEM team) and improved on the Imperial cipA by removing the illegal EcoRI site within the gene. CipA is attached to a monomeric streptavidin instead of the wild-type tetramer streptavidin to prevent the chance of protein aggregation, which can inhibit protein function [4]. We thus, sacrifice a small decrease in binding affinity from the monomeric streptavidin for an increase in functional activity of the overall hybrid protein. Together, the cipA + monomeric streptavidin gene made up the cellulose attachment part of our uCBD sytem (Part I). The second part of the system involves molecular cloning any protein of interest onto the bifunctional ligase/repressor (birA) protein attached to an acceptor peptide. BirA is found in e. coli and it catalyzes attachment of biotin onto the biotin acceptor peptide such as the Avitag [5]. Because the acceptor peptide is connected to the protein of interest, this protein can be extracted and purified from the cells and can then attach to Part I via streptavidin-biotin interaction.

Experiment Engineering E. coli to produce polystyrene

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Data and Results Optimizing the production of biological PHA

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Protocols

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References

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