Team:StanfordBrownSpelman/Cellulose Acetate
From 2014.igem.org
Biomaterials
The body of the UAV is designed to consist a styrofoam-like filler consisting of fungal mycelia, coated with a cellulose acetate covering. The skin will be biologically waterproofed.
Material Waterproofing,
Biosensors can be linked to the cellulose acetate skin (see
Amberless Hell Cell),
through a biological cross-linker
(see
Cellulose Cross Linker.)
Cellulose acetate is a biodegradable thermoplastic polymer used for a variety of industrial applications [1]. The monomer of cellulose acetate is glucose with one or more of its available hydroxyl groups replaced with acetyl groups. Cellulose acetate is industrially produced by treating cellulose, typically from wood or cotton, with acetic anhydride and sulfuric acid at high temperatures [1]. Our aim is to engineer bacterial cells to produce industrial-grade cellulose acetate biologically, allowing this plastic to be produced anywhere that bacterial colonies can be grown (i. e. in space). This material could then be used as a basis or coating for a biodegradable UAV. Many species of bacteria produce cellulose fibers; however, Gluconacetobacter hansenii has been identified as species producing the highest yield of cellulose [2]. Another strain of bacteria, the SBW25 isolate of the species Pseudomonas fluorescens, produces a biofilm containing cellulose fibers with a small degree of acetylation (.14 acetyl groups per glucose monomer) [3]. Industrial-grade cellulose acetate must have at least 1.71 acetyl groups per glucose monomer [4]. In order to engineer a bacterium to efficiently produce cellulose acetate, our strategy is to transform G. hansenii with the four genes, wssF, wssG, wssH, and wssI, that have been identified [3] as being involved in cellulose acetylation in P. fluorescens, and to use directed evolution to further increase percent acetylation of the polymer.
In addition, we seek to create a streptavidin/cellulose-binding-domain fusion protein which will have the capacity to both cross-link bacterial cellulose acetate polymers (improving material properties) and allow the modular addition of cells (e.g. biosensors). This will be accomplished through the expression on the cells of a biotinylated membrane protein. This will allow biological sensors to be added directly to our cellulose acetate fibers, allowing bacterial sensors to be attached directly to the body of our UAV.
Approach & Methods
Our goal was to turn bacterial cellulose into cellulose acetate.
Fig. 1: Cellulose on the left transformed into cellulose acetate on the right.
To accomplish this we looked to transform the genes responsible for the acetylation of cellulose in P. fluorescens, wssF-I [3], into our model cellulose-producing organism G. hansenii.
Fig. 2: Acetylation genes. Image via [7].
However, G. hansenii is not a well-characterized organism for standard synthetic biology lab procedures and consequently cannot use the standard pSB1C3 backbone. Instead, we utilized the multi-host shuttle vector pUCD4 [6], which allowed us to grow the plasmid to large quantities in E. coli before transforming it into G. hansenii. For the transformation we adapted the electroporation protocol found in [5].
Fig. 3: Design of pUCD4 plasmid
Note: pUCD4 differs from pUCD2 in only one restriction site.
Note: pUCD4 differs from pUCD2 in only one restriction site.