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Team:StanfordBrownSpelman/Cellulose Acetate
From 2014.igem.org
Biomaterials
The body of the UAV is designed to consist of a styrofoam-like filler made of fungal mycelia, coated with a cellulose acetate covering. The skin will be biologically waterproofed. Biosensors can be added to the cellulose acetate skin through a biological 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 produced industrially 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 (e.g., 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 the 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 (0.14 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. In particular, the standard pSB1C3 backbone does not contain a suitable origin of replication for G. hansenii. 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]. Note that [5] gives a transformation protocol for a cellulose-negative strain of G. hansenii. However, we were able to successfully transform cellulose-producing bacteria using the same procedure.
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.
Results
We were able to extract total DNA from P. fluorescens and amplify the four acetylation genes.
Fig. 4: Gel verification confirming amplification of four acetylation genes from total P. fluorescens DNA.
The prefix and suffix were added onto these PCR products, and then they were inserted (separately) into pSB1C3 for biobricking (see Submitted Bricks).
Fig. 5: wssF sequencing alignment.
We were also able to show that the pUCD4 shuttle vector was effective in making G. hansenii a suitable chassis for carrying synthetic information, an important step in the process of studying cellulose derivate polymers. By plating both transformed and untransformed cells on antibiotic selection plates and using colony PCR to screen for the presence of the plasmid, we found that pUCD4 was effective at providing resistances to multiple antibiotics.
Fig. 6: pUCD4 verification gel.
The first step in working towards producing our building material was to grow cultures of cellulose producing bacteria in sterile trays. After these cultures grew for 1-2 weeks, we removed the produced cellulose sheet from the culture to test various methods of drying. We experimented with drying the sheet in an oven, to produce an extremely thin layer of cellulose. We also wrapped fungal mycelium blocks provided by Ecovative, which we intend to be the body of our UAV, with wet cellulose, and allowed the cellulose to dry on its own. This will provide the platform for us to alter the biomaterial for flight, by, for example, making it waterproof.
Fig. 7: Production of dried cellulose. a) A wet cellulose sheet, soaking in 50% alcohol solution. b) The cellulose was placed between two acrylic gel casters and left in a 75ºC oven for 2 days. c) A thin, dry cellulose sheet. d) Fungal mycelium wrapped in dry cellulose.
One alteration we intend to make in order to produce a functional UAV is to draw circuits on the biomaterial to conduct electricity. In order to produce a biodegradable circuit, we worked with a company called AgiC, which prints circuits out of silver nano particles (see our Building a UAV page for a full circuit). By taking silver ink and painting it on to our bacterial cellulose, we were able to test the conductive capabilities of our building material.
