Team:StanfordBrownSpelman/Cellulose Acetate

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Stanford–Brown–Spelman iGEM 2014 — Cellulose Acetate

Biomaterials

Introduction ● Methods ● Results ● References ● BioBricks

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.

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.

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 used the electroporation protocol found in [5].

Note: pUCD4 differs from pUCD4 in only one restriction site.

The first step in working towards producing our building material was to grow cultures of cellulose producing bacteria. 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 extreme thin layer of cellulose. We also wrapped fungal mycelium, 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 making it waterproof, for example.

Figure 1: 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.

Figure 2: Making cellulose electrically conductive. a) The silver ink used to paint cellulose. b) Silver nano particles painted onto cellulose covered mycelium. c) Positive Control: Aluminum foil has a resistance of 0.5 ohms. d) Negative Control: Unaltered cellulose has no resistance, and thus no conductivity. e) Experimental: Cellulose painted with silver nano particles has a resistance of 1.6 ohms.

Results

We were able to extract total DNA from P. fluorescens and amplify the four acetylation genes.

The prefix and suffix were added onto these PCR products, and then they were inserted (separately) into pSB1C3 for biobricking (see Submitted Bricks).

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.

Additional Information

Interested in what we've been working on and want to find more relevant information? Check out some of the following sites, companies, and people who either aided us in our production of biomaterials or collaborated with us in working to produce a viable biological unmanned aerial vehicle.
● Click here for a list of submitted biobricks, the four genes found in P. fluorescens responsible for the acetylation of the cellulose polymer. These genes are all found individually in the biobrick backbone and are fully biobrick compatible.

● Click here for more information on the specific protocols we used for working with G. hansenii and bacterial cellulose, including our recipe for Acetobacter Media, an electroporation transformation protocol, and instructions on generating, cleaning, and drying bacterial cellulose.

Building a Biological UAV

Our team modeled, prototyped, and collaborated with Ecovative Design to grow a mycelium-based chassis for our biological drone. Below you'll find process photos, part designs, and links to open source model files for downloading and additively manufacturing your own biological or bio-inspired unmanned aerial vehicle. Finally, you can see images of the biological, biodegradable UAV that we built and flew! Check our more about the material, design, and construction aspects of our biomaterial project here.

References

1. Fischer S et al. (2008) Properties and Applications of Cellulose Acetate. Macromol. Symp. 262: 89-96. DOI: 10.1002/masy.200850210

2. Ross P et al. (1991) Cellulose Biosynthesis and Function in Bacteria. Microbiological Reviews 55: 35-58. PMID: 2030672 

3. Spiers AJ et al. (2003) Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Molecular Microbiology 50: 15-27. PMID: 14507360 

4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013. 

5. Hall PE et al. (1992) Transformation of Acetobacter xylinum with Plasmid DNA by Electroporation. Plasmid 28: 194-200.  PMID: 1461938 

6. Close TJ et al. (1984) Design and Development of Amplifiable Broad-Host-Range Cloning Vectors: Analysis of the wir Region of Agrobacterium tumefaciens Plasmid pTiC58. Plasmid 12: 111-118. PMID: 6095350 

7. Spiers AJ et al. (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in Cellulose - Medical, Pharmaceutical, and Electronic Applications. DOI: 10.5772/53736

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