Team:StanfordBrownSpelman/Cellulose Acetate

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    <title>Stanford–Brown–Spelman iGEM 2014 — Cellulose Acetate</title>
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  			<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Acetate">Biomaterials</a></h3>
  			<div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#CA">BioBricks</a></h7></div>
 
<h6><center><iframe width="420" height="315" src="//www.youtube.com/embed/o6hQ5_nTMQc" frameborder="0" allowfullscreen></iframe></center><br>		

<h6 id="int">
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.
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Material_Waterproofing" target="_blank">Material Waterproofing, </a>

Biosensors can be linked to the cellulose acetate skin (see 
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Amberless_Hell_Cell" target="_blank">Amberless Hell Cell), </a>
through a biological cross-linker
(see
<a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Cross_Linker" target="_blank">Cellulose Cross Linker.) </a>


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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, <i>Gluconacetobacter hansenii</i> has been identified as species producing the highest yield of cellulose. [2] Another strain of bacteria, the SBW25 isolate of the species <i>Pseudomonas fluorescens</i>, 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 <i>G. hansenii</i> with the four genes, wssF, wssG, wssH, and wssI, that have been identified [3] as being involved in cellulose acetylation in <i>P. fluorescens</i>, and to use directed evolution to further increase percent acetylation of the polymer.

</p>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. 

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  			<h5><center>Approach & Methods</h5>
<h6>Our goal was to turn bacterial cellulose into cellulose acetate.</h6>
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<h6><center>Cellulose on the left transformed into cellulose acetate on the right.</center></h6>
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<h6>To accomplish this we looked to transform the genes responsible for the acetylation of cellulose in <i>P. fluorescens</i>, wssF-I [3], into our model cellulose-producing organism <i>G. hansenii.</i></h6>
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<h6><center>Acetylation genes. Image via [7].</center></h6>
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<h6>However, <i>G. hansenii</i> 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 <i>E. coli</i> before transforming it into <i>G. hansenii.</i> For the transformation we used the electroporation protocol found in [5].</h6>
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<h6><center><b>Note:</b> pUCD4 differs from pUCD4 in only one restriction site.</center></h6>
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  			<h6>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.</h6> 

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<h6><center>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.</center></h6>
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<h6>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 <a href="http://agic.cc" target="_blank">AgiC</a>, which prints circuits out of silver nano particles (see our <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Building a UAV</a> 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.</h6> 

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<h6><center>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. </center></h6>
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<h6>More methods here. 	
  			
<div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="work/PUT-PDF-REFERENCE-HEREpdf">Click here to go to our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.</a></div>
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                        <h5><center>Results</h5>
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  				Results go here.

  			
  			<br><br><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone"><b>Building a Biological UAV</b></a></center><br>
  			
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! <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">Check our more about the material, design, and construction aspects of our biomaterial project here.</a>
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  			<h5><center>References</h5>
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  				<div class="sub5">1. Fischer S <i>et al.</i> (2008) Properties and Applications of Cellulose Acetate. <i>Macromol. Symp.</i> 262: 89-96. DOI: <a href="http://blogs.unpad.ac.id/evyerna/files/2010/11/ca.pdf" target="_blank">10.1002/masy.200850210</a></div>
  				<div class="sub5">2. Ross P <i>et al.</i> (1991) Cellulose Biosynthesis and Function in Bacteria. <i>Microbiological Reviews</i> 55: 35-58. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2030672" target="_blank">2030672 </a></div>
  				<div class="sub5">3. Spiers AJ <i>et al.</i> (2003) Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. <i>Molecular Microbiology</i> 50: 15-27. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/14507360" target="_blank">14507360 </a></div>
  				<div class="sub5">4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013. </div>
  				<div class="sub5">5. Hall PE <i>et al.</i> (1992) Transformation of Acetobacter xylinum with Plasmid DNA by Electroporation. <i>Plasmid</i> 28: 194-200.  PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/1461938" target="_blank">1461938 </a></div>
  				<div class="sub5">6. Close TJ <i>et al.</i> (1984) Design and Development of Amplifiable Broad-Host-Range Cloning Vectors: Analysis of the wir Region of Agrobacterium tumefaciens Plasmid pTiC58. <i>Plasmid</i> 12: 111-118. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/6095350" target="_blank">6095350 </a></div>
<div class="sub5">7. Spiers AJ <i>et al.</i> (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in <i>Cellulose - Medical, Pharmaceutical, and Electronic Applications</i>. DOI: <a href="http://cdn.intechopen.com/pdfs-wm/45637.pdf" target="_blank">10.5772/53736</a></div>
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