Team:StanfordBrownSpelman/Cellulose Acetate

From 2014.igem.org

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   <div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="data">Methods</a> ● <a href="#" id="methods">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBricks</a></h7></div>
   <div class="boxedmenu"><h7><center><a href="#" id="intro">Introduction</a> ● <a href="#" id="data">Methods</a> ● <a href="#" id="methods">Results</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks">BioBricks</a></h7></div>
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  The goal of this subproject is to engineer <i>Gluconacetobacter hansenii,</i> which produces large quantities of bacterial cellulose (BC), to use the acetylation machinery found in the wrinkly spreader isolate SBW25 of <i>Pseudomonas fluorescens</i> to produce bacterial cellulose acetate (BCOAc) towards its ultimate application as the foundation of a fully biological UAV. Industrially-produced cellulose acetate has many uses as a synthetic fiber and has advantages over pure BC in terms of material properties.   However, its production presents some issues in that it requires harsh chemical processes, making the biological manufacturing method highly valuable. Using directed evolution, we plan to select for those organisms which produce the polymer with highest acetate content. In addition, we seek to create a fusion protein which will have the capacity to both cross-link BCOAc polymers (improving material properties) and allow the modular addition of any cell (e.g. a biosensor). This will be accomplished through the expression of a biotinylated membrane protein, through the protein’s streptavidin domain, making the UAV highly programmable.
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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.
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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>Links & References</h5>
   <h5><center>Links & References</h5>
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   <div class="sub5"><a href="work/PUT-PDF-REFERENCE-HEREpdf">● Have a link or reference? Put it here!</a></div>
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   <div class="sub5">● 1. Fischer, et al. Properties and Applications of Cellulose Acetate. Macromol. Symp., 262, 89-96</div>
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   <div class="sub5"><a href="work/PUT-PDF-REFERENCE-HEREpdf">● Link, acknowledgement, or reference 2</a></div>
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   <div class="sub5">● 2. Ross, P., Mayer, R., & Benziman, M. Cellulose Biosynthesis and Function in Bacteria. Microbiological Reviews, 55, 35-58.
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   <div class="sub5"><a href="work/PUT-PDF-REFERENCE-HEREpdf">● Additional links, acknowledgements, and references</a></div>
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  <div class="sub5">● 3. Spiers, A. J., Bohannon, J., Gehrig, S. M., & Rainey, P. B. 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.</div>
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   <div class="sub5">● 4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013.
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  <div class="sub5">● 5. Hall, P. E., Anderson, S. M., Johnston, D. M., Cannon, R. E. Transformation of Acetobacter xylinum with Plasmid DNA by Electroporation. Plasmid, 28, 194-200.
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   <div class="sub5">● 6.  Close, T. J., Zaitlin, D., & Kado, C. I. Design and Development of Amplifiable Broad-Host-Range Cloning Vectors: Analysis of the wir Region of Agrobacterium tumefaciens Plasmid pTiC58. Plasmid, 12, 111-118.</div>
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Revision as of 03:05, 13 October 2014

Stanford–Brown–Spelman iGEM 2014 — Cellulose Acetate

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Results
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Methods & Safety
Fusce venenatis, justo id luctus dictum, turpis libero tincidunt mauris, sit amet tempor lectus tortor ut ante. Pellentesque egestas felis et est venenatis, eget lobortis dui adipiscing. Suspendisse volutpat sem eu ornare tincidunt. Mauris pharetra sed justo vitae sodales. Nulla in sodales tortor, placerat tempor dui.
Click here to download our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.
Links & References
● 1. Fischer, et al. Properties and Applications of Cellulose Acetate. Macromol. Symp., 262, 89-96
● 2. Ross, P., Mayer, R., & Benziman, M. Cellulose Biosynthesis and Function in Bacteria. Microbiological Reviews, 55, 35-58.

● 3. Spiers, A. J., Bohannon, J., Gehrig, S. M., & Rainey, P. B. 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.
● 4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013.

● 5. Hall, P. E., Anderson, S. M., Johnston, D. M., Cannon, R. E. Transformation of Acetobacter xylinum with Plasmid DNA by Electroporation. Plasmid, 28, 194-200.

● 6. Close, T. J., Zaitlin, D., & Kado, C. I. Design and Development of Amplifiable Broad-Host-Range Cloning Vectors: Analysis of the wir Region of Agrobacterium tumefaciens Plasmid pTiC58. Plasmid, 12, 111-118.
Additional Information
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