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="cellulose_acetate.html">Cellulose Acetate</a></h3>
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   <h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Acetate">Biomaterials</a></h3>
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   <div class="boxedmenu"><h7><center><a href="#" id="intro">Intro</a> ● <a href="#" id="methods">Methods</a> ● <a href="#" id="data">Results</a> ● <a href="#" id="moreinfo">Links</a> ● <a href="#" id="links">References</a> ● <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#CA">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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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 <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Material_Waterproofing" target="_blank">waterproofed</a>. <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Amberless_Hell_Cell" target="_blank">Biosensors</a> can be added to the cellulose acetate skin through a biological <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Cross_Linker" target="_blank">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 produced industrially by treating cellulose, typically from wood or cotton, with acetic anhydride and sulfuric acid at high temperatures [1].
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</p>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, <i>Gluconacetobacter hansenii</i> has been identified as the 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 (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 <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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</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>
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<h6>Our goal was to turn bacterial cellulose into cellulose acetate.</h6>
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<h6><center>Fig. 1: 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>Fig. 2: 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. In particular, the standard pSB1C3 backbone does not contain a suitable origin of replication for <i>G. hansenii</i>. 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 adapted the electroporation protocol found in [5]. Note that [5] gives a transformation protocol for a cellulose-negative strain of <i>G. hansenii.</i> However, we were able to successfully transform cellulose-producing bacteria using the same procedure. </h6>
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<h6><center>Fig. 3: Design of pUCD4 plasmid<br /><b>Note:</b> pUCD4 differs from pUCD2 in only one restriction site.</center></h6>
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   We were able to extract total DNA from <i>P. fluorescens</i> and amplify the four acetylation genes.
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<h6><center>Fig. 4: Gel verification confirming amplification of four acetylation genes from total <i>P. fluorescens</i> DNA.</center></h6>
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<h6>The prefix and suffix were added onto these PCR products, and then they were inserted (separately) into pSB1C3 for biobricking (see <a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#CA" target="_blank"><u>Submitted Bricks</u></a>).</h6>
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<h6><center>Fig. 5: wssF sequencing alignment.</center></h6>
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<h6>We were also able to show that the pUCD4 shuttle vector was effective in providing antibiotic resistance to <i>G. hansenii</i>, showing that our transformation protocol was effective. 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 tetracycline and streptomycin.</h6>
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<h6><center>Fig. 6: pUCD4 verification gel.</center></h6>
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  <h6>Ultimately, we were unsuccessful in our attempts to ligate the wss genes into the pUCD4 plasmid: we were therefore unable to produce cellulose acetate biologically in <i>G. hansenii</i>. Future attempts could be made, possibly using a different shuttle vector. However, we continued to investigate the use of pure, unmodified bacterial cellulose as a building material. 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.</h6>  
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<h6>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.</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"><u>Building a UAV</u></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>Fig. 8: Making cellulose electrically conductive. a) The silver filamentous ink offered by silicon valley start up AgIC Inc., is used to coat the surface of 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 measurable conductivity. e) Experimental: Cellulose painted with silver filamentous ink has a resistance of 1.6 ohms. </h6>
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   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.
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   <div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf"><img src="images/download.png"></a><a href="work/PUT-PDF-REFERENCE-HEREpdf">Click here to download 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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   <div class="sub5"><a href="https://2014.igem.org/Team:StanfordBrownSpelman/BioBricks#CA">● Click here for a list of submitted biobricks, the four genes found in <i>P. fluorescens</i> responsible for the acetylation of the cellulose polymer. These genes are all found individually in the biobrick backbone and are fully biobrick compatible.</a></div>
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  <div class="sub5"><a href="https://static.igem.org/mediawiki/2014/d/d7/SBSiGEMCelluloseAcetateProduction.pdf">● Click here for more information on the specific protocols we used for working with <i>G. hansenii</i> and bacterial cellulose, including our recipe for <i>Acetobacter</i> Medium, an electroporation transformation protocol, and instructions on generating, cleaning, and drying bacterial cellulose.</a></div>
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<h5><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone"><b>Building a Biological UAV</b></a></center></h5>
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Our team modeled, prototyped, and collaborated with Ecovative Design to grow a mycelium-based chassis for our biological drone. For more information on this process, including part designs, links to open source model files, and photographs of the biological and biodegradable UAV we built and flew, <a href="https://2014.igem.org/Team:StanfordBrownSpelman/Building_The_Drone">click here</a>!
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   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><br><br>
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  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><br><br>
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  3. Spiers AJ <i>et al.</i> (2003) Biofilm formation at the air–liquid interface by the <i>Pseudomonas fluorescens</i> 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><br><br>
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  4. The United States Pharmacopeial Convention. Cellulose Acetate. USP-NF. 2013.
<br><br>
 +
  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><br><br>
 +
  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><br><br>
 +
7. Spiers AJ <i>et al.</i> (2013) Cellulose Expression in <i>Pseudomonas fluorescens</i> 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><br>
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Latest revision as of 04:15, 9 January 2015

Stanford–Brown–Spelman iGEM 2014 — Cellulose Acetate

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.

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 providing antibiotic resistance to G. hansenii, showing that our transformation protocol was effective. 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 tetracycline and streptomycin.


Fig. 6: pUCD4 verification gel.
Ultimately, we were unsuccessful in our attempts to ligate the wss genes into the pUCD4 plasmid: we were therefore unable to produce cellulose acetate biologically in G. hansenii. Future attempts could be made, possibly using a different shuttle vector. However, we continued to investigate the use of pure, unmodified bacterial cellulose as a building material. 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.


Fig. 8: Making cellulose electrically conductive. a) The silver filamentous ink offered by silicon valley start up AgIC Inc., is used to coat the surface of 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 measurable conductivity. e) Experimental: Cellulose painted with silver filamentous ink has a resistance of 1.6 ohms.

Building a Biological UAV
Our team modeled, prototyped, and collaborated with Ecovative Design to grow a mycelium-based chassis for our biological drone. For more information on this process, including part designs, links to open source model files, and photographs of the biological and biodegradable UAV we built and flew, click 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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