Team:StanfordBrownSpelman/Cellulose Cross Linker

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   <h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Cross_Linker">Cellulose Cross-Linker</a></h3>
   <h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Cellulose_Cross_Linker">Cellulose Cross-Linker</a></h3>
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   <h7><center><a href="#" id="pics">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>
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   <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#CCL">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 goal of this subproject is to create a cellulose cross-linking protein to increase material strength and allow for the modular attachment of biological sensors.  This fusion protein contains two distinct cellulose-binding domains[1] on either side of a streptavidin domain. The cellulose-binding domains cross link the cellulose fibers while the streptavidin serves as a binding domain for biological sensors. The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in nature [2]. Therefore a cell expressing an outer membrane protein that has been biotinylated will bind tightly to this domain. This will allow our UAV to make use of a number of biological sensors.
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<h6><b>Figure 1.</b> An illustration of cellulose binding domains cross-linking cellulose fibers with a streptavidin domain in the middle. The biosensing cell is expressing a biotinylated AviTag which will bind to the streptavidin .</center></h6>
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<h6><b></b>Our initial approach was to use the cellulose binding domains from <i>C. cellulovorans </i>  <a href="http://parts.igem.org/Part:BBa_K863111">(part BBa_K863111)</a></a> on either side of the streptavidin domain <a href="http://parts.igem.org/Part:BBa_K283010">(part BBa_K283010) under a T7 promoter in the PSB1A3 backbone.</a></a>We also included a His-Tag for protein purification. The protein is then expressed in <i> E. coli </i>. Once purified, the cross-linking protein is tested on bacterial cellulose we grew in our lab from the organism <i>G. hansenii</i>. By dotting the protein on the cellulose, the cellulose binding domains will bind to the cellulose fibers and leave the streptavidin domain unbound and ready to bind biotin.</center></h6>
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<div class="sub4"><a href=https://static.igem.org/mediawiki/2014/2/2b/Cross-LinkingAdapter.pdf" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://static.igem.org/mediawiki/2014/2/2b/Cross-LinkingAdapter.pdf">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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   Using the same cellulose binding domain on either side of the streptavidin caused problems that lead us to revaluate our approach. Due to the repetitive nature of the sequence and potential homologous recombination, we had many issues with molecular cloning. We changed our approach to using two different cellulose-binding domains with different sequences. The first cellulose binding domain remained the same, but rather than repeating that same sequence on the other side of the streptavidin, we instead used the cellulose anchoring protein cohesin from the organism <i>C. cellulovorans </i>This allowed us to successfully conduct the molecular cloning.
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<h6><b>Figure 2.</b> Sequencing data for the cross-linking protein. The solid green bar indicates a perfect match between our sequence and the expected sequence.The first 1000 base pairs are sequenced in this forward sequence.</center></h6>
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<h6><b>Figure 3.</b> Sequencing data for the cross-linking protein. The solid green bar indicates a perfect match between our sequence and the expected sequence.The last 1000 base pairs are sequenced in this reverse sequence. This in combination with the perfect sequencing of the first 1000 base pairs shows our construct matches the CBD-Streptavidin-CBD protein exactly.</center></h6>
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   After obtaining a good sequence and inducing the protein with IPTG, we used a procedure similar to a western blot to test for functionality. After dotting the protein on bacterial cellulose and incubating with skim milk, unbound proteins were washed off with TBS buffer. The cellulose was then incubated with Biotin (5-fluorescein) conjugate which would  bind to the streptavidin domain. While our results are still inconclusive, if we can detect fluorescence on the cellulose sheet, we will know that the cellulose binding domains are functional. The biotin conjugate will only bind to the streptavidin domain which will only be present on the cellulose sheet if the binding domains are functional.  
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  <div class="sub4"><a href="work/PUT-PDF-REFERENCE-HEREpdf"><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 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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1. Linder M <i>et al.</i> (1996) The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose. <i>PNAS</i> 93: 12251-12255. PMID: <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC37976/?page=1" target="_blank">PMC37976</a>.<br><br>
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2. Chivers CE <i>et al.</i> (2011) How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer. <i>Biochem.J.</i> 435: 55-63. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21241253" target="_blank">21241253</a>.</div>
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Built atop Foundation. Content &amp; Development &copy; Stanford–Brown–Spelman iGEM 2014.
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Latest revision as of 00:31, 18 October 2014

Stanford–Brown–Spelman iGEM 2014 — Cellulose Acetate

Approach & Methods


Figure 1. An illustration of cellulose binding domains cross-linking cellulose fibers with a streptavidin domain in the middle. The biosensing cell is expressing a biotinylated AviTag which will bind to the streptavidin .


Our initial approach was to use the cellulose binding domains from C. cellulovorans (part BBa_K863111) on either side of the streptavidin domain (part BBa_K283010) under a T7 promoter in the PSB1A3 backbone.We also included a His-Tag for protein purification. The protein is then expressed in E. coli . Once purified, the cross-linking protein is tested on bacterial cellulose we grew in our lab from the organism G. hansenii. By dotting the protein on the cellulose, the cellulose binding domains will bind to the cellulose fibers and leave the streptavidin domain unbound and ready to bind biotin.
Results
Using the same cellulose binding domain on either side of the streptavidin caused problems that lead us to revaluate our approach. Due to the repetitive nature of the sequence and potential homologous recombination, we had many issues with molecular cloning. We changed our approach to using two different cellulose-binding domains with different sequences. The first cellulose binding domain remained the same, but rather than repeating that same sequence on the other side of the streptavidin, we instead used the cellulose anchoring protein cohesin from the organism C. cellulovorans This allowed us to successfully conduct the molecular cloning.


Figure 2. Sequencing data for the cross-linking protein. The solid green bar indicates a perfect match between our sequence and the expected sequence.The first 1000 base pairs are sequenced in this forward sequence.


Figure 3. Sequencing data for the cross-linking protein. The solid green bar indicates a perfect match between our sequence and the expected sequence.The last 1000 base pairs are sequenced in this reverse sequence. This in combination with the perfect sequencing of the first 1000 base pairs shows our construct matches the CBD-Streptavidin-CBD protein exactly.
After obtaining a good sequence and inducing the protein with IPTG, we used a procedure similar to a western blot to test for functionality. After dotting the protein on bacterial cellulose and incubating with skim milk, unbound proteins were washed off with TBS buffer. The cellulose was then incubated with Biotin (5-fluorescein) conjugate which would bind to the streptavidin domain. While our results are still inconclusive, if we can detect fluorescence on the cellulose sheet, we will know that the cellulose binding domains are functional. The biotin conjugate will only bind to the streptavidin domain which will only be present on the cellulose sheet if the binding domains are functional.
References
1. Linder M et al. (1996) The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose. PNAS 93: 12251-12255. PMID: PMC37976.

2. Chivers CE et al. (2011) How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer. Biochem.J. 435: 55-63. PMID: 21241253.
Built atop Foundation. Content & Development © Stanford–Brown–Spelman iGEM 2014.

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