Team:StanfordBrownSpelman/Cellulose Cross Linker

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<div class="sub4"><a href="https://docs.google.com/document/d/1-BS2AXdxk_gbYPC2qc5T6e1L7qtnXg4DxsOF-aoxIQg/edit?usp=sharing" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="https://docs.google.com/document/d/1-BS2AXdxk_gbYPC2qc5T6e1L7qtnXg4DxsOF-aoxIQg/edit?usp=sharing">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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<div class="sub4"><a href="http://docs.google.com/document/d/1-BS2AXdxk_gbYPC2qc5T6e1L7qtnXg4DxsOF-aoxIQg/edit?usp=sharing" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/25/SBS_iGEM_2014_download.png"></a><a href="http://docs.google.com/document/d/1-BS2AXdxk_gbYPC2qc5T6e1L7qtnXg4DxsOF-aoxIQg/edit?usp=sharing">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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Revision as of 05:44, 17 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 .


Results
Our initial approach was to include two identical cellulose-binding domains on either side of the streptavidin domain. 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. 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.
References
1. M Linder and T T Teeri (1996) The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose. PNAS 122251 PMID: 24136966.

2. Claire E. CHIVERS, Apurba L. KONER, Edward D. LOWE and Mark HOWARTH (2011) How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramerBiochem.J. 55 PMID: 2981802.
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