"
Team:StanfordBrownSpelman/Cellulose Cross Linker
From 2014.igem.org
(Difference between revisions)
| Line 79: | Line 79: | ||
<h6> | <h6> | ||
</h6> | </h6> | ||
| + | |||
</div></div> | </div></div> | ||
<div class="small-9 small-centered columns"><br><center><img src=""><br> | <div class="small-9 small-centered columns"><br><center><img src=""><br> | ||
| - | <h6>< | + | <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 then express the cross-linker protein in <i> E. coli </i>. We included a His-Tag to purify the protein. 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, but ready to bind biotin.</center></h6> |
</div> | </div> | ||
<div class="row"> | <div class="row"> | ||
| Line 89: | Line 90: | ||
<h6> | <h6> | ||
| + | |||
| + | |||
| + | |||
| + | <h6> | ||
<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> | <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> | ||
</h6> | </h6> | ||
| Line 153: | Line 158: | ||
<!-- ====== Additional Information ====== --> | <!-- ====== Additional Information ====== --> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<!-- ====== Cell Spacer 6 ====== --> | <!-- ====== Cell Spacer 6 ====== --> | ||
Revision as of 16:08, 17 October 2014
Cellulose Cross-Linker
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.
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.
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 then express the cross-linker protein in E. coli . We included a His-Tag to purify the protein. 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, but ready to bind biotin.
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.
