Team:StanfordBrownSpelman/Cellulose Cross Linker

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.

Approach & Methods

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.

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.

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.