Once we produced all of our enzymes, we realised that they freely diffused throughout the paper. This meant that we could not concentrate the colour change or completely control where the bioactive zone would be. With this in mind we thought about Cellulose Binding Domains (CBDs) as they can be fused to proteins while also binding to the cellulose in paper1.
The problem with this method of immobilisation is that upon fusion to CBDs, we cannot be sure that the enzyme will fold correctly. The CBDs are large proteins which, when fused to an enzyme may interfere with its normal folding pattern. In an attempt to maximise the chances of producing correctly folded proteins, we fused our suite of 5 CBDs to both the N and C terminals of each of the genes for the detection enzymes.
In order to visualise the fusion arrangements that allow correct protein folding, each CBD was fused to each terminal of RFP. The cultures which had red fluorescence were assumed to have correctly folded proteins, and the cultures with no fluorescence were the result of either no protein or mis-folded protein. By confirming the presence of protein of the correct size on an SDS PAGE gel we could conclude which CBDs at which positions are likely to interfere with protein folding.
The use of the RFC25 biobrick assembly method made the fusion of these proteins possible. Unlike RFC10, using the RFC25 assembly standard creates a 6-nucleotide scar that keeps the second protein in the correct reading frame2. This means that protein fusions where both proteins are transcribed and translated correctly can be engineered.
The CBDs which used were sourced from the 2014 Imperial iGEM team.
The Cellulose Binding Domain of Endo-beta-1,4-glucanase A (CBDcenA) from Cellulomonas fimiwith an endogenous C terminal linker sequence3.
The double cellulose binding domain (dCBD) consists of two CBDs from the cellobiohydrolases of Trichoderma reesei held together by an internal linker sequence4. This CBD also has an endogenous N terminal linker sequence.
The Cellulose Binding Domain of Exoglucanase (CBDcex) from Cellulomonas fimiwith an endogenous N terminal linker sequence5.
The Cellulose Binding Domain of from the cellulose binding protein gene of Clostridium cellulovorans (CBDclos)6.
The Cellulose Binding Domain from the Cellulosomal-scaffolding protein (CBDcipA) from Clostridium thermocellum. It includes both an N and C terminal linker sequence7.
The sequence for CBDcipA is an improvement on Imperial’s CBDcipA (BBa_K1321014) since the illegal sites making it RFC25 incompatible were removed.
Overall, this design of a suite of CBDs fused to detection enzymes at both terminals allows for modularity, reusability and interchangeability of standard parts.
1Boraston, A. B., Bolam, D., Gilbert, H., & Davies, G. J. (2004). Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem. J, 382, 769-781.
2 Anderson, J., Dueber, J. E., Leguia, M., Wu, G. C., Goler, J. A., Arkin, A. P., & Keasling, J. D. (2010). BglBricks: A flexible standard for biological part assembly. Journal of biological engineering, 4(1), 1-12.
3Din, N., Forsythe, I.J., Burtnick, L.D., Gilkes, N.R., Miller, R.C. Jr, Warren, R.A., Kilburn, D.G. (1994). The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding. Molecular Microbiology, 11, 747-755.
4Linder, M. and Teeri, T.T. (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.
5Ong, E., Gilkes, N.R., Miller, R.C.Jr., Warren, R.A. & Kilburn, D.G.(1993). The cellulose-binding domain (CBD(cex)) of an exoglucanase from Cellulomonas fimi: production in Escherichia coli and characterisation of the polypeptide. Biotechnology and Bioengineering, 42, 401-409.
6Shoseyov, O., Takagi, M., Goldstein, M.A. & Doi, R.H. (1992). Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A. PNAS, 89, 3483-3487.
7Carrard, G., Koivula, A., Soderlund, H. & Beguin, P. (2000). Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose. PNAS, 97, 10342-10347.