Difference between revisions of "Part:BBa K1321340"

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Double CBD (dCBD) with N-terminal linker

Double cellulose binding domain (dCBD) using two cellulose binding domains from Trichoderma reesei cellobiohydrolases, with an N-terminal linker and internal linker sequence between the two domains which is derived from the endogenous cellobiohydrolase linker sequence.

Please see below for AutoAnnotator information about the final protein product.

Usage and Biology

This part is based on the double cellulose-binding domain construct (CBDcbh2-linker-CBDcbh1) synthesised and characterised by Linder et al [http://dx.doi.org/10.1074/jbc.271.35.21268 (1)] who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.

The two CBDs are from the fungus T. reesei (Hypocrea jecorina) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID [http://www.uniprot.org/uniprot/P62694 P62694]; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID [http://www.uniprot.org/uniprot/P07987 P07987] (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder et al [http://dx.doi.org/10.1074/jbc.271.35.21268 (1)]. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the [http://www.cazy.org/CBM1.html CBM family 1]. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder et al [http://dx.doi.org/10.1074/jbc.271.35.21268 (1)]; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.

The binding ability of this CBD to bacterial cellulose was characterised when fused to sfGFP, relative to other CBDs fused to sfGFP. The binding ability was represented by the percentage fluorescence remaining from the sfGFP-CBD fusions bound to bacterial cellulose discs, when subjected to various washes (protocol [http://2014.igem.org/Team:Imperial/Protocols here]). It was determined that the dCBD-sfGFP fusion had the greatest binding ability in comparison to four other CBDs fused to sfGFP after three washes with both dH2O and 70% EtOH (see first two graphs). When washed with PBS and 5% BSA it had on average the third greatest ability to bind bacterial cellulose (third and fourth graphs below).

As part of our project, we needed to assay the metal binding capability of Phytochelatin (a general metal binding protein) fused to different CBDs (cellulose binding domains). The parts used for this assay are Phytochelatin+CBDcex, Phytochelatin+dCBD, CBDcipA+Phytochelatin, Phytochelatin alone and sfGFP+dCBD wash (only one wash).

Phytochelatin fused to our CBDs were bound onto cellulose that were dried in the bottom of 96-well plates and tested against 3 different metals (nickel, copper, zinc). First, the fusion protein cell lysate was incubated overnight in the cellulose wells. Following this, the metal salt solutions are added in excess into the wells. Finally, an EDTA step removes the bound metal ions into solution, and the metal concentration in solution is quantified by mass spectrometer. Multiple washes with PBS and water were done between each binding step, ensuring that the metal ions that are measured were released from the phytochelatin.

To evaluate if these CBD fusions were reusable, we re-applied metal ion solutions onto the same wells with the CBD fusions adhered. We washed the wells as before, then eluted with EDTA in the same manner. The results from the each elutions are shown below, with the final graph comparing between the first and second wash. We found along the way our bacterial cellulose alone had some metal chelating properties, as was also confirmed by our filtering set up with the dCBD-phytochelatin, but the second elution seems to show less background noise as the EDTA disrupts cellulose's natural binding to the metal ions. The full table of results are shown below as well. The full assay protocol can be found as ‘Metal binding assay protocol’ at this link: http://2014.igem.org/Team:Imperial/Protocols

To read more about this assay and explanation of these results, please see: http://2014.igem.org/Team:Imperial/Water_Filtration

References

1. Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full