Revision as of 15:15, 21 July 2019

Introduction

CBDcipA-GS+Thrombin linker
A cellulose binding domain (CBDcipA) from Clostridium thermocellum Cellulose scaffolding protein (CipA) which can be used to purify or attach proteins to cellulose, this part has a sfGFP fused to the CBDcipA. The part also has a flexible GS-linker (-GGGGSGGGGS-) with a thrombin site (-LVPRGS-, thrombin RS) added at the end, clevage with thrombin will add one glycine and one serine to the N-terminal of the C-terminal fusion protein of the CBDcipA.

This part was heavily inspired by iGEM Imperial 2014 and iGEM Ecuador 2018 which both confirmed terminal choice for the fusion protein and buffers which could be used to purify this part.

Assembly techinique used

Added a BamHI recognition sequence (BamHI RS) to enable changeable fusion protein to the CBDcipA. BamHI was chosen because its RS codes for one glycine and one serine, fitting it to the end of the thrombin site, BamHI can also be used on dam/dcm methylated DNA, which is crucial to this part.

For our method "pink-white screening" utilizing a chromoprotein to assemble this part with fusion proteins please see: BBa_K3182100.
The expressional system used to characterize this part was a combination of: BBa_I719005 BBa_K1758100.

Mechanism of action

Figure X. Crystal structure of CBDcipA with a resolution of 1.75 Å which were solved by [http://www.ncbi.nlm.nih.gov/pmc/PMC452321 Tormo et al. 1989]. PDB code 1NBC. In red, W118, R112, D56, H57 and Y67, thought to be the surface which interacts strongly with cellulose.

Figure Y. Mechanism of action

Usage and Biology