Revision as of 09:03, 1 August 2019

Introduction

pT7-CBDcipA-Magainin 2

Figure Y. Mechanism of action

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.

Also 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 with methylated DNA.

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CBDcipA and sfGFP 3D structure

Figure Y. 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 from the left, W118, R112, D56, H57 and Y67, thought to be the surface which interacts strongly with cellulose.

Figure Y. Crystal structure of sfGFP with a resolution of 1.4 Å which were solved by [http://www.ncbi.nlm.nih.gov/pubmed/?term=16369541 Pédelacq et al. 2006]. PDB code 2B3P. In red the chromophore can be seen. Excitation wavelength: 485 nm, emission wavelength: 510 nm

Expression system

The part has a very strong expression with a T7 promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). Both this part and the part were sfGFP was changed for AsPink (BBa_K3182000) showed great expression.

Figure B. Benchling screenshot of the expression system. This expression system leads to a high expression and therefore protein yield.

Cellulose binding domain from Clostridium thermocellum fused to Homo sapiens antimicrobial peptide LL-37. Expression of this biobrick showed no toxic effects towards the host.

Cellulose binding domain from Clostridium thermocellum fused to Staphylococcus genus phage endolysin.

Sequence and Features