Revision as of 16:14, 25 August 2019

Introduction

pT7-CBDcipA-AsPink

Figure Y. Mechanism of action. The CBDcipA-fusion is attached to cellulose. By adding thrombin from any source the fusion protein will be cleaved and the C-terminal fusion protein will be released into the solution. By changing the fusion protein to an antimicrobial peptide/enzyme, and using the cellulose as a bandage, the peptide/enzyme can be released into a wound by native human thrombin.

This part consists of a cellulose binding domain (CBD) from Clostridium thermocellum cellulose scaffolding protein (CipA) and is a central part Clostridium thermocellum's cellusome. The CBD-fusion were fused using a flexible GS-linker (-GGGGSGGGGS-). A thrombin cleavage site (-LVPRGS-) was added to the end of the linker and its breakage will leave a glycine and serine attached to the N-terminal of the fusion protein.

An internal BamHI recognition sequence (RS) has been added to enable changeable fusion proteins. BamHI was chosen because its RS codes for glycine and serine, fitting it to the end of the thrombin site. It is also cost-effective enzyme and is unaffected by methylated DNA.

This part uses an expression system 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]).

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.

Expression system

The part has a very strong expression with a T7-RNA-polymerase 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. The T7-RNA-polymerase promotor is followed by a T7 g10 leader sequence which enhances the binding to the 16S ribosomal RNA. After the leader sequence a poly A spacer is found, which has been shown to increase translation in vitro. Before the start codon a strong RBS, g10-L, followed by an AT-rich spacer can be seen, which will slightly increase translation of the following gene.

Usage and Biology

Figure 1. E. coli BL21 cells expressing this biobrick, incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.

Figure 2. Lysated (via sonication) BL21s which are expressing the biobrick. It is lysate from the culture above in figure 1. Incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.

Figure 3. Centrifuged lysate of BL21 culture which express the biobrick. It is the same culture as figure 1 and 2. A pellet of non-lysated bacteria can be observed.

Figure 4. Lysate (via sonication) from BL21 E. coli was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol.

Figure 5. Lysate (via sonication) from BL21 E. coli was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol. To the right is Epiprotect that has not been incubated with AsPink-CBD lysate.