Revision as of 13:40, 6 October 2019

Introduction

pT7-CBDcipA-LysK

Figure 1. Mechanism of action. The CBDcipA-fusion is attached to a polysaccaride material. 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 material as a bandage, the peptide/enzyme can be released into a wound by native human thrombin.

This part consists of a carbohydrate binding domain (CBD) from Clostridium thermocellum (C. 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.

Protease site and use

The thrombin site was added to enable the ability to release the fusion protein down into skin wounds. Because of our integrated human practice we learned that infection span much deeper into wounds that we thought. Simply attaching the CBD-fusion protein to a carbohydrate material wouldn't make the fusion protein reach far into the wound. The thrombin site was also chosen because of thrombins endogenous occurrence in humans.

Assembly compabilities

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 a cost-effective enzyme and is unaffected by methylated DNA.

CBDcipA crystal structure

Figure 1. 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 polysaccarides.

Important molecular faces

CBDcipA is composed of a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. It further contains conserved residues exposed on the surface which map into two clear surfaces on each side of the molecule. One of faces mainly contains planar strips of aromatic and polar residues which may be the carbohydrate binding part. Further aspect are unknown and unique with this CBD such as the other conserved residues which are contained in a groove.

Carbohydrate binding domain specificity

Since the CBD is from the cellusome of C. thermocellum some researches call it a cellulose binding domain. However, iGEM19 Linköping noticed that this domain could also bind to different sources of polysaccaride materials. This serves as a domain for iGEM19 Linköpings modular bandage, where the polysaccaride material can be changed for anything and not exclusively cellulose.

The choice of carbohydrate binding domain

iGEM Linköping 2019 choose CBDcipA due to many other iGEM teams exploring the possibilities of this domain. Our basic design was influenced by [http://2014.igem.org/Team:Imperial iGEM14 Imperial], [http://2015.igem.org/Team:edinburgh iGEM15 Edinburgh] and [http://2018.igem.org/Team:ecuador iGEM18 Ecuador]. Purification and where to place the fusion protein (N- or C-terminal) was determined by studying the former projects. CBDcipA also originates from a thermophilic bacteria which further increases the domains applications.

Expression system

The part has a 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]).

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.

Antimicrobial Agent - LysK

Bacteriophage endolysins digest the peptidoglycan wall of bacteria. The LysK is an endolysin targeting gram+ bacteria. It has two catalytical domains known as CHAP (cysteine-and histidine dependent amidohydrolase/peptidase) and a central amidase-2 domain together with a cell binding domain. The CHAP domain which is the truncated part expressed in this year’s LiU iGEM (more specifically CHAP C1-C162) team still has lytic activity against staphylococcus. The C1 to C162 amino acids are required for full endopeptidase activity

The endolysin is designed to battle the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. family of pathogens (ESKAPE). ESKAPE is a family(ies) of bacteria which has multiple understrains that has evolved resistance to the most commonly used antibiotics.

Antimicrobial activity of CHAP

Sequence and Features