Revision as of 11:03, 20 October 2019

Introduction

pT7-CBDcipA-LL-37

Figure 1. Mechanism of action for Novosite. 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). This binding domain is a central part of Clostridium thermocellum's cellusome and has a strong affinity for cellulose. The CBD was fused to another protein using a flexible GS-linker (-GGGGSGGGGS-)in order to attach this complex to a polysaccaride material. 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. The main mechanism of iGEM19 Linköping's project can be seen in Figure 1.

Protease site and use

The thrombin site was added to enable the ability to release the fusion protein down into skin wounds. Thanks to our integrated human practice we learned that infections span much deeper into wounds that we thought. Simply attaching the CBD-fusion protein to a carbohydrate material would not enable the fusion protein to reach far into the wound. The thrombin site was also chosen because of thrombin's endogenous existence in humans.

Assembly compabilities

An internal BamHI recognition sequence (RS) has been added to enable interchangeable fusion proteins to the CBD. 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. BamHI is a part of the RFC21 standard.

CBDcipA crystal structure

Figure 2. 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, which can be seen in Figure 2. It further contains conserved residues exposed on the surface which map into two clear surfaces on each side of the molecule. One of the faces mainly contains planar strips of aromatic and polar residues which may be the carbohydrate binding part. Further aspects are unknown and unique to 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 research labeled 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 exchanged for other/similar materials and not exclusively cellulose.

The choice of carbohydrate binding domain

iGEM Linköping 2019 chose CBDcipA due to the fact that many other iGEM teams had explored 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 domain's applications.

Expression system

The part has a very strong expression with a T7-RNA-polymerase promotor (BBa_I719005), seen in Figure 3, as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in Escherichia coli (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 3. 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.

Fusion protein bound to the carbohydrate binding domain

LL-37 is an antimicrobial peptide found in humans, and consists of 37 amino acids. LL-37 can create aggregates in solution and lipid bilayers and is therefore somewhat protected from protease degradation. Due to LL-37’s positive charge, it can easily associate with negatively charged membranes containing phospholipids. LL-37’s 37 amino acids form an alpha helix when interacting with membranes. When the α-helix is formed the hydrophobic residues are segregated unilaterally which activates the membrane penetrating ability to take action. The penetration results in transmembrane pores which destabilize the outer membrane of gram-negative bacteria.

The peptide 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 substrains that has evolved resistance to the most commonly used antibiotics.

Usage and Biology

The desired function of this construct is that the CBD will allow a more efficient expression of antimicrobial peptides by being a hydrophilic anchor to the bactericidal peptide or enzyme. The CBD can be used for easy purification of the fusion protein and via thrombin cleavage of the linker, yield the peptide or enzyme only. In iGEM19 Linköping's project the thought was to create an antimicrobial bandage by having a polysaccharide based bandage with CBD-antimicrobial agents bound to it. When applying the bandage to a patient, the patient's blood (containing thrombin) will release the peptide or enzyme into the wound (thrombin sprays used in hospitals can be used as well) leading to the activation of the peptides/enzymes and enabling them to reach the bacteria, thus eliminating the infecting pathogens.

Expression, purification and protease treatment

The antimicrobial agents which iGEM19 Linköping used were all expressed the same way. Early expression experiments showed great promise but with low yields. The early experiments were done with standard E. coli BL21 (DE3) expression, cultures were grown to an optical density at 600 nm (OD₆₀₀) of 0.4-1.0 in varying volumes and induced with 0.5 mM IPTG, and let to express the agents for 16 hours in 18 °C. Harvest of the cells was done at 3500 rpm for 30 minutes.

After harvest the cells were re-suspended in phosphate buffered saline (PBS, NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 10 mM, KH₂PO₄ mM) buffer or carbohydrate binding module buffer (CBM-buffer, Tris–HCl pH 7.0, 20 mM NaCl, 5 mM CaCl₂) and sonicated for 6 minutes, 30 % amplitude and 30 seconds on, 30 seconds off. The sonicated bacteria were then centrifuged at 12000 g for 15 minutes. The soluble fraction was then purified by attaching the CBD-fusion to cellulose. This showed very faint bands on SDS-PAGE analysis.

Antimicrobial activity agents in different states

The experiment below used E. coli BL21 (DE3) in concentrations of 10000 CFU/mL. This was done in order to get starting cultures of 0 OD₆₀₀. Using spectrometry to measure the time until the bacteria started growing, instead of showing the killing capability on high optical density cultures, mimicked early stage wounds. This also showed many other interesting things, which can be seen in the results below. One being that an equilibrium settled in lower than for the negative control, meaning even though the antimicrobial agents could not inhibit the growth fully, it was slowed and the stationary phase was reached earlier (Figure 5, 6B).

Figure 4 CBD-LL37 was tested against an E. coli BL21 (DE3) 0 OD₆₀₀ culture. Unfilled boxes is a negative control containing 160 µL E. coli BL21 (DE3) and 40 µL water. The rest of the samples in the graph represent a dilution series of CBD-LL37 at the same volumes as the negative control. The media used was low salt LB (0.4 g/L NaCl). The error bars represent the mean ± SD from six technical replicates.

CBD bound agents

An experiment to test the antimicrobial activity of our agents still bound to the CBD was conducted, which can be seen in Figure 4. This was done in order to see if the absence of thrombin cleavage would yield inactive agents. The CBD-bound agents were purified like above method mentions. Six technical replicates of E. coli BL21 (DE3)(160 µL in low salt LB-media, 0.4 g/L NaCl) were added to a 96-well plate by Eppendorf. To this 40 µL water was added to function as a negative control (called negative control in Figure 4). To 6x6 wells a concentration gradient was added in the same way, always adding 160 µL (in low salt LB-media) E. coli BL21 (DE3) and 40 µL of the unbound agents in water, diluted to the concentrations in Figure 5. The experiment was run for 16 hours in 37 °C and before each measurement a quick shake (200 rpm, 10 seconds) was done. The absorbance at 600 nm was measured every hour.

Importance of testing the CBD-bound agents

High amounts of the CBD-bound agent could still inhibit growth of E. coli BL21 (DE3), meaning if thrombin is not present in the wound the bandage would still yield a antimicrobial effect, this can further be seen in Figure 4. This also explains the trouble when expressing the CBD-agents and why they can be found in the insoluble fraction.