Latest revision as of 19:23, 14 October 2020

Introduction

pT7-CBDcipA-AsPink

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.

Usage and Biology

Figure 4. A: E. coli BL21 (DE3) cells expressing the biobrick, incubated for 16 hours at 16 °C at 80 rpm in 1 L of LB-miller. B: Lysated (via sonication) BL21s which are expressing the biobrick. It is lysate from the culture above in figure 4. Incubated for 16 hours at 16 °C at 80 rpm in 1 L of LB-miller. C:Centrifuged lysate of BL21 (DE3) culture which expresses the biobrick. It is the same culture as figure 4 and 5. A pellet of non-lysated bacteria can be observed.

A pink colony from an agar plate was selected and cultured in 10 mL LB-miller for 16 h at 37 °C, 180 rpm. This culture was afterwards added to 1 L of LB-miller containing appropriate antibiotics which was grown for 16 h, 80 rpm at 16 °C. This result can be observed in figure 4. Approximately 45 mL of the culture was poured to a 50 mL Falcon tube and sonicated which is displayed in figure 5. A 50 mL Falcon tube was later filled and centrifuged a pink pellet was formed (seen in figure 6).

AsPink bound to cellulose bandage

Epiprotect® (microbial cellulose bandage, S2Medical) was incubated in AsPink lysate from BL21 (DE3) Gold for 30 min. Epiprotect® was later washed with 70 % ethanol three times. To the left is AsPink incubated Epiprotect® and to the right is plain Epiprotect®.

Thrombin Cleavage

In figure 9 bacterial lysate of sonicated E. coli BL21 (DE3) was incubated with cellulose (Whatman CF-11 Fibrous Medium Cellulose Powder) for 1 h on an end-to-end rotator at R.T. The cellulose was saturated with AsPink lysate. The samples were afterwards centriuged at 3000 g in 1 min and the supernatant was removed. In all samples cleavage buffer (20 mM Tris-HCl pH 8.4, 150 mM NaCl and 2.5 mM CaCl₂) was added and three different dilutions of thrombin (Novagen) were added. The dilutions were 0.02, 9.94 and 0.08 units (U). One unit refers to the amount of thrombin needed to cleave 1 mg of protein for 16 h at R.T. in a 200 µL reaction containing the cleavage buffer. The samples were put on an end-to-end rotator overnight (ca 16 h) and left at R.T. The control with only cleavage buffer has a transparent supernatant and a pink pellet of cellulose. The most diluted (0.02 U) has a slightly pinker supernatant and less pink pellet, however the 0.04 U and 0.08 U dilutions have a pinker supernatant and more pale pellet, with 0.08 U dilution being the sample with the most cleaved AsPink displayed by the more intense supernatant.

Figure 6. Lysate (via sonication) from E. coli BL21 (DE3) was incubated with Epiprotect® for 1 h and washed thrice with 70 % ethanol. To the right is Epiprotect® that has not been incubated with AsPink-CBD lysate.

Figure 5. Lysate (via sonication) from E. coli BL21 (DE3) was incubated with cellulose fibers (Whatman CF-11 Fibrous Medium Cellulose Powder). Saturated cellulose was incubated with different units (U) of thrombin (Novagen).The leftmost is cellulose with cleavage buffer and the control contains bound AsPink with cleavage buffer. All tubes have been incubated overnight in R.T on an end-to-end rotator.