Demonstrating our Concept

Introduction

The purpose of this demonstrate page is to demonstrate that our project’s concept of an antimicrobial bandage containing a drop mechanism and a polysaccharide material, in this case cellulose (Figure 1). These are condensed results to prove our project. To read further about our other various and more detailed results below, please visit our results page and to further read about our biobricks please visit our parts page.

About our Project - Novosite

The base of our project is to reduce the need for systemically administered antibiotics. Attaching antimicrobial agents in the form of peptides and enzymes to a bandage treats the wounds locally and avoids a systemic antibiotic dose. For this, a carbohydrate binding domain (CBD) was used to bind our antimicrobial fusion protein to a cellulose bandage. Due to the CBD’s various binding capacities it can also be bound to future candidates, such as chitinosan, alginate and cotton. However, when researching the currently used bandages in wound care we came across a study performed by the Burn Care Center (BRIVA) in which they concluded that cellulose was just as effective as silver bandages, it was also reported to reduce patient's pain scoring [1]. By meeting with and integrating BRIVA in our project we continued to discuss with them the different aspects of cellulose. During one visit, we were allowed to watch an operation in which the bacterial cellulose Epiprotect®2117 (S2Medical) cellulose bandage was used. We also spoke to staff and patients of BRIVA and learned that when changing wound dressings the dressings tended to stick to the wound in later stages, causing pain. This was further strengthened in our interview with Lasse Gustavsson, a survivor of a burn trauma. BRIVA also provided us with Epiprotect®2117 which we used in multiple experiments. We also talked to the Center for Disaster Medicine and Traumatology (KMC), currently testing the same Epiprotect®2117 cellulose bandage. Further on, we payed a visit to the production company S2Medical and met with their COO, Mårten Skog.

After meetings with BRIVA and KMC we realized that bacteria were prominent deep in the wounds. By incorporating a thrombin cleavage site in our construct, the agents could be released, thereby increasing their activity and helping them reach microorganisms in different depths of the wound. Thrombin can be given via recombinant sources or from the patient's own blood. The antimicrobial agents would then reduce the need for antibiotics and fight off antibiotic resistant strains of bacteria. Our chosen agents were based on the discussions with our community sources.

Figure 1. Mechanism of action for Novosite. The CBD_cipA-fusion protein is attached to a polysaccaride material. By adding thrombin 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 thrombin cleavage.

Expression of antimicrobial agents

SDSpage — **Figure 2:** The theoretical deactivation mechanism. The carbohydrate binding domain (CBD) will act as a hydrophilic anchor inhibiting/deactivating the harmful effect of the agent in its host. This will allow a higher expression of the desired product.

Expression of antimicrobial agents in bacteria is a big hurdle we aimed to overcome. Expression of these types of proteins is difficult due to their toxic effect on the bacteria. By analyzing previous iGEM teams which have used CBDs for various reasons (Imperial 2014, Edinburgh 2015 and Ecuador 2018) together with scientific articles we realized that the use of a hydrophilic domain, such as a CBD, reduced the agent’s toxicity [2]. By choosing to use a CBD, we could also use the CBDs binding capacities. We could bind our agents to a polysaccharide material not only for purification purposes but also in order to create a bandage. The CBD would therefore act both as an anchor to retain the agents from harming the chassis, as well as a binding domain for purification and binding of the agents to the cellulose bandage.

Our CBD of choice, CBD_CipA, was inspired by the previously referenced iGEM-teams: Imperial 2014, Edinburgh 2015 and Ecuador 2018. CBD_CipA originates from Clostridium thermocellum's cellusome. We also used sfGFP fused to the CBD as a reporter of affinity (BBa_K3182108) during our first pilot studies. We could therefore use the sfGFP to follow the CBD’s binding capacity and all the purification steps before moving on to our agents. We discovered that the CBD binds strongly and that we could wash our bandage in sterilizing solutions such as ethanol.

Demonstrate

Verifying our construct

Here we provide proof of successful expression of agents in E. coli. The agents could be found in small amounts in the soluble fraction after sonication. Most of the fusion proteins could be found in the insoluble fraction (bacterial membranes), where we used Triton X-100 1% (non-ionic detergent) to solubilize the pellets and release our agents.

Figure 3 proves that the agents could be expressed (Figure 3C), that the agents could be bound to cellulose and cleaved from the cellulose with thrombin, (Figure 3A and B). Our agents can be seen in the insoluble fraction in Figure 3C.

In Figure 3B, CBD-Pln1 (BBa_K3182107) had been purified with cellulose and afterwards eluted with water, proving its binding capacity and its relative purity.

Figure 3A, CBD-Pln1 has been cleaved by thrombin and the cellulose was run in as well the cellulose had both cleaved and uncleaved CBD-proteins bound to it. The figure also displays Pln1 not bound to the CBD after thrombin cleavage, i.e. released from the CBD domain.

Attatching our Construct

As discussed earlier, a bandage consisting of bacterial cellulose (Epiprotect, S2Medical) was chosen because of its positive characteristics, its existing use in the relevant medical fields, criteria it has fulfilled as a novel bandage and based on information from integrated human practice. Our description page further explains the subject regarding bacterial cellulose and describes it as being more efficient in wound healing than the commonly used silver bandages. This was further strengthened through the human practice feedback where we understood silver bandages to be negatively viewed and expensive. Cellulose is cheap to produce and existing domains with high affinity for cellulose has been discovered and used already.

The CBD has been studied in great detail and has been featured in research in- and outside of iGEM. The fluorescent protein sfGFP fused to the CBD was employed as a reporter of affinity during the first pilot studies, so that the CBD binding capacity and all the purification steps could easily be observed and followed before moving on to our agents. There a clear binding of CBD-sfGFP to the cellulose bandage which can be observed in Figure 4 and 5. In figure 4, an almost transparent negative control cellulose bandage (Epiprotect, S2Medical) can be compared to the same bandage that has been incubated in CBD-sfGFP.

In figure 5, the cellulose bandage (Epiprotect, S2Medical) was incubated with CBD-sfGFP (Figure 5A-C) leading to the CBD-sfGFP being bound to the cellulose. This is confirmed by the narrow/green spectrum capture (Figure 5B) in which the laser excites green fluorophores (485 nm) resulting in a clearly green cellulose. Measurements were done (1-4, Blank) in the green spectrum capture and plotted in the Figure 5C graph proving that the CBD-sfGFP was successfully bound by clear emission peaks at 510 nm. The negative control (Figure 5D-F) was not incubated with CBD-sfGFP. It did not emit green fluorescence as seen in Figure 5E where only noise can be seen. The measurements in the spectra was plotted in Figure 5F where no emission peak at 510 can be seen.

Figure 5: Upper row, Figure 5A-C: CBD-sfGFP incubated cellulose. Bottom row, Figure 5D-F: Negative control. Figure 5A and D are broad spectrum captures of the CBD-sfGFP incubated bandage (A) or the negative control which has not been incubated (D). A capture of only the green spectra at 485 was performed in Figure 5B and E where five points were measured, including one blank at the corner where the bandage was not present (1-4, Blank). These five points were plotted against wavelength in C and F to measure emission.

Verification of our mechanism of action

In order to investigate if the thrombin site was functional we utilized our CBD-sfGFP construct (BBa_K3182108). Below (Figure 6) is both a visual and kinetic experiment proving successful thrombin cleavage. After incubating the cellulose, it was washed three times with 70 % ethanol, a step that has been implemented in all experiments in which the bandage has been used.

In the kinetic reading (Figure 6A, thrombin +) cleavage can be confirmed whilst the negative control (Figure 6A, thrombin -) had no release. Here, the maximum effect of the thrombin added was reached after approximately 8 hours with a steep increase. The thrombin concentration used is similar to concentrations found in early wound stages (0.6 µM thrombin).

In Figure 6B, both samples have been treated equally, both in relation to incubation time and buffer storage (except thrombin added in thrombin +). The cellulose and the related 1.5 ml Eppendorf tube (thrombin -) is a negative control containing no thrombin. The 1.5 ml Eppendorf tube that the cellulose was kept in is not fluorescent, meaning that there was no passive release of the CBD or indirect cleavage of the linker. The bandage next to the tube has a significantly higher intensity than the experiment to the right. The experiment containing thrombin (thrombin +) shows a clear cleavage of the linker, which can be observed in the 1.5 ml Eppendorf tube's supernatant, whilst the negative control does not have any sfGFP in its supernatant. Not all the sfGFP has been cleaved from the cellulose bandage, possibly because active thrombin molecules ran out. However, there is still an decrease when compared to the negative control. The experiment containing thrombin (thrombin +) shows a clear cleavage of the linker, which can be observed in the tube's supernatant, whilst the negative control does not have any sfGFP in its supernatant. Not all the sfGFP has been cleaved from the cellulose bandage, possibly because active thrombin molecules ran out.

Figure 6: Control of thrombin protease activity. A and B are two separate experiments but represent the same result, that being the thrombin cleavage mechanism of our construct.A: Kinetic experiment of thrombin's protease activity. Bacterial cellulose, with CBD_CipA-sfGFP attached, were analyzed spectrophotometrically. B:Bacterial cellulose was incubated with CBD-sfGFP for 30 minutes on an end-to-end rotator in room temperature. The samples were incubated either in only thrombin cleavage buffer (negative control) or thrombin and thrombin cleavage buffer (thrombin (+)) over-night on an end-to-end rotator in room temperature.

Activity of unbound agents

In order to verify the agents' efficiency after their release from a bandage subsequent to cleavage with thrombin (i.e. the agents are no longer bound to the CBD) we purified them and tested them accordingly in Figure 7 below. This can be compared to the last step in mechanism of action in Figure 1. E.coli and B. subtilis were used as an infection model since they are classified as gram negative and gram positive, respectively.

The experiment presented in Figure 7 acted as a positive control to ensure the antibacterial activity of Novosite’s agents when released from the bandage. The results confirm our theory that the antimicrobial agents will have effect against bacteria after the peptides are cleaved off. Pln1, PlyF307 and CHAP exhibited a high activity against E.coli. Whilst CHAP had a much higher activity against B. subtilis compared to the other agents.

All of the agents tested in Figure 7 had a strong activity against at least gram positive or negative bacteria. This proves that the agents can be used to kill bacteria. The different activities against gram- positive or negative bacteria also show that the Novosite construct can be used modularity to preserve one or the other of these groups, according to preference and goal. By getting promising results from this "positive control" we could further move on to the proof of concept.

To see the result of CBD-bound agent's antimicrobial activity, please see our results page.

Figure 7: Antimicrobial activity of unfused agents. Agents that has been cleaved from the CBD was tested on E.coli (gram negative) and B.subtilis (gram positive). The graph show OD₆₀₀ after 16 hours of growth. The negative control contains thrombin (0.5 U, final concentration 0.6 µM) and thrombin cleavage buffer (40 µL, 20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2) in addition to the cellulose bandage. The agents contain the same thrombin the thrombin cleavage buffer. MIC = minimum inhibitory concentration.

Comparison of Agent Efficacy to Antibiotics

In Table 1 an approximation of our agents efficiency compared to three different types of commonly used antibiotics can be seen. These values are based on the EUCAST's ECOFF database and reflect E.coli antibiotic MIC (minimum inhibitory concentration). As seen in Table 1, all of our agents are more effective than the compared antibiotics and the most effective agents are the lysins.

Table 1. Comparison between the agent's efficiency to antibiotics. The table refers to how many times (x times more efficient) more efficient the agents are to three different antibiotics. Antibiotic MIC values were taken from EUCAST, ECOFF.

Immobilization and release on a cellulose bandage

To prove that the concept of iGEM19 Linköping's project worked, the antimicrobial agents were fused to a cellulose bandage (Epiprotect by S2Medical). The Epiprotect bandage, developed by S2Medical, is used clinically for both chronic wounds and burns, further displaying Novosite's potential as a usable product. By simulating a newly infected wound we tested the cellulose bandage on E.coli BL21 (DE3) at 0 OD₆₀₀.

Thrombin cleavage buffer was added to all wells. The negative control in addition to this, contained a cellulose bandage without any bound agents and with thrombin. In the Pln1/PlyF307 bandage (triangles, Figure 8A and B) the cellulose with bound agents were added without thrombin. The filled boxes in Figure 8 below (Pln1/PlyF307 bandage + thrombin) contained the agents bound to cellulose together with thrombin, recreating the mechanism of action.

Noticeably, the thrombin released agents inhibited the E.coli growth the most in comparison to the other combinations tested. In Figure 8A the non-released agents bound to cellulose exhibited antimicrobial activity. Pln1 fused and unfused had a similar activity with the unfused being slightly more effective. PlyF307 had a strong antimicrobial activity both in a fused and unfused state. The unfused, released PlyF307 completely inhibited the growth of E.coli. These results demonstrate that the of concept of iGEM19 Linköping's main mechanism is functional from start to finish and that these types of wound dressings could be used clinically.

Figure 8. A bacterial cellulose bandage (Epiprotect, S2Medical) was incubated with E.coli BL21 (DE3) 0 OD₆₀₀ cultures. The unfilled boxes indicate a negative control (In both A and B) which contain thrombin, thrombin cleavage buffer and cellulose bandage. The filled boxes indicate a cellulose bandage which has CBD-Pln1 (A) or CBD-PlyF307 (B) bound to it, thrombin and thrombin cleavage buffer. The triangles indicate a cellulose bandage which has CBD-Pln1 (A) or CBD-PlyF307 (B) bound to it and thrombin cleavage buffer. The media used in all wells was low salt LB (0.4 g/L NaCl). In A the error bars represent the mean ± SD from two independent experiments. In B the error bars represent the mean ± SD from three technical replicates.

Thrombin accessibility in wounds

The thrombin used for releasing the agents into the solution had a concentration of 0.6 µM, this was to mimic the concentration found in blood from wounds. According to K.G Mann et al. 2003 the concentration of active thrombin during clotting is several hundred nanomolar, depending on the age of the wound. In the experiment above, the thrombin concentrations used was 600 nM [7]. This hopefully mimics a real wound as closely as possible, while working in vitro.

Conclusion

We demonstrated that our concept; an antimicrobial bandage (Novosite) with bound fusion proteins along with a drop mechanism was successful from start to finish. We could successfully express our fusion proteins by using a CBD and then successfully continue to bind the CBD fusion protein to cellulose. By adding thrombin, we were able to demonstrate that the thrombin cleavage drop mechanism worked and that subsequent to cleavage from the CBD the activity of the agents was increased. Next, we proved that our agents had antimicrobial activity towards both gram positive and gram negative bacteria. Thus, demonstrating that the complete concept of the Novosite bandage worked. We were able to observe the activity of the agents bound to the bandage and released from the bandage. Furthermore, by binding these agents to a CBD, the fusion proteins can easily be expressed and purified directly onto the cellulose, which can in turn be utilized as an antimicrobial bandage when thrombin cleaves and releases the agents - leading to the elimination of bacteria in the wound.

References

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