Results

The results of our project are mainly divided into two sections. These two sections reflect the two main concepts we wanted to try during the project. First, we present the results from our experiments where we attach our carbohydrate binding domain to cellulose as well as other potential wound dressing carbohydrate materials. Secondly, we present the results of how well our antimicrobial agents affect bacterial growth. In this section we also show results of how well the mechanism of action works from start to finish and thereby proving our concept.

Domain affinity

The binding capacity of the CBD was tested and observed in multiple experiments. In order to be able to follow and observe the binding mechanism of our CBD-construct the green fluorescent protein (sfGFP) was used as a replacement for the antimicrobial agents. Thus, a fusion protein containing sfGFP and CBD was created - mimicking the CBD-peptide/enzyme construct.

The binding strength was confirmed by testing different buffers. The results can be viewed in Figure 1A. According to the results, a highly hydrophilic buffer will generate more dissociation between CBD-sfGFP and the cellulose, compared to other less hydrophilic buffers. Figure 1B exhibits a large culture of E. coli BL21 (DE3) expressing CBD-sfGFP. Lysate containing CBD-sfGFP (via sonication, soluble fraction) from E. coli BL21 (DE3) was incubated with Epiprotect for 1 hour and washed thrice with 70 % ethanol. The result can be seen in Figure 1C, showing a strong green color for the cellulose which has been incubated with CBD-sfGFP. This indicates that the CBD has a strong affinity for cellulose.

Figure 1. A: Binding studies of the CBD_cipA-sfGFP bound to bacterial cellulose. Washed three times with either 70 % ethanol, PBS or deionized water. B: Induced culture after 16 hours. E. coli BL21 (DE3) cells were grown in the presence of 25 ug/mL chloramphenicol until an OD600 of 0.8 at 37 °C, and later induced with 0.5 mM IPTG. The induced culture was then incubated in 18 °C for 16 hours. C: Left: CBD_cipA-sfGFP bound to bacterial cellulose (Epiprotect®2117, S2Medical) in form of a thin film, right: bacterial cellulose (Epiprotect®2117, S2Medical) reference. Binding of CBD_cipA-sfGFP was done the same way as the pictures below.

The cellulose bandage (Epiprotect) was incubated with CBD-sfGFP and Figure 2A shows the unwashed bandage. Figure 2B shows the bandage where the lysate containing CBD-sfGFP has been removed. Figure 2C depicts the bandage after one wash with 70 % ethanol for 30 min on an end-to-end rotator. These results show that the CBD has a strong affinity for cellulose.

Figure 2. A: Lysate containing CBD_cipA-sfGFP with bacterial cellulose (Epiprotect®2117, S2Medical) before incubation. B: Lysate (CBD_cipA-sfGFP) bound to bacterial cellulose after incubation at room temperature for 30 minutes on an end-to-end rotator. C: Bacterial cellulose after incubation with 70 % ethanol at room temperature for 30 minutes on an end-to-end rotator. All pictures were taken on a 302 nm UV-table for better visualization of the result.

In Figure 3, a cellulose bandage (Epiprotect, S2Medical) was incubated for 1 h at R.T in CBD-sfGFP lysate from BL21 (DE3) and afterwards washed thrice with 1 mL ethanol 70 %. The Negative control (Figure 3D-F) was washed thrice with 70 % ethanol. The bandages were mounted on a microscopy slide and photographed with a epifluorescent microscope. The relative fluorescent units (RFU) were measured in five places with one being a blank (bottom left corner, Figure 3B and E). The excitation was 485 nm and emission could be plotted in the treatment compared to the negative control (Figure 3C and F). The negative control did not emit due to no CBD-sfGFP being bound, whilst the CBD-sfGFP positive treatment had a clear emission peak matching sfGFP's 510 nm. Figure 3A and D is broad spectrum photos while figure 3B and E is only the green spectrum captured.

Figure 3. Figure 3A 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 3B 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.

In Figure 4, the same BL21 CBD-sfGFP lysate was used and the samples were incubated 30 minutes in room temperature. Both the CBD-sfGFP bound agarose and agarose that was not incubated with CBD-sfGFP were washed in 70 % ethanol once. The agarose with bound CBD-sfGFP displayed a yellow tint in comparison to the agarose without bound CBD-sfGFP which was the original transparent color.

Figure 4. The negative controls refer to the agarose and agarose powder not being exposed to the CBD-sfGFP containing lysate. A & B: Agarose 2,2%. In A the agarose can be observed in normal white light and in B the agarose was put on an UV-table (302nm). C & D: Agarose powder was incubated in CBD-sfGFP containing lysate.

Figure 5. The cotton (A) and Jelonet +paraffin and -paraffin (B) without sfGFP refer to as negative controls. Both pictures were taken with an UV-table (302nm). A:Cotton was incubated in bacterial lysate containing CBD-sfGFP for 30 minutes and both cotton samples were washed with 70 % ethanol before analyzed on the UV-table. B: The Jelonet bandage was either washed with detergents to remove paraffin (-paraffin) or not (+paraffin). CBD-sfGFP -parafin was incubated for 30 minutes and washed with 70 % ethanol three times.

The samples in Figure 5 were incubated with the same BL21 (DE3) lysate for 30 minutes and then washed three times with 70 % ethanol. In Figure 5A cotton was incubated with sfGFP whilst in Figure 5B a bandage called Jelonet was used. Jelonet contains cotton threads that are covered in paraffin and we discovered that the paraffin inhibited the binding of CBD-sfGFP. Therefore, by using detergents the paraffin could be removed prior to the CBD-sfGFP incubation.

Figure 1-5 illustrate the modularity of the CBD-construct proving that it not only binds to cellulose, as proved in Figure 1 and Figure 2, but also to other polysaccharide based materials. By binding CBD-sfGFP to Jelonet, Figure 5B, we aimed to prove that the construct can be bound to already existing materials in the medical field. Jelonet is commonly used for open wounds such as burn wounds. The bandage both provides some antibacterial barrier as well as preventing the wounds from sticking to gauze used on top of Jelonet.

The bacterial cellulose bandage used in Figure 1-3 is named Epiprotect®2117 and is produced by S2Medical. Epiprotect®2117 is currently used for wounds such as burn wounds and chronic wounds. These tests were also performed to prove that the CBD-construct can be used in existing medical products. Epiprotect®2117 was also used in later experiments to observe the thrombin cleavage mechanism.

Purification of CBD-sfGFP

CBD-sfGFP was purified with 2 g cellulose fibers medium length (Whatman, #CF11 medium length cellulose fibers) in a column. In Figure 6 the fluorescence data of all purification steps are collected. The first striped bar is the flowthrough after adding BL21 (DE3) Gold lysate. This is followed by 8 bars in black representing ethanol washes and thereafter 10 blue bars representing water elution steps are depicted. Lastly, two final steps with ethanol and water are illustrated in red and grey respectively.

The different washes of 70 % ethanol were performed to remove unspecifically bound CBD-sfGFP, leading to only bound CBD-sfGFP remaining on the bandage. This can be seen in the bar graph in Figure 6 due to the drastic drop of sfGFP elution which means that 70 % ethanol is not useful for CBD-sfGFP elution. The water washes (dH₂O, blue bars) indicate that an efficient elution of CBD-sfGFP can be performed within the first 4-6 elutions of dH₂O. The last two bars (red - ethanol 70 % and grey - dH₂O) indicate the endogenous fluorescent abilities of each solution. It is minimal for ethanol and none for dH₂O.

The washes and elutions were later analyzed using SDS-PAGE to verify their pureness. These results can be seen under "SDS-PAGE analysis of cleavage and expression"

Figure 6.Purification with 2 g cellulose (Whatman, #CF11 medium length cellulose fibres) in a column. The first striped bar is the flowthrough after adding BL21 (DE3) Gold lysate. The black bars are the RFU of the 70 % ethanol washes. The blue bars are the distilled water (dH₂O) elutions in which 1 mL dH₂O was added until the column stopped dropping. The red and grey ethanol 70 % and dH₂O bars illustrate the possible interfering RFU values of each fluid.

Binding assay of CBD-sfGFP

Figure 7. This graph depicts the fluorescence of CBD-sfGFP lysate after incubation with cellulose CF11. The volume of bacterial lysate remained constant at 700 ul and the mass of cellulose CF11 was varied between 0-100 mg, which can be seen on the x-axis. The fluorescence was plotted on the y-axis and was measured at emission 510 nm with a gain of 40.

Figure 7 illustrates that increasing mass of cellulose CF11 fibers leads to elevated of CBD-sfGFP.

To make the binding assay, Eppendorf tubes with varying mass of cellulose (Whatman, #CF11 medium length cellulose fibres) between 0-100 mg was mixed with a constant volume of CBD-sfGFP lysate (700 uL) and vortexed. The tubes were then attached to an end-to-end rotator for 30 minutes. Thereafter, they were placed in a tube holder until the cellulose powder settled in the bottom and the supernatant containing lysate was clear of cellulose. 100 µL from each of the supernatants was then applied on a plate reader measuring the fluorescence of CBD-sfGFP at 485 nm.

Reporter of successful cleavage and release from the cellulose binding domain

In Figure 8 the release of sfGFP from our bacterial cellulose bandage (Epiprotect®2117, S2Medical) is visualized over time. The approximate area of the cellulose was 1 cm2. The cellulose-CBD-sfGFP were attached to the sides of a 96-well plate using strings. Allowing the spectrophotometer to measure the supernatant in the center of the well, bottom up (ex. 485 nm, em. 510 nm) for 16 hours. The well further contained 200 uL 1X thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl₂). In the positive control experiment, measuring thrombin cleavage (blue), an amount of 0.03 units of human thrombin (Novagen, #69671-3) was added. In the graph (blue), successful release of sfGFP from the CBD can be seen. The maximum amount of fluorescence is reached after about 8 h. In red the negative control experiment can be seen where thrombin buffer was added and the well did not contain any thrombin. No significant amount of fluorescence can be observed. The temperature was set to 37 °C. A visual representation of this can be observed below in Figure 9.

Figure 8. A kinetic experiment of thrombin protease activity. Bacterial cellulose with attached CBD_cipA-sfGFP were analyzed spectrophotometrically.

Figure 9. Visual control of human thrombin protease activity. Bacterial cellulose was incubated with CBD-sfGFP for 30 minutes on an end-to-end rotator in room temperature. The samples were incubated with either only thrombin cleavage buffer (negative control) or thrombin and thrombin cleavage buffer (thrombin (+)) overnight on an end-to-end rotator in room temperature.

To the left in Figure 9 a visual experiment with this part can be seen. After unbound protein had been removed the cellulose was washed three times with 70 % ethanol. To test the activity, 200 uL thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl₂) were added along side 0.03 units of human thrombin (Novagen, #69671-3) to the bacterial cellulose (Epiprotect®2117, S2Medical). To the right in Figure 9, the successful cleavage of CBD-sfGFP can be seen. The cellulose is to the left of the tube where free (cleaved at the thrombin site) sfGFP can be seen. To the left, the control sample can be seen where no sfGFP can is detected in the supernatant. The picture is taken on a 302 nm UV-table for a better visualization of the results. Both samples were incubated in room temperature over-night on an end-to end rotator.

The sample containing thrombin (thrombin (+)) has 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 sfGFP has been cleaved from the cellulose bandage and this is probably due to the thrombin buffer running out of needed content for the thrombin cleavage.

SDS-PAGE analysis of cleavage and expression

E. coli BL21 (DE3) Gold cells were grown in the presence of 25 ug/mL chloramphenicol until an OD600 of 0.8 at 37 °C was reached, and later induced with 0.5 mM IPTG. The induced culture was then incubated in 18 °C for 16 hours. The bacteria was then lysed with sonication at 30 % for 6 minutes. Most of this part could be found in the soluble fraction. The lysate (1 mL) was then incubated with cellulose (CF11) for 30 minutes in room temperature. Four washes with 70 % ethanol was then conducted, all with a volume of 1 mL. Elution of CBD_cipA-sfGFP was done with 1 mL fractions of dH₂O. One replicate was cleaved with thrombin instead. Thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl₂) was added to the Eppendorf tube with the cellulose at a volume of 500 uL, and 0.03 units was then added to the solution. The cleavage was done in room temperature over 16 hours, with inversion of the tube.

These different solutions were applied to an SDS-PAGE to analyze the effectiveness of the purification as well as of the thrombine cleavage. In Figure 10 the resulting gel can be observed.

Figure 10. SDS-PAGE analysis of CBD-sfGFP and thrombin cleavage product. The gel to the left is a 4–20% Mini-PROTEAN® TGX™ by BioRad and the ladder is a PageRuler™ Prestained Protein Ladder. The gel was run at 200 V for 30 minutes. Lane 1 contains the ladder, lane 2 bacterial lysate with CBD-sfGFP, lane 3-6 contain four washes with 70 % ethanol, lane 7-10 contain four elution fractions with deionized water. Lane 11 contains sfGFP subsequent to cleavage with thrombin.

Other bacterial proteins are washed away with the ethanol which can be observed by the other fragments observed in lanes 3-6, all of the proteins can also be observed in the bacterial lysate lane 2. In lanes 7-10 water (dH₂O) elutions can be observed, they also contain some of the other bacterial proteins though they are very faint and the last lane is almost only CBD-sfGFP. These elutions were the same as those in Figure 6. Lane 11 only contains sfGFP which has been cleaved from the CBD-construct, proving a successful cleavage of the linker. The effectiveness of this cleavage is depicted in Figure 9.

Expression in Vibrio natriegens

The antibiotic concentrations used were taken from iGEM18 Marburg. For more information of chemical competent cells, transformation and medium please see the original article: Gibson et al. 2016.

In order to see if CBD-sfGFP worked in Vibrio natriegens using the strain Vmax, pUC19-CBD-sfGFP BBa_K3182108 and pUC19-CBD-pCons-AsPink BBa_K3182100 was heat shocked into Vmax. Thereafter, the bacteria was spread onto LB-Miller V2 agar plates with 200 µg/ml carbenicillin and incubated in 37 °C for 16 hours. Both plates were illuminated on an UV-table at 302 nm light (Figure 11 A). Figure 11 A shows CBD-sfGFP in Vmax, emitts a strong green fluorescence, in comparison to the control CBD-pCons-AsPink in Vmax. This indicates that pUC19-CBD-sfGFP was able to replicate and the protein could be expressed. To measure the protein expression of this part in different bacteria and carbenicillin concentrations. E. coli BL21 (DE3) and Vibrio natriegens , using the strain Vmax, was grown in Falcon tubes to 0.5 OD₆₀₀. Vmax was grown with two different carbenicillin concentrations, 200 and 600 µg/mL, while BL21 (DE3) had 100 µg/mL carbenicillin. The bacteria was induced with 1 mM IPTG and placed in a 96-well plate in 4 replicates with 200 µL per well. A spectrometry experiment was conducted and measured the fluorescence (excitation 470 nm, emission 515 nm) during 28 hours in 37 °C. The results seen in Figure 11 B shows that expression in Vmax with 600 µg/mL carbenicillin gave the highest protein yield. Another important factor was the use of an optimal concentration of carbenicillin (600 µg/mL) for Vmax which retained the plasmid more efficiently than Vmax at 200 µg/mL carbenicillin.

Figure 11. A This biobrick expressed in V.natriegens Vmax was incubated for 16 h in 37 °C on an LB-Miller V2 Agar plate. The control expressed the biobrick BBa_K3182100 and express no fluorescent protein. The figure is illuminated on an UV-table in 302 nm light. B CBD-sfGFP expression in different chassis. The orange line represent Vmax at 600 µg/mL carbenicillin, blue represents Vmax at 200 µg/mL carbenicillin, and green represents E. coli BL21 at 100 µg/mL carbenicillin. The y-axis depicts relative fluorescent units and the x-axis represents the time over 28 hours.

Results of antimicrobial effect

The goal of our project was to functionalize a cellulose wound dressing with antimicrobial agents to help fight infections. We engineered E. coli and V. natriegens to express antimicrobial peptides and enzymes linked to a carbohydrate binding domain (CBD). These fusion proteins were then purified by attaching them to a polysaccharide based material (e.g. cellulose) which after the purification could act as an antimicrobial bandage. The CBD enabled the attachment of antimicrobial agents to the polysaccharide material while it also inactivated the agents when being expressed. Later, when the bandage came in contact with thrombin the antimicrobial agents would be activated. This is due to thrombin cutting the linker between the antimicrobial agents and CBD which contains a thrombin cleavage site.

Expression

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. Our agents can be seen in the insoluble fraction in Figure 12C. Figure 12 proves that the agents could be expressed (Figure 12C), that the agents could be bound to cellulose and cleaved from the cellulose with thrombin, (Figure 12A and Figure 12B.

In Figure 12B CBD-Pln1 has been purified with cellulose and then eluted with water, proving its binding capacity and its relative purity.

In Figure 12A CBD-Pln1 has been cleaved by thrombin and the cellulose was run in this lane which still contained both uncleaved and cleaved CBD-Pln1. The figure also displays Pln1 not bound to the CBD after thrombin cleavage.

Figure 12: SDS-PAGE gel showing bacterial expression. A: CBD-Pln1 purified and cleaved with thrombin run on an SDS-PAGE 4-20 % gel. Lane 1 contains CBD-Pln1 previously bound to cellulose and treated with thrombin, which explains the multiple bands. Lane 2 contains the cleaved Pln1 from lane 1, which was found in the supernatant from the cleavage along with thrombin (not visible). Lane 3 contains a protein ladder called LMW by GE Healthcare. B: CBD-Pln1 run on an SDS-PAGE 4-20 % gel after purification. Lane 1 contains a protein ladder called Precision Plus Dual color by BioRad. Lane 2 contains purified CBD-Pln1 in fraction 1 after elution with water. Lane 3 contains purified CBD-Pln1 in fraction 2 after elution with water. Lane 4-5 are empty. Lane 6 contains E. coli BL21 (DE3) lysate from induced cells which carrie the plasmid expressing CBD-Pln1. C: Bacterial pellet of BL21 (DE3) expressing PlyF307 and Pln1 were lysated and the insoluble fraction was centrifuged and analyzed in this result. Lane 1 contains a GE healthcare LW protein ladder, lane 2 contains the insoluble fraction of PlyF307 expressing culture and lane 3 contains the insoluble fraction of bacteria expressing Pln1.

Antimicrobial activity agents in different states

All experiments below used Escherichia coli BL21 (DE3) and/or Bacillus Subtilis in concentrations of 10 000 CFU/mL to test antimicrobial activity. The usage of the two bacteria verified and explored the agent's activity to both gram negative and positive bacteria, respectively. This dilution was performed in order to get starting cultures of 0 OD₆₀₀. This method used spectrometry to measure the time until the bacteria started growing, mimicking early stages of a wound, instead of showing the killing capability on high optical density cultures.

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 13. This was done in order to test our hypothesis that the presence of a CBD would yield inactive agents. 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 (Eppendorf). To this 40 µL water was added to function as a negative control (Figure 13) due to the CBD-bound agents being eluted in water. To 6x6 wells a concentration gradient was added in the same way, constantly adding 160 µL (in low salt LB-media) E. coli BL21 (DE3) and 40 µL of the unbound agents including additional water for diluted agents in order to achieve 40 µL as a final volume (Figure 1). The experiment was run for 16 hours at 37 °C, and before each measurement a quick shake of 200 rpm, 10 seconds was done. The absorbance at 600 nm was measured, from bottom-up in the center of the well every hour.

Figure 13:Antimicrobial effect of CBD-bound agents on E. coli. A:CBD-Pln1 antimicrobial activity in various concentrations. B: CBD-PlyF307 antimicrobial activity in various concentrations. C: CBD-LL37 antimicrobial activity in various concentrations. D: Magainin 2 antimicrobial activity in various concentrations. The CBD-bound agents were tested against an E. coli BL21 (DE3) 0 OD₆₀₀ culture. Clear boxes represent the 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-bound agents 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.

Importance of testing the CBD-bound agents

Our hypothesis, regarding the inactivation of the antimicrobial agent bound to the CBD, was only partially true. The results shown in Figure 13 showed many interesting findings. Firstly, that the agents still had antimicrobial activity. This result indicated that if the bandage is applied to a patient who is not bleeding or is supplied by extrinsic thrombin, the bandage will still have a partial antimicrobial activity. Another conclusion that can be drawn from this, is that an equilibrium of the growth phase occurs and settles in lower than for the negative control. This means that even though the antimicrobial agents could not inhibit the growth fully, the growth was slowed and the stationary phase was reached earlier (Figure 13). The results also explain the difficulty that was experienced during expression of the CBD-agents, the CBD was not effective enough to inactivate the agents completely, leading to a toxic effect on the bacterial chassis. It also explains why they can be found in the insoluble fraction because the agents are still bound to bacterial membranen.

Unbound agents

To approximate the agents' efficiency after they were released from a bandage (thus not bound to the CBD) we purified the agents. CBD-agents were bound to microcrystalline cellulose and thrombin together with thrombin cleavage buffer was added. E. coli and B. subtilis was used as test subjects since they are gram negative and gram positive, respectively. After over-night incubation in 37 °C at 200 rpm, the microcrystalline cellulose was centrifuged down, creating a supernatant containing thrombin and the unbound agent. This concept and solution is what was tested below in Figure 14.

This experiment acts as a positive control before starting our experiments that would show the actual proof of concept with the Novosite CBD-agent-bandage. The experimental setup was the same as the earlier experiment but instead of adding water to the negative control, 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) was added. This was because the agent solution contained thrombin and cleavage buffer. The negative control showed to not affect the bacterial growth, but rather increased it due to the added salt in in the cleavage buffer.

The results confirm our theory that the antimicrobial agents will have an increased effect against bacteria after the CBD is cleaved. Pln1, PlyF307 and CHAP had a high activity against E. coli. Whilst CHAP had a much higher activity against B. subtilis. Pln1 is an antimicrobial peptide (AMP) which has been reported to have effect against both gram positive and gram negative bacteria [1]. According to our results, Pln1 was more effective against the gram negative E. coli in comparison to B. subtilis which is gram positive. This may be due to the gram negative wall being more easy to penetrate and therefore creating pores, whereas the gram positive wall is thicker and can act as a partial trap for Pln1 [2]. PlyF307 is an antimicrobial enzyme (lysin) derived from an A. baumannii bacteriophage [3]. On its C-terminal is an AMP allowing the lysin to easily find its target [3]. PlyF307 is more effective against A. baumanii than E.coli [3], however, because both bacteria are gram negative a high activity was still retained as seen in Figure 14C. CHAP is also a lysin, but it is from an S. aureus bacteriophage [4]. S. aureus is a gram positive bacteria and due to lysins' generally specific nature, it can be seen in Figure 14D and 13E that CHAP is much stronger against B. subtilis (13E) in comparison to E.coli (13D). Figure 14E shows that CHAP completely inhibited B. subtilis growth, while still being effective against E. coli (likely due to it being able to affect the bacteria during cell division). All of the agents tested in Figure 14 had a strong activity against at least gram positive or gram negative bacteria. This proved that the agents can be used to kill bacteria. The different activities against gram positive or gram negative bacteria also shows that the Novosite construct can be used modularly to preserve one of these groups if wished. By getting promising results from this "positive control" we could further move on to the proof of concept.

Figure 14: Unbound agents' antimicrobial activity. A: Pln1 tested against E. coli BL21 (DE3). B: Pln1 tested against B. subtilis. C: PlyF307 tested against E. coli BL21 (DE3). D: PlyF307 tested against B. subtilis. E: CHAP tested against E. coli BL21 (DE3). F: CHAP tested against B. subtilis. Unfilled boxes indicate negative control containing thrombin and thrombin cleavage buffer. Filled boxes indicate an agent present. The E. coli BL21 (DE3) and B. subtilis culture started with 0 OD₆₀₀. The media used in all wells was low salt LB (0.4 g/L NaCl). The error bars represent the mean ± SD from three independent experiments.

Parseq peptide activity

The Parseq peptides derived from our developed Parseq model was also tested against the same bacteria as described previously in Figure 14. These two Parseq-α and β peptides were chemically synthesised by CASLO through a sponsorship. They were therefore not thrombin cleaved. As a control, the peptide buffer (negative control) Urea was used. the results of these tests can be seen in Figure 15. The two peptides performs best against E.coli where they both totaly inhibit growth at 30 µg/ml for α and 100 µg/ml for β which is shown in A and B. In C and D their effect on B.subtilis is shown. Neither peptide were able to totaly inhibit the growth at 300 µg/ml even thou the growth is decreased so the peptides have had a desired effect. In all four graph, especially A, C and D, the concentrations to low to totaly inhibit bacerial growth have delayed the exponential growth phase showing that the peptides still are affecting the bacteria.

Figure 15: Parseq model peptides' antimicrobial activity. A: Parseq-α antimicrobial activity on E. coli BL21 (DE3) in various concentrations. B: Parseq-β antimicrobial activity on E. coli BL21 (DE3) in various concentrations. C: Parseq-α antimicrobial activity on B. subtilis in various concentrations. D: Parseq-α antimicrobial effect on B. subtilis in various concentrations. Unfilled boxes represent the negative control containing the same volume but only contains the peptide buffer as the added peptides. The E. coli BL21 (DE3) and B. subtilis culture started at 0 OD₆₀₀. The media used in all wells was low salt LB (0.4 g/L NaCl). The error bars represent the mean ± SD from three independent experiments.

Immobilization and release on a cellulose bandage - Proof of concept

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.

The cellulose was first incubated with E. coli BL21 (DE3) lysate (20 mL) containing CBD-Pln1 or CBD-PlyF307 for 2 hours in 4 °C. After immobilization of the agent on cellulose, the cellulose was washed three times with carbohydrate binding module buffer (CBM-buffer, 10 mL, Tris–HCl pH 7.0, 20 mM NaCl, 5 mM CaCl₂). After washing, small pieces of bandage were cut out to fit into a well in a 96-well plate. Added to these wells was also low salt LB-media (160 µL, 0.4 g/L NaCl) containing E. coli BL21 (DE3) at 0 OD₆₀₀.

Thrombin cleavage buffer (40 µL, 20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl₂) was added to all the wells. The negative control, in addition to thrombin cleavage buffer, contained a cellulose bandage without any bound agents. In the Pln1/PlyF307 bandage (triangles, Figure 15A and 14B) the cellulose with bound agents was added. The filled boxes in Figure 15 below (Pln1/PlyF307 bandage + thrombin) contained the agents bound to cellulose together with thrombin (0.5 U thrombin, final concentration in well 0.6 µM), recreating the mechanism of action.

Noticeably, the thrombin-released agents inhibited E. coli growth the most, in comparison to the other combinations tested. In both Figure 15A and 14B the non-released agents bound to cellulose exhibited an antimicrobial activity. This correlates with the results in Figure 13 which indicated that bound agents exhibited a moderate antimicrobial effect. Pln1 bound and unbound had a similar activity with the unbound being slightly more effective. PlyF307 showed a strong antimicrobial activity in both a bound and unbound state. The unbound/released PlyF307 completely inhibited the growth of E. coli. These results proved that the of concept of iGEM19 Linköping's main mechanism worked and that these types of wound dressings have the potential to be used clinically.

Figure 15: A bacterial cellulose bandage (Epiprotect, S2Medical) was incubated with E. coli BL21 (DE3) 0 OD₆₀₀ cultures. The clear boxes represent a negative control (in both A and B) which contained thrombin, thrombin cleavage buffer and cellulose bandage. The filled boxes represent a cellulose bandage which had CBD-Pln1 (A) or CBD-PlyF307 (B) bound to it, thrombin and thrombin cleavage buffer. The filled triangles represent a cellulose bandage which had 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.

References

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