Results

Our project transFERRITIN aimed to synthesize a modular drug delivery system, a CPP-Ferritin-Container that allows the specific introduction of antibiotic molecules into bacterial pathogens by using nanobodies bound to a modified codon in the L-loop of the ferritin subunits using click-chemistry. During our journey we managed to accomplished the following aspects:

• Deletion of SapI and BsaI restriction recognition sites to enable future iGEM teams to continue working with the ferritin construct
• Successful introduction of CPP TAT (BBa_K1202006), R9 (BBa_K4669001) or R12 (BBa_K4669002) on the N-terminus to ferritin (BBa_K4669027).
• Optimization of the purification protocol and successful purification of WT-Ferritin
• Implementation of Amber codon into the L-loop of WT-Ferritin (BBa_K4669007) and TAT-Ferritin (BBa_K4669008)
• Analysis of the penetration efficiency of CPP R9, R12 and TAT2 (BBa_K4669016)
• Visualizing the structural integrity of purified WT-Ferritin by negative staining EM

Click here for an overview of our workflow

To commence with our project Prof. Dr. Tobias Beck provided us with the plasmid pET22b(+) WT-ferritin (fig. 1) containing the conding reagion for human heavy chain ferritin. In order to provide a plasmid for future iGEM teams that wish to proceed with our project or want to develop their own ferritin project, we needed to eliminate the SapI and BsaI restriction recognition sites (RRS) by using classic PCR mutagenesis.

Workflow:

• PCR mutation ro remove RRS
• PCR purification
• Ligation
• Transformation of the ligated product into DH5α
• Miniprep
• double digest for control
• PCR mutation to remove the second RRS
• PCR purification
• Ligation
• Transformation of the ligated product into DH5α
• Miniprep
• double digest for control
• Sequencing

In the initial phase of this process, we began with a PCR mutagenesis to excise the first RRS, the success of which got subsequently confirmed by agarose gelelectrophoresis (fig. 2 A). Following this confirmation, we purified the product, ligated the mutated DNA and transformed via heat shock into DH5α for further mutation.
After a Miniprep of the transformed clones, we performed a double digest assay using BsaI and SapI restriction enzymes. With an agarose gel, we checked if the elemination of the first RRS was achieved. In theory, due to the absence of either the SapI or BsaI RRS, the plasmid should be sliced at only one site, yielding a linear product with 6,000 bp in size. Due to premature termination of the double digest assay after 15 min, not all plasmids could undergo complete cleavage. As a consequence, the agarose gel analysis exhibited two visible bands, as depicted in figure 2 B. The upper band showed the successfully digested plasmid, while the lower band indicated the supercoiled uncut plasmid. We used the plasmids with positive digestion results for the next PCR mutagenesis to delete the second RRS.

After the second PCR mutagenesis we checked the outcome again through an agarose gel (fig. 3 A). We repeated the methodology as in the previous mutation process and conducted again the double digest to verify if both RRS got eliminated. However, this time we extended the time for the digest from 15 min to 1 h in order to ensure complete enzymatic activity.
We used plasmid of the initial mutagenesis step for which one RRS is still intact to compare the result with the double mutated pasmid. As shown in figure 3 B and C, the SapI Mut and BsaI Mut plasmids underwent digestion which resulted in a band at approximately 6,000 bp. In contrast the double mutated plasmids (SapI  (BsaI) Mut and BsaI (SapI) Mut) did not exhibit any observable digestion, leading to a lower base pair indicative of supercoiled plasmid.

As a final product we obtained the pET22b(+) WT-Ferritin plasmid without SapI and BsaI RRS (BBa_K4669027) (fig. 4), which was verified by sequencing and therefore could be used for further modifications.

We employed our previously mutated plasmid, pET22b(+) WT-ferritin without SapI | BsaI as a starting point to establish a protocol for fusing cell-penetrating peptides (CPPs) TAT, R9, or R12 to the N-terminus of ferritin through site directed mutagenesis.

Workflow:

• PCR mutation for fusing CPPs with ferritin
• PCR purification
• Ligation
• Transformation of the mutated product into DH5α
• Colony PCR
• Miniprep
• Sequencing

Following the PCR mutagenesis, agarose gelelectrophoresis was once again utilized to asses the success of the reaction, as present in figure 5. Within the gel, the faint upper band corresponded to the lineralized PCR product, representing the fusion of CPP with ferritin, measuring approximately 6,000 bp in size. Nevertheless, the presence of additional bands in the gel suggests deviations from the expected outcome, indicating an abnormal reaction
Furthermore, it is important to note that the extended length of the forward primers for TAT (74 bp), R9 (65 bp), and R12 (80 bp) may lead to the formation of secondary structures within the oligonucleotides themselves. These secondary structures may have a negative impact on the PCR mutagenesis process.

The DNA samples with positive agarose gel results and so exhibiting indications of CPP mutation underwent purification and subsequent ligation. Following this, a transformation via heat shock was performed into DH5α bacterial cells.
To have a verification for a successful mutation we checked the transformed colonies through colony PCR. The primers employed in this analysis were designed to anneal upstream of the CPPs and downstream of the ferritin DNA. So by checking the results via agarose gelelectrophoresis we expected intense bands at approximately 600 bp signifying the success of the mutation. The successful sequence amplification was achieved for each CPP mutation, although not for all of the individual samples used in the analysis (fig. 6).

The colonies that resulted in a successful colony PCR amplification were used for Miniprep and subsequently submitted for sequencing to final verify the insertion of the CPPs into our plasmid.
We managed to fuse each CPP with our plasmid (fig. 7). Following this, E.coli BL21 (DE3) star cultures were transformed via heat shock to introduce the CPP-Ferritin clones for subsequent expression.

To continue with our work we needed to produce and extract our proteins.

Workflow:

• Transformation of plasmid into BL21 (DE3) star
• Preparation of expression culutre
• IPTG induction

After the successful transformation of the various CPP-Ferritin constructs into BL21 (DE3) star, we initiated the preparation of expression cultures. These cultures were allowed to grow until reaching an OD600 of 0.6 before inducing the expression. Since the T7 promoter is used in the plasmid construct, 0.25 mM isopropyl β-D-1 thiogalactopyranoside (IPTG) was utilized as an inducer for T7 RNA polymerase production followed by incubating the cultures at 18 °C and 180 rpm for 48 h
To check if our target protein got expressed after induction, we separated the proteins in a 15 % polyacrylamide gel with samples collected both prior to and 48 h after the induction with IPTG. As shown in figure 8 after the induction (I), compared to the non induced samples (N), intense bands were evident at the approximate weight of 21 kDa for WT-Ferritin (WT-Ftn), 23.6 kDa for TAT-Ferritin and 23.1 kDa which corresponds to R9-Ferritin. Even though we did not obtain the expected band for R12-Ferritin at the approximate weight of 23.6 kDa, we proceeded with our experiments.

In order to validate the expression of our protein of interest, we performed SDS-PAGE and western blot analysis with our cell culture normalized to OD600 values of 0.2 and 0.1. Notably, bands corresponding to WT-Ferritin and the respective CPP-Ferritin construct could be detected in each lane, as illustrated in figure 9 A.
After performing the transfer of the proteins to the membrane, the membrane got stained with Ponceau S to assess the efficiency of the transfer process. Figure 9 B depicts successfully transferred proteins to the membrane, however the band which represents R12-Ferritin at approximately 23.6 kDa is hardly recognizable.
Following the brief imaging process of the membrane stained with Ponceau S, the staining got removed during two wash steps with water and we continued with the blotting protocol using the ferritin antibody with conjugated peroxidase to detect the ferritin construct.

The application of the ferritin antibody revealed a protein in the lanes of TAT-Ferritin and R9-Ferritin with a molecular weight of about 23 kDa which correlates with the molecular weight of TAT-Ferritin and R9-Ferritin (fig. 10). However, it is noteworthy that no detectable signal was obtained for R12-Ferritin. Additionally, in the lane containing WT-Ferritin with OD600 1.0, a faint signal was observed. The faint bands with higher molecular weight in R9-Ferritin OD600 2.0 and in both R12-Ferritin samples could be an indicator for completely disassembled ferritin protein, due to its heat-stability.

We successfully expressed our CPP-Ferritin protein and validated this expression through the utilization of SDS-PAGE and western blot. Furthermore, the ferritin antibody demonstrated the capability to recognize and bind our target protein, despite its modification with CPPs.

In order to move forward with the subsequent analysis, it was essential that we purify our protein constructs. For this purpose, we used a purification protocol for WT-Ferritin provided by Professor Dr. Tobias Beck, which we adapted to our specific needs.

Workflow:

• Heat precipitation and ammonium sulfate precipitation
• Ion exchange chromatography
• Size exclusion chromatography run 1
• Size exclusion chromatography run 2

Heat Precipitation and Ammoniumsulfate Precipitation

Initially in the purification process, we began with heat precipitation at 65 °C and ammonium precipitation, which can be used due to the great heat stability of ferritin.

After finishing the first part of the purification we checked the results via gelelectrophoresis using a 15 % SDS-gel and western blot just like for the expression analysis. Each lane was loaded with either 20 µg or 10 µg of total protein.
Since the gels were stained after blotting, it is difficult to detect the bands for small proteins like the CPP-Ferritin constructs because they are already transfered on the membrane (fig. 11). The prominent band observed in the WT-Ferritin lane with an approximate molecular weight of 21 kDa is indicative of the presence of this specific protein.
However it can be noted that, after heat precipitation and ammoniumsulfate precipitation, a quite clear protein solution was expected. In both SDS-PAGEs with the protein solution after the precipitation, a lot of other proteins could be detected that indicated that the heat precipitation alone is not very effective for high purity.

After the blotting process the membrane got stained with Ponceau S to assess the success of the blotting process (fig. 12). Using this staining, it is possible to detect WT-Ferritin with the predominant band with approximately 21 kDa, as well as TAT-Ferritin, R9-Ferritin and R12-Ferritin with the bands of approximately 23 kDa. When the Ponceau S imaging was finished we continued with the antibody staining.

However, no signal was detected in both western blots.
Even if the heat precipitation and ammoniumsulfate precipitation did not work as well as expected and the western blot experiment resulted with no signal, we assume that we were able to obtain WT-Ferritin and CPP-Ferritin after the first purification step.

Ion Exchange Chromatography (IEC)

For ion exchange chromatography we used the HiTrap Q HP 5 mL column and injected 5 mL of heat and ammonium precipitated protein solution.

For the elution we used 20 CV and 60 % of elution buffer. The following figures 13 to 21 show the chromatograms of the proteins. According to the protocol of Beck we expected to detect WT-Ferritin and CPP-Ferritin constructs at nearly 35 mS/cm. Indeed, for nearly all samples we could detect a peak around 30 mS/cm to 35/nbsp/cm except for sample WT-Ferritin 1, where no peak could be detected indicating for WT-Ferritin protein

Click here for Chromatograms

After the chromatography process we collected the samples of the peak sections and concentrated the solution by using 30 MWCO filter units. Subsequently SDS-PAGE was used to check if the WT-Ferritin, TAT-Ferritin, R9-Ferritin and R12-Ferritin got purified, as shown in figure 19. Nonetheless, beside the intense bands in the WT-Ferritin samples at approximately 20 / 21 kDa, that indicate the presence of WT-Ferritin protein, no other bands in the CPP-Ferritin samples could be found. This implies that no CPP-Ferritin construct could be purified. Probably, the proteins were already washed out of the column during the washing process, but this would need further investigation. Besides that, it must be noted that even after the purification step, a lot of unspecified proteins are still present.

To improve the IEC for purification of R9-Ferritin and TAT-Ferritin, we adjusted the pH of the lysis buffer (sample buffer) and elution buffer from pH 7.5 to pH 9 according to the isoelectric point (pI) to provide enhanced binding affinity to the stationary phase of the column. Although we expected a better separation and therefore a better signal with this adjustment, no peak indicating the presence of our proteins was detected in the R9-Ferritin and TAT-Ferritin samples (fig. 23).

We managed to successfully purify the WT-protein, however it was not possible to perform the purification method with the CPP-Ferritin proteins, even though the pH was adjusted according to the pI.

Size Exclusion Chromatography (SEC)

Since it was not possible to purify the CPP-Ferritin proteins by IEC, SEC was performed only with WT-Ferritin. As described in the original protocol, two SEC runs were performed in order to discard larger proteins. For this, the Superdex 200 Increase 10/300 GL coloumn was used and 500 µL of post IEC concentrated protein solution was injected. According to the standard protein injection, and the original protocol we expected the ferritin protein to elute after 11 mL.
In the chromatogram of the first SEC run (fig. 24 A) two peaks can be identified. The first peak indicates wrong assembled ferritin that must be discarded. The second peak represents the ferritin monomer. Compared to the second run, the signal for both peaks is strongly reduced (fig. 24 B).

To analyze if the WT-Ferritin is still present after the SEC runs, we performed a SDS-PAGE (fig. 25). After both runs one band is visible at approximately 21 kDa. That indicates the presence of WT-Ferritin. However, after the second run the signal in the gel is much less intense compared to the first run. Therefore, it can be assumed that significant amounts of protein were lost between the runs.

Since no further bands can be detected after the first run, it can be concluded that WT-Ferritin was successfully purified in the first run.
The purified protein can now be used for further experiments.

To analyze the successful assembly of the ferritin complex after protein purification, we used the technique of negative staining with uranyl acetate. This method plays a crucial role in transmission electron microscopy (TEM) as it increases the contrast of biological samples that would otherwise be difficult to detect.
Negative staining involves depositing the staining agent around the specimen, leaving the specimen unstained. This makes the sample appear bright against a dark background, which facilitates the identification and analysis of its structures.
We used the Talos TEM L120C to examine the sample. The microscope operated at an accelerating voltage of 120 kV (HT 120 kV). A 70 µm aperture was employed to produce a magnification of 92000x by 4096 pixels. These settings were carefully calibrated to ensure optimal representation of the sample's microstructure.
This method allowed us to capture high-resolution, clear and easily interpretable images of the specimen. Figure 26 shows a structural representation of WT ferritin, annotated with a scale bar of 100 nm.

Under the TEM, ferritin appears as a spherical protein whose 24 subunits are arranged in a characteristic way to form a symmetrical hollow sphere. This cavity appears under the microscope as a darker area in the centre of the molecule. At the current resolution, the surface of the ferritin molecule appears smooth.

Workflow:

For the nanobody recognition to integrate the Amber Codon

• PCR mutagenesis to intgerate the Amber Codon
• PCR purification
• DpnI digestion as control
• Miniprep
• Sequencing

For the concentration of the helper plasmid

• PCR of the p15A backbone for linearization and addition of BsaI recognition site for the Golden Gate Assembly
• Golden Gate Assembly of our three fragments
• Transformation into DH5α and plating onto chloramphenicol agar plates
• Colony PCR
• Miniprep
• double digest as control
• Sequencing

Amber Codon

As we settled for click chemistry to add the nanobodies onto our ferritin, we were in need of a binding site. For that, we mutated the outwards facing lysine in the L-loop at position 88 Using QuikChange mutagenesis, we transformed our ferritin plasmid and successfully introduced the mutation K88TAG, as proofed by the sequencing report.
After the mutagenesis, we performed a DpnI restriction and transformed DH5α competent cells with the plasmid. Since the negative control did not grow any colonies when plated out on agar, we can assume, that the DpnI restriction worked and we only introduced plasmid that was produced in our mutagenesis PCR (fig.27). This result is backed up by the sequencing results.

GGA/Helper plasmid

To actually utilize the introduced Amber codon as binding site for the click-chemistry, the bacteria needed a helper plasmid to produce the corresponding tRNA as well as the aminoacyl tRNA synthetase to incorporate the ncAA that will be added later. To achieve that, we tried to assemble the p15A plasmid via Golden Gate Assembly with three fragments. These fragments code for the synthetase and two copies of the tRNA (fig. 28).

Afterwards, competent DH5α cells were transformed and plated out. From these plates, we picked colonies for a colony PCR. With our primers designed to anneal in front of the second araBAD promotor (4948-49726 bp) and inside the ori (62-84 bp) to flank our tRNA inserts. The PCR product is expected to have a size of 900 bp. We used the colonies with positive results to induce overnight cultures. From these overnight cultures, we extracted the DNA via Miniprep and sent them in for sequencing. We managed to get only one colony so far with a positive result in both the colony PCR as well as the sequencing report (fig. 29, red marked lane), but could not manage to re-trafo the plasmid in time to use for further experiments.

We also attempted a double digest with two restriction sites (EcoRI and Cfr9I), that should only be present in our GGA product. Sadly, this assay failed presumably cause of to little amount of DNA, since in the negative control you can clearly see a band, but no band is visible in the lane with our digested product (fig. 30).

Co-transformation

We also performed a co-trafo of our complete and finished WT- and TAT-Ferritin plasmid with our supposed to be GGA transformed helper-plasmid, which resulted in only a handful of mini colonies to grow. Afterwards, as we received the sequencing results, it showed that the helper-plasmid we used did not got mutated correctly and therefore does not carry the tRNA and synthetase as wanted. Although, since we used two different resistances in our two plasmids and both antibiotics on the agarplate, somehow the resistance must have been successfully implemented into our helper-plasmid. Nontheless, since we had at least one successful introduction of all fragments into the helper-plasmid as suggested by the sequencing report, we still have to validate this result and then retry the co-trafo of our two plasmids.

The apparently successful GGA product needs to be re-transformed into bacteria culture. Afterwards, we can try the double digestion again to double proof the correct insertion of our fragments. If this is succesful, we can continue with the co-trafo of both of our plasmids and try if our nanobodies can bind to our ncAA on the Ferritin L-Loop. If they do so, all of our parts are complete and we can undergo research on their selective target penetration and cargo release. If you want to know more in detail, how we can proceed with our project after the iGEM competition, have a look at our Outlook page

In order to test the capability of selected CPPs to penetrate the plasma membrane of gram-negative prokaryotes, CPP-eGFP fusion proteins were incubated with E. coli DH5α cells and analyzed by fluorescence microscopy.
E. coli DH5α cells were cultured until OD600 0.7-0.8 and 1.5 · 10⁸ cells in 500 µL PBS were incubated with 10 µg of fusion protein - R9-eGFP, R12-eGFP, TAT2-eGFP, or eGFP without CPP as negative control - for 1 hour. After incubation, cells were washed with 1X PBS, fixed with 4 % formalin and mounted with Mowiol mounting medium. The samples were analyzed under the Leica Dmi8 Wide Field Microscope and Leica SP8 Confocal Microscope.
Two different thresholds were set to determine the efficiency of CPP penetration. By looking at the negative control, the mimimum of threshold 1 was set to the value of 40, corresponding to the autofluorescence of the bacterial cells. A second threshold was set to a mimimum value of 250 to include cells within the focal plane and exclude GFP signals from outside the focal plane to determine the fluorescences intensity of the TAT2-eGFP incubated cells.
Cells incubated with TAT2-eGFP showed significantly increased GFP signals compared to the negative control of cells incubated with eGFP. TAT2-eGFP incubated cells showed an approximately 10-fold increase in GFP signal with a value of approximately 627 compared to the autofluorescence of approximately 67 (table 1). Thus, TAT2-eGFP has either bound or penetrated the plasma membrane of E.coli DH5α.
In addition to fluorescence intensity measurements, the comparison of the DAPI and GFP channel of TAT2-eGFP incubated cells show that all cells stained with DAPI have bound GFP and thus TAT2. The binding coverage seems to be at approximately 100 %.

Table 1: Fluorescence measurements of E. coli DH5α cells incubated with eGFP (negative control) and TAT2-eGFP for 1 hour.
Area of measurement ("Area"), average fluorescence ("Mean"), minimum values ("Min") and maximum values ("Max"). Measurements: Background, threshold 1 with MinThr=40 for differentiation of cells from background in negative control, threshold 2 (for TAT2-eGFP sample only) with MinThr=250 for more accurate detection of cells and exclusion of GFP signals from outside the focal plane.

		Area	Mean	Min	Max	MinThr	MaxThr
eGFP	Background	1122.650	23.833	8	36	0	65535
	Threshold 1 (cells, autofluorescence)	275.081	67.218	40	280	40	65535
TAT2-eGFP	Background	1054.966	7.466	0	24	0	65535
	Threshold 1	334.852	237.112	40	3635	40	65535
	Threshold 2	92.244	627.443	250	3635	250	65535

R9 and R12 showed different results in the penetration tests. R12-eGFP incubated cells show high GFP signals indicating that R12 has bound or penetrated the plasma membrane. R9-eGFP incubated cells did not show any fluorescence intensity higher than autofluorescence.
The fluorescence intensities of R9-eGFP incubated cells and eGFP-incubated cells were measured by a threshold with a minimum value of 140. The fluorescence intensity of with R9-eGFP incubated cells of approximately 145 is not higher than the fluorescence of cells in the negative control (table 2).
R12-eGFP incubated cells show both visually (fig. 32) and by the measured fluorescence intensity of approximately 1098 (table 3) that R12 binds or penetrates the plasma membrane. As with TAT2-eGFP (fig. 31), all cell groups stained with DAPI are labeled with R12-eGFP (fig. 32) indicating a binding coverage of approx. 100 %.

Table 2: Fluorescence measurements of E. coli DH5α cells incubated with R9-eGFP, R12-eGFP, and eGFP (negative control) for 1 hour.
Area of measurement ("Area"'), average fluorescence ("Mean"), minimum values ("Min") and maximum values ("Max"). Measurements: Background, threshold 1 with MinThr= 140 for differentiation of cells from background, threshold 2 (for R12-eGFP sample only) with MinThr=400 for more accurate detection of cells and exclusion of GFP signals from outside the focal plane.

		Area	Mean	Min	Max	MinThr	MaxThr
eGFP	Background	1635.836	106.377	80	139	0	65535
	Threshold 1	0.211	164.020	140	235	140	65535
R9-eGFP	Background	935.111	106.325	66	129	0	65535
	Threshold 1	0.946	145.826	140	176	140	65535
R12-eGFP	Background	1954.789	106.471	79	150	0	65535
	Threshold 1	881.204	674.414	140	4416	140	65535
	Threshold 2	463.816	1098.096	400	4416	400	65535

In order to evaluate the capability of the CPP-eGFP fusion proteins to penetrate the plasma membrane, it is necessary to differentiate whether the GFP signal is located at the plasma membrane or in the cytoplasm. For this, the penetration experiments were repeated and examined on the SP8 Leica Confocal Microscope with Lightning Deconvolution.
Cells incubated with TAT2-eGFP and with R12-eGFP again showed increased fluorescence compared to the negative control (fig. 33). In Z-stack images of single cell groups (fig. 34 to 36), GFP signals are low, possibly indicating a high level of photobleaching. For each Z-stack, a single focal plane of high DAPI signal - to detect a cytoplasm-rich interface - and a projection of maximum intensity are shown. Most Z-stacks do not show distinct fluorescence confirming CPP penetration. Z-stacks 2 and 4 from R12-eGFP incubation (fig. 34) show GFP signal both within a cytoplasm-rich focal plane and in the projection of maximum intensities. It can be assumed that R12 penetrated the plasma membrane. Further imaging is required. TAT2 shows minimal GFP signals, which is only visible in the projections of the maximum intensities (fig. 35). Thus, differentiation between plasma membrane and cytoplasmic localization is not possible for TAT2-eGFP.

Further penetration experiments were performed with calcium-competent E. coli DH5α cells. The images obtained do not show increased GFP signals compared to the negative control. Lower binding efficiency of GFP-CPP fusion proteins in 50 mM CaCl₂ treated cells can be assumed. The results are shown in figure 37. To validate this assumption, the experiment will be repeated. A possible explanation could be the the binding of Ca²⁺ to the negatively charged plasma membrane, inhibiting the binding of the cationic CPPs used. DAPI signals are also of low intensities suggesting that fluorescence microscopy may have produced insufficient results.

To determine the localization of CPPs, additional controls besides the negative control - eGFP without fused CPP - will be implemented in the future. Additionally, a positive control for cytoplasmic fluorescence could be used, for instance an E.coli DH5α mutant expressing an abundant cytosolic protein in fusion with GFP. A suitable expression level resulting in GFP signal intensities similar to those expected upon successful CPP penetration could be tested by different promoters. Another negative control that exhibits fluorescence at the plasma membrane but not in the cytoplasm could be used. Additionally, a longer incubation period of three hours will be tested. The use of another microscope like the Nikon Spinning Disc Confocal Microscope will be considered.

With a agar diffusion test with simple E.coli on an agar plate, we wanted to test the efficiency of antibacterial plant substances in various concentrations. We plated out 100 µL E.coli BL21 (DE3) star bacteria on an regular agar plate and placed filter paper soaked with flavone, quercetin, rutin and ethylparaben individually or in combination in following concentrations in different parts of the plate as shown in figure 38: 1, 10, 25, 50, 75, and 100 µg/m. As controls, we used 70 % ethanol. We then incubated the plates for 20-30 h at 37 °C. While we did this disk diffusion test several times, we only observed significant and reproducable inhibition of cell growth for 70 % ethanol. The other compounds inhibited bacterial growth not significantly.