The control strain W168 (wild type) and control 1, will presumably not show any luminescence output, while the positive control 2 is expected to show a steady luminescence signal regardless of the presence of any antibiotic compound. Generally, the P_xyl promoter is activated by adding xylose as an inducer to the medium. In the plate reader experiments, 0.2% xylose was added to the cells (beginning with the day culture) to activate expression of blaR1.

Further we propose biosensor strains carrying the genotype remark penP::kan^R to give a stronger signal in presence of β-lactam compounds, as they cannot be degraded by the PenP enzyme.

As shown in Figure 3, the wildtype W168 (black with white dots) shows no increase in RLU/OD₆₀₀ values when induced with the different β-lactam antibiotics and controls. Control 1 (black tight stripes) behaves similarly to the wild type strain. The slight decrease of control 2 (light grey) in the bar chart where induction with ampicillin and carbenicillin happened is mostly explained by the high growth inhibition caused by the chosen concentrations for W168 (with functional PenP). Most of the times, the constitutive expression of the lux operon resulted in an RLU/OD₆₀₀ of over 1.3 million for control 2 (see Figure 3).

Biosensor 1 gives an overall good signal for all β-lactam antibiotics tested, but also shows a higher basal activity in absence of the β-lactam compounds of 40.000- 90.000 RLU/OD₆₀₀ (see Figure 3, bar chart with bacitracin and dH₂O). Further, we could observe a difference in signal intensity dependent on the β-lactam antibiotic tested. Therefore, biosensor 1 gives the highest signal in presence of penicillin G, cefoxitin and cefoperazone with up to 2.7 million RLU/OD₆₀₀. Ampicillin and penicillin G again show a weaker increase in signal produced by biosensor 1, which could be due to the same reason as for control 2 (see Figure 3).

For biosensor 2, the detection range and sensitivity is comparable to biosensor 1. This strain strongly senses cefoxitin, ampicillin and cefoperazone reaching up to 2.4 million RLU/OD₆₀₀. Even the basal activity of the P_blaZ promoter in biosensor 2, as shown in the bar charts with bacitracin and dH₂O, conforms with the one from biosensor 1.

To determine whether the results obtained in the plate reader assays under liquid conditions can be reproduced on solid agar plates, we performed disk diffusion assays. This assay visualises the detection range of the biosensor by a glowing halo at the edge of the growth inhibition zones, where bacteria are exposed to sub-lethal antibiotic concentrations. We tested the same β-lactam antibiotics and controls as in the previous experiments, but chose higher concentrations since B. subtilis tends to be more resistant against antibiotics when tested on solid media. By increasing the antibiotic concentrations we still obtained slight growth defects but remained in the detection range of the biosensor. The concentrations used in the disk diffusion assays can be extracted from Table 3.

We expected the control substances (water and bacitracin) to not cause any luminescence signal at the edge of the inhibition zones. The β-lactam antibiotics should lead to a glowing halo when tested with the three different biosensor versions. The wildtype strain and control 1 should not show any signal, since both strains are lacking the lux operon. In the case of control 2, a luminescence signal should be spread over the whole plate, due to the constitutive expression (P_veg) of luciferase. Figure 4 sums up the results of the disk diffusion assay for all strains tested. After 24 hours of incubation at 37°C, plates were photographed under daylight conditions and under a chemiluminescence dock (with two minutes exposure time).

As expected, the wildtype and control 1, show no luminescence signal, while control 2 leads to a strong luminescence signal spread across the entire plate (Figure 4, Panel B). Neither bacitracin nor dH₂O lead to a detectable output, accounting for all strains tested. While in liquid medium biosensor 1 behaves similar compared to biosensor 2, there is a tremendous difference in the detection capability. Biosensor 2 showed detection for all β-lactams tested (Figure 4, Panel D). Au contraire, biosensor 1 only showed a luminescence signal for cefoperazone, cefoxitin, and cefalexin (Figure 4, Panel D). Further, the luminescence halo around the cefoxitin disk is quite broad compared to the others, indicating an increased diffusion of the compound into the lawn. Although biosensor 1 was activated by penicillin G in liquid medium, we could not observe an induction on plate (Figure 4, Panel D).

Biosensor 2 was activated by all of the β-lactam compounds tested (Figure 4, Panel D). Ampicillin, cefoxitin, cefalexin, and cefoperazone strongly activate the system, while penicillin G and carbenicillin just show a weak induction of the signal on the plate. These findings go along with the results obtained in liquid medium in the previous experiments. On the plate with the lawn of biosensor 3 (Figure 4, Panel D), all β-lactams could be detected efficiently when 0.2% xylose was added. In contrast to biosensor 1 and 2, there is a very weak luminescence halo around the cefalexin disk. Also, this halo seems not to de directly at the edge where the cells are in contact with the antibiotic, but rather a bit further off the inhibition zone. Without induction of biosensor 3 with 0.2% xylose, we could not detect any luminescence signal, demonstrating that the receptor (BlaR1) is crucial for detection and signal transduction, standing in line with results obtained in liquid medium (data not shown). The following Table 4 contains the measured diameters of all inhibitions zones caused by the antibiotic. As expected, no inhibition zones around the negative control dH₂O were observed.

3. Examining the dose-response relationship

As a final characterization of our biosensor, we carried out Dose Response assays in order to investigate the detection sensitivity. In this experiment, eleven different concentrations of the β-lactam antibiotics and bacitracin (control) have been tested together with the biosensor 2. This biosensor was chosen due to its reliable performance in liquid and on solid medium and because all necessary components are constitutively expressed without the addition of any further substances (as with biosensor 3).

The obtained results (see Figure 5) indicate that biosensor 2 shows the highest dynamic range for cefoperazone and is capable to even sense very low concentrations of this compound. Biosensor 2 shows the weakest dose response towards cefalexin, which is in agreement with all previous experiments (see Figure 5). The antibiotics cefalexin, ampicillin and penicillin G lead to a decrease in luminescence signal for concentrations higher than 10^-1, due to the growth inhibition at these concentrations (see Figure 5). Overall, a high dynamic range upon antibiotic exposure can be detected for all compounds starting at concentrations above 10^-3.

4. Encapsulation of the Biosensor into Peptidosomes – Proving the Application Potential

After evaluation of the biosensor, we probed its activity when encapsulated in Peptidosomes. An overnight culture was inoculated in Fmoc-FF-Solution with a final OD600=10. Peptidosomes were prepared containing no bacteria (A), W168 (B), Control 2 (C) and Biosensor 2 (D) (see Figure 6 below) and underwent 3 washing steps. Afterwards, the Peptidosomes were transferred to a 12-well plate, incubated at 37˚C and luminescence was detected every hour. Induction with 0.2µg µl^-1 cefoperazone happened after 1 hour of growth.

The Peptidosomes without cells and the wild-type W168 are expected to show no luminescence signal at all times (A and B). We estimate control 2 to reach a luminescence signal under non-induced as well as under induced conditions (C). This signal should be weaker than that of the induced biosensor 2 (D, +AB). No signal is expected for the encapsulated biosensor in absence of cefoperazone (D, -AB).

In this experiment, we successfully encapsulated biosensor 2 into Peptidosomes and demonstrated its ability to sense the β-lactam cefoperazone diffusing into the Peptidosome. Already 2 hours post induction, there is a luminescence signal detectable for control 2 and the encapsulated biosensor 2 (see Figure 6, middle, C and D). Thereby we could validate the hypothesis that antibiotic compounds can enter the Peptidosomes and trigger the activation of the biosensor. We also showed, that the performance of the biosensor is not compromised by the encapsulation.