Team:TU Dresden/Project/Biosensor

Luminescence Alert!
β-Lactam Biosensor

At a Glance


Demonstrate that encapsulated bacteria could respond to the environment that surrounds the peptidosomes.


Develop a novel whole-cell biosensor that responds to a variety of β-lactam antibiotics (input) with bioluminescence (output).


(I) A fully functional BlaR1I-PblaZ system from Staphylococcus aureus was heterologously established in Bacillus subtilis. (II) The resulting whole-cell biosensor responded specifically to the presence of a diverse range of β-lactam antibiotics in a dose-dependent manner. (III) Encapsulated biosensor cells could respond to antibiotics provided to the outside. (IV) 8 novel basic BioBrick parts were generated and fully evaluated for functionality.

Short Description

Worldwide, multidrug-resistant bacteria are on the rise and provoke the intensive search for novel effective compounds. To simplify the search for new antibiotics and to track the antibiotic pollution in water samples, whole-cell biosensors constitute a helpful investigative tool. In this part of EncaBcillus, we developed a functional and independent heterologous β-lactam biosensor in Bacillus subtilis. These specialised cells are capable of sensing a compound of the β-lactam family and will respond by the production of an easily measurable luminescence signal. We analysed the detection range and sensitivity of the biosensor in response to six different β-lactam antibiotics from various subclasses. The evaluated biosensor was then encapsulated into Peptidosomes to prove the concept of our project EncaBcillus. The encapsulation of engineered bacteria allows a simplified handling and increased biosafety, potentially raising the chances for their application e.g. sewage treatment plants.


Figure 1 β-Lactam Compounds
Figure 1: Commonly used β-lactam antibiotics and their chemical structure. All of them share the so-called β-lactam ring structure (here shown as square structure containing nitrogen).[4]

Antibiotics represent the most effective treatment against bacterial infections. Since the discovery of penicillin by Alexander Fleming in 1928, many new antibiotics have been constantly developed and were successfully applied to treat life-threatening diseases. This significant advancement in medicine saved millions of lives and still does today. However, fighting microorganisms has never been a completed task, but rather an ongoing race between drug discovery and pathogens developing resistances. Thus, multi-drug resistant bacteria still constitute a major threat for humanity, as infectious diseases represent the second leading cause of death worldwide.[1][2]

Table 1: β-lactams and controls tested in this project and their respective classification.[4]
Table 1 β-Lactam Compounds Classification

One major reason, for the steady increase of antimicrobial resistances is the “inappropriate use of antimicrobials”. Due to excessive prescription and application in livestock farming, little amounts of antibiotics are nearly found everywhere, even in drinking water. These low-dose and non-lethal concentrations containing habitats, allow bacteria to adjust and develop resistances.[2][3]

As β-lactams make up a large percentage of all antibiotics used, the project preferentially focused on this class of broad-spectrum antibiotics. Carbapenems, penicillin derivatives, cephalosporins and monobactams represent the four main classes of the β-lactams that sum up to over 100 different active substances. All compounds of this particular group can be easily identified by their common chemical structure: the β-lactam ring (see Figure 1).[4]

To address the increasing development of multi-drug resistant bacteria our iGEM Team aims at developing a novel β-lactam biosensor in Bacillus subtilis based on the genetics of the bla-operon found in Staphylococcus aureus (for a detailed description consider our Design section below). The genetically engineered biosensor will help to (I) reliably detect even minimal antibiotic concentrations of compounds from the β-lactam family in waste and drinking water and (II) unravel producer strains of yet unknown β-lactam related antibiotics. After extensive characterization of the detection range and sensitivity, the greater goal is to combine the functional β-lactam biosensor with our Peptidosomes. Thereby, we would prove the applicability of EncaBcillus as a completely new cultivation platform. Encapsulation of this whole-cell biosensor, will allow an easier and safer handling of the bacteria and thus making them more appealing for field applications, like for example in sewage treatment plants. As a proof of principle we used six β-lactams and two controls (water and bacitracin) to evaluate our biosensor (Table 1).


Genetic engineering

To achieve our goal of encapsulating bacteria into Peptidosomes that can sense antibiotics of the β-lactam family, we first needed to develop a reliable biosensor strain. In Staphylococcus aureus the bla-operon encodes a one-component system, which is responsible for sensing and mediating resistance against β-lactam antibiotics. The idea was to transfer the regulatory elements of this operon to Bacillus subtilis and replace the native output – being the β-lactamase BlaZ – by an easy detectable signal. Thus, making Bacillus subtilis a β-lactam sensing biosensor. (see Figure 2).

Figure 2 Molecular mechanism of the Biosensor
Figure 2: Overall concept showing the components and the molecular mechanism of the biosensor in B. subtilis. Upon binding of a β-lactam to the receptor BlaR1 (1), due to the receptors c-terminal proteolytic activity, the repressor BlaI is degraded and frees the target promoter (2) enabling the expression of an easy detectable reporter (3).
Figure 3 Genetic constructs constituting the biosensor.
Figure 3: Genetic constructs necessary for the functional biosensor strain. (A) blaR1 under control of the constitutive promoter Pveg or the inducible promoter PxylA, (B) blaI downstream of the promoter PlepA, and (C) the promoters PblaZ or PblaR1I controlling the expression of the luxABCDE operon.

For the creation of our biosensor in B. subtilis, the bla-operon from S. aureus was split into three genetic constructs: (A) The Receptor gene blaR1 under control of a strong constitutive promotor (Pveg), (B) the repressor gene blaI under control moderate strong constitutive promoter (PlepA) and (C) the target promoter region of the bla-operon (PblaZ and PblaR1I) in front of the lux-operon (luxABCDE) (see Figure 3). In addition, an inducible version of the blaR1 construct was made by inserting the PxylA promoter upstream of the blaR1 gene (A).[5]

All genetic constructs and plasmids have been created using the RFC10 and/or RFC25 cloning standard. Enzymes used were obtained from New England BioLabs©. Cloning procedures were carried out according to the manufacturer`s protocols.

For submission of our parts to the registry, all Biobricks were cloned into the pSB1C3 backbone. The created genetic constructs were verified by sequencing (Eurofins or GATC sequencing services). All designed plasmids were stored in Escherichia coli DH10β (see Experiments and Protocols for details). In this project, we used integrative single-copy B. subtilis specific vectors that stably integrate into the genome at designated loci.[6]

Table 2: Overview of the basic parts designed for the biosensor project in the pSB1C3 backbone .
Part in pSB1C3 backboneBioBrick Number
pSB1C3-blaR1 BBa_K2273108
pSB1C3-blaZ BBa_K2273109
pSB1C3-blaI BBa_K2273110
pSB1C3-PblaZ BBa_K2273111
pSB1C3-PblaR1I BBa_K2273112
pSB1C3-PpenP(short) BBa_K2273113
pSB1C3-penP BBa_K2273114
pSB1C3-PpenP(long) BBa_K2273116

Biosensor Characterization

First, we investigated the detection range towards different β-lactam families as well as the sensitivity of the created biosensor. Therefore, we conducted plate reader experiments and disk diffusion assays to test our biosensor in liquid as well as on solid conditions. We recorded the luminescence signal and growth behavior (see Experiments and Protocols for details) of our biosensor strains in the presence of six different β-lactam antibiotics. We also included physiological controls that lack one or two of the genetic constructs of the complete biosensor machinery. Furthermore, we analyzed the impact of deleting the Bacillus subtilis gene penP - encoding a β-lactamase (which has not been studied intensively yet) - on the luminescence output. The strain W168 penP::kanR was created via Long-Flanking Homology PCR (see Experiments and Protocols for details). We also investigated, if the different β-lactam antibiotics induce the promoter driving penP.[7]

Encapsulation into Peptidosomes

Graphic-Biosensor in Peptidosomes

Finally, we could demonstrate a fully functional biosensor encapsulated in Peptidosomes. For this we used the strain showing the strongest response, broadest detection range and the highest viability when tested with different β-lactams. Peptidosomes containing the biosensor, were exposed to different β-lactams and development of the luminescence signal was obtained every hour over a time period of 5 hours.


1. Determination of Inhibitory Antibiotic Concentrations

Before starting the actual tests regarding the functionality of the β-lactam biosensor, several pretests have been conducted to determine the optimal antibiotic concentration for the subsequent experiments. Therefore, we analyzed the concentration dependent effect of six different β-lactam antibiotics on the growth of Bacillus subtilis W168 and a strain lacking the B. subtilis native β-lactamase PenP (W168 penP::kanR). We decided to test the following β-lactams in our assays: ampicillin, carbenicillin, cefoperazone, cefoxitin, cefalexin and penicillin G. As controls we chose water (dH2O) and the peptide antibiotic bacitracin, which does not belong to the group of β-lactams.

Table 3: Antibiotic concentrations in [µg µl-1] (final concentration in the well) used in the plate reader assay.
Table 3: β-Lactam concentrations tested in preliminary assays

In pre-tests, we investigated the growth of B. subtilis wild type and the penP mutant, upon exposure to different concentrations of each tested β-lactam (data not shown). After that, we narrowed these down to two concentrations per antibiotic (see Table 3). Since we did not want to kill our biosensors, we focused on β-lactam concentrations which result in a slight inhibition of growth. These concentrations were used for all following experiments to further characterise our biosensors and the effect of the B. subtilis native β-lactamase (PenP) (see Figure 4). Plate reader experiments were performed in Mueller Hinton (MH) media, induction with the antibiotics was carried out after one hour of incubation at 37˚C in the plate reader. Growth was monitored every five minutes for at least 18h.

We expected a higher growth inhibition with rising antibiotic concentrations. In the penP mutant we expected an increased sensitivity towards the β-lactam antibiotics. Addition of water to the culture should not show any effect on the growth and serves as a control. We included a non β-lactam (the peptide antibiotic bacitracin) in all our assays to demonstrate the specificity of the biosensor.

Figure 4: Results from growth inhibition experiment
Figure 4: Growth curves of W168 (1a-f) and W168 penP::kanR (2a-f) showing an effect after treatment with the tested antibiotics indicated in the legend above the graphs. Samples were induced after 1 hour (indicated by the black line) with (a) ampicillin, (b) carbenicillin, (c) cefoperazone, (d) cefalexin, (e) cefoxitin as well as with (f) penicillin G. Bacitracin and dH2O serve as controls. Number 1 and 2 represent the two different concentrations tested of each antibiotic, referring to Table 3. Mean values and standard deviation are depicted from at least three biological replicates.
Table 4: Antibiotic concentrations in [µg µl-1] (final concentration in the well) used in all further plate reader experiments.
Table 2: β-Lactam concentrations tested all subsequent assays

As expected, the data from the pretest performed in triplicates in Figure 4 show, that the presence of the β-lactamase PenP plays a key role in facilitating survival at higher antibiotic concentrations. The wild type strain B. subtilis W168 is therefore able to grow at higher antibiotic concentrations as the mutant W168 penP::kanR when treated with ampicillin, carbenicillin, cefoperazone, cefoxitin and penicillin G (see Figure 4). However, this was not the case for cefalexin and bacitracin. Here, we could not observe any difference in growth inhibition between the wild type and the penP mutant in regard to the tested concentrations (see Figure 4). Cefalexin showed a very strong inhibitory effect on the growth of both wildtype and the mutant. For this reason, we chose a relatively weak final concentration (see Table 4 below). Furthermore, we noticed a growth inhibition of the mutant W168 penP::kanR during the stationary phase, especially when treated with carbenicillin, cefalexin and penicillin G (data not shown). For this reason, we selected weaker antibiotic concentrations for the upcoming experiments with this mutant.

From these first experiments, we selected the final antibiotic concentrations for the upcoming plate reader experiments with the biosensor strains (see Table 4).

2. Analysis of Detection Range and Sensitivity

2.1 Assessing the Detection Range via Plate Reader Assays

During this project, we generated several strains to investigate the functionality of the heterologous constructs constituting the biosensor in Bacillus subtilis. This result section though will focus on the evaluation of the strains shown in Table 5 as these represent the most interesting ones.

In our first experiment, we performed plate reader assays in a 96 well plate format and measured growth (OD600) and luminescence output for 18 hours every 5 minutes. Induction with the β-lactam antibiotics occurred after 1 hour. All strains have been tested in triplicates under the same conditions. Strains with the genotype penP::kanR have been induced with lower concentrations compared to the wild type strain W168 (see Table 4 above).

After induction, we anticipate a strong increase in luminescence signal for strains containing the full set of constructs (PblaZ_lux or PblaR1I_lux, Pveg_blaR1 or Pxyl_blaR1, PlepA_blaI), thus representing functional biosensors. Besides the biosensor constructs, we also tested all physiological controls missing one essential composite of the biosensors' heterologous one-component system (data not shown). 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.

Table 5: Strains of interest with their names and important genotype remarks for differentiation.
Table 5: Genotype remarks of the Strains

As the β-lactamase PenP confers resistance to β-lactam antibiotics in B. subtilis, a rising luminescence signal is estimated for the PpenP_lux constructs post induction. Generally, the Pxyl 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::kanR to give a stronger signal in presence of β-lactam compounds, as they cannot be degraded by the PenP enzyme.

We could not observe a substantial activation of the PblaR1I promoter by the β-lactam compounds, which is why we are not taking it into account in the evaluation below. The bar charts in Figure 5 illustrate the best biosensor constructs identified in the plate reader experiments and compare the RLU/OD600 values of the strains 2 hours post induction with the antibiotics.

Figure 5: Bar Charts showing the detection range of the Biosensors
Figure 5: RLU/OD600 values of the different biosensors and the controls are shown 2 hours after induction with the six β-lactams, bacitracin and dH2O. Graphs show the Wild-type (black), control 1 (light gray), control 2 (dark gray), biosensor 1 (pink), biosensor 2 (purple), biosensor 3 (white and black) and biosensor 3 Xylose induced (dark blue). Luminescence (RLU/OD600) output is shown two hours after β-lactam antibiotic induction. Mean values and standard deviation are depicted from at least three biological replicates.

As shown in Figure 5, 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 5).

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 5, 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 5).

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 PblaZ promoter in biosensor 2, as shown in the bar charts with bacitracin and dH2O, conforms with the one from biosensor 1.

Figure 6: Promoter activity of P<sub><i>penP</i></sub> in the Presence of antibiotics
Figure 6: Luminescence (RLU/OD600) output of the PpenP promoter versions, two hours after β-lactam induction. Strains carrying different PenP promoter versions (long or short / 1 and 2) or with varying genetic backgrounds (either W168 or penP::kanR / a and b). Mean values and standard deviation are depicted from at least three biological replicates.

The activity of biosensor 3 can be induced by adding xylose (0.2% final concentration) to the sample. In absence of xylose, the signal is very weak as can be seen in the bar with the dark blue stripes (see Figure 5). This is due to the fact, that without xylose, nearly no receptor molecules are localized in the bacteria`s inner cell membrane and the signal cannot be detected and amplified. For induction with cefoperazone though, the weak receptor density on the surface because of the leaky expression of blaR1, seems to be enough to cause a quite powerful signal already. From this can be concluded, that the receptor seems to have a high affinity to cefoperazone. In the bar charts of bacitracin and dH2O (see Figure 5), biosensor 3 shows the lowest basal promoter activity compared to the other two biosensors tested. Here, we could also observe a high sensitivity for penicillin G, cefoxitin, cefoperazone and ampicillin. In general, all biosensors show good performance in liquid MH-Medium. They able to detect the six β-lactams reliably by giving a signal way stronger than the basal luminescence.

Furthermore, the analysis of the induction of PpenP by different β-lactam antibiotics unfolded that this promoter seems to be constitutively active during exponential phase (see Figure 6). As the exact promoter length and potential regulatory regions upstream are still unidentified, two versions (short and long) of the promoter have been examined.

The RLU/OD600 values shown in Figure 6 indicate a moderate promoter activity during exponential growth. There is no noticeable difference between the strains with a functional PenP enzyme (a) and the penP mutant (b) (see Figure 6). We could not observe a particular activation by β-lactam antibiotics, which suggests that this enzyme is produced might have other functionalities, too.

2.2 Analyzing the biosensors' behavior on solid medium conducting Disk Diffusion assays

Table 6: Antibiotic concentrations in [µg µl-1] (final concentration in the well) used in the Disk Diffusion assays.
Table 4: β-Lactam concentrations tested Disk Diffusion Assay

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 6.

Figure 7: Disk Diffusion Assay showing the biosensors' activity on solid agar plates
Figure 7: Photographs of the plates from the disk diffusion assay. The upper rows (Panel A and C) show pictures of the plates with the strains under daylight conditions, while the row beneath (Panel B and D) shows the plate after detection of bioluminescence (2 minutes exposure time). At the bottom in Panel E, the disk layout and the most important remarks of the genotype of all strains are indicated.

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 (Pveg) of luciferase. Figure 7 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 day light 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 7, Panel B). Neither bacitracin, nor dH2O lead to an 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 7, Panel D). Au contraire, biosensor 1 only showed a luminescence signal for cefoperazone, cefoxitin and cefalexin (Figure 7, 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 7, Panel D).

Biosensor 2 was activated by all of the β-lactam compounds tested (Figure 7, 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 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 (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 be 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 7 contains the measured diameters of all inhibitions zones caused by the antibiotic. As expected, no inhibition zones around the negative control dH2O were observed.

Table 7: Measured diameters in mm of the inhibition zones around the disks with the different antibiotics on the plates from the disk diffusion assays.
Table 7: Disk Diffusion Assay - Inhibition zone sizes

Figure 8: Disk Diffusion Assay showing the Promoter of activity of P<sub><i><b>penP(long)</b></i></sub> on solid agar plates
Figure 8: Results from the Disk Diffusion Assay with the PpenP(long) reporter strains. Daylight pictures (top row) and luminescence detection using a chemiluminescence dock (with two minutes exposure time). Top right scheme shows the disk layout together with the antibiotics applied in the assay. Please note: only results for the long version of the promoter are shown, as the short version behaves accordingly.

After evaluating our biosensor versions, we were also curious to follow up on the native B. subtilis β-lactamase PenP. Thus, we performed disk diffusion assays with the PPenP (short and long version) reporter strains and checked if any of the β-lactams would lead to a luminescence signal (Figure 8). Unfortunately, none of the tested substance lead to a notable luminescence. We again could only observe a weak basal promoter activity (as in liquid) with both reporter strains. The measured diameters of the inhibition zones are summarised in Table 8. Taking these results together with the observations in liquid (Figure 6), we can state that the native β-lactamase in B. subtilis dose not respond to any of our tested β-lactams. Yet, we could observe increased sensibility of our biosensors, when penP, is knock-out, accounting for ampicillin in liquid conditions (Figure 5) and for all antibiotics tested on solid agar plates (Figure 7). We could also clearly demonstrate increased sensitivity in terms of resistance against β-lactams, when B. subtilis is lacking PenP (Figure 4).

Table 8: Measured diameter of the inhibition zones in [mm] from the Disk Diffusion Assay with the PpenP(long) reporter strains.
Table 8: Inhibition zone sizes in mm  from the Disk Diffusion Assay with the P<sub><i><b>penP(long)</b></i></sub> promoter on solid agar plates

2.3 Examining the Sensitivity of the Biosensor

Figure 9: Disk Diffusion Assay showing the Biosensor`s activity on solid agar plates
Figure 9: Dose-Response Curves of the six different β-lactam antibiotics of biosensor 2. Observed luminescence signal (two hours after antibiotic exposure) was plotted according to each tested antibiotic concentration. Please note both axes are depicted logarithmic. Mean values and standard deviation are depicted from at least three biological replicates.

As a final characterisation 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 (se Figure 9) 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 9). 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 9). Over all, a high dynamic range upon antibiotic exposure can be detected for all compounds starting at concentrations above 10-3.

3. 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 prepare containing no bacteria (A), W168 (B), control 2 (C) and biosensor 2 (D) (see Figure 10 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 0.2 µg 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).

Figure 10: Encapsulation of the biosensor into peptidosomes
Figure 10: Encapsulation experiment with biosensor 2. The pictures in the upper row show the distribution of the Peptidosomes at the time point of luminescence detection, which was immediately performed afterwards using a chemiluminescence dock (bottom row). Pink arrows indicate Peptidosomes with a luminescence signal deriving from the encapsulated biosensor. Upper row of well plates contain non-induced samples. Lower row of well plates were induced with cefoperazone (0.2 µg µl-1).

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 10, 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.


In this part of EncaBcillus we successfully created and analyzed three biosensors that are able to detect six different β-lactam compounds in liquid and on solid medium. We recommend biosensor 2 as our final β-Lactam biosensor, as its performed best: showing the highest detection range of different β-lactams and having the highest sensitivity. In addition to the evaluation of the different biosensors, we even demonstrated the possibility to encapsulate the biosensor in Peptidosomes. By doing so, we confirmed our hypothesis that the antibiotics can diffuse into the Peptidosome and activated the biosensor, which was visible by the luminescence signal. Finally, our encapsulated biosensor is now ready to be used in many potential field applications.


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