Revision as of 09:54, 30 October 2017

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 Beta-lactam biosensor in Bacillus subtilis. These specialised cells are capable of sensing a compound of the beta-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 Beta-lactam antibiotics from various subclasses. The evaluated Biosensor was then encapsulated into Peptidosomes to proof the concept of our project EncaBcillus. The encapsulation of engineered bacteria allows an simplified handling and increased biosafety, potentially raising the chances for their application in e.g. sewage treatment plants.

Background

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

Table 1 Beta-Lactam Compounds Classification — **Figure 1: Commonly used Beta-lactam antibiotics and their chemical structure.** All of them share the so-called Beta-lactam ring structure (here shown as square structure containing nitrogen).

Design

Genetic engineering

To achieve our goal of encapsulating bacteria into Peptidosomes that can sense antibiotics of the beta-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 beta-lactam antibiotics. The idea was to transfer the regulatory elements of this operon to Bacillus subtilis and replace the native output – being the beta-lactamase BlaZ – by an easy detectable signal. Thus, making Bacillus subtilis a beta-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 beta-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 P_veg or the inducible promoter P_xylA, **(B)** *blaI* downstream of the promoter P_lepA, and **(C)** the promoters P_blaZ or P_blaR1I 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 (P_veg), (B) the Repressor gene blaI under control moderate strong constitutive promoter (P_lepA) and (C) the target promoter region of the bla-operon (P_blaZ and P_blaR1I) in front of the lux-operon (luxABCDE) (see Figure 3). In addition, an inducible version of the blaR1 construct was made by inserting the P_xylA 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]

Biosensor Characterization

First, we investigated the detection range towards different beta-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 beta-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 beta-lactamase (which has not been studied intensively yet) - on the luminescence output. The strain W168 penP::kan^R was created via Long-Flanking Homology PCR (see Experiments and Protocols for details). We also investigated, if the different beta-lactam antibiotics induce the promoter driving PenP.[7]

Encapsulation into 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 beta-lactams. Peptidosomes containing the biosensor, were exposed to different Beta-lactams and development of the luminescence signal was obtain every hour over a time period of 5 hours.

Results

1. Determination of Inhibitory Antibiotic Concentrations

Before starting the actual tests regarding the functionality of the beta-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 beta-lactam antibiotics on the growth of Bacillus subtilis W168 and a strain lacking the B. subtilis native beta-lactamase PenP (W168 penP::kan^R). We decided to test the following beta-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 beta-lactams.

Table 3: Antibiotic concentrations in [µg µl^-1] (final concentration in well) used in the killing assay.

In pre-tests, we investigated the growth of B. subtilis wild type and the PenP mutant, upon exposure to different concentrations of each tested beta-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 beta-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 beta-lactamase (PenP) (see Figure 4). Plate reader experiments were performed in Mueller Hinton 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 beta-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 beta-lactam (the peptide antibiotic bacitracin) in all our assays to demonstrate the specificity of the biosensor.

Figure 4: Growth curves of W168 (1a-f) and W168 penP::kan^R (2a-f) showing an effect after treatment with the tested antibiotics indicated in the legend above the graphs. Samples were induced after 1 hour with (a) ampicillin 1 and 2, (b) carbenicillin 1 and 2, (c) cefoperazone 1 and 2, (d) cefalexin 1 and 2, (e) cefoxitin 1 and 2 as well as with (f) penicillin G 1 and 2. Every graph shows also the controls, where we induced with bacitracin 1 and 2 and dH₂O. Number 1 and 2 represent the two different concentrations tested of each antibiotic. The concentrations refer to table 3.

Table 4: Antibiotic concentrations in [µg µl^-1] (final concentration in well) used in all further plate reader experiments.

As expected, the data from the pretest performed in triplicates in Figure 4 show, that the presence of the beta-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::kan^R 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::kan^R 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

Table 4: Antibiotic concentrations in [µg µl^-1] (final concentration in well) used in all further plate reader experiments.

During this project, we generated several strains to investigate the functionality of the heterologous constructs constituting the biosensor in Bacillus subtilis [file strains?]. 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 Beta-lactam antibiotics occurred after 1 hour. All strains have been tested in triplicates under the same conditions. Strains with the genotype penP::kan^R 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 (P_blaZ_lux or P_blaR1I_lux, P_veg_blaR1 or P_xyl_blaR1, P_lepA_blaI), thus representing functional biosensors. Besides the biosensor constructs, we also tested all physiological controls missing one essential composite of the biosensor`s 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.

As the beta-lactamase PenP confers resistance to Beta-lactam antibiotics in B. subtilis, a rising luminescence signal is estimated for the P_penP_lux constructs post induction. 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 Beta-lactam compounds, as they cannot be degraded by the PenP enzyme.

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: RLU/OD₆₀₀ values of the different biosensors and the controls at 2 hours after induction with the 6 Beta-lactams, bacitracin and dH₂O. The legend above defines the coloration of the single bars in each chart. Error bars indicate the standard deviation. The white text boxes contain the maximal RLU/OD_{600 that was reached reached 2 hours post induction.}

As shown in Figure 5, the wildtype W168 (black with white dots) shows no increase in RLU/OD₆₀₀ values when induced with the different Beta-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 Beta-lactam antibiotics tested, but also shows a higher basal activity in absence of the Beta-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 Beta-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 P_blaZ promoter in biosensor 2, as shown in the bar charts with bacitracin and dH₂O, conforms with the one from biosensor 1.

Figure6: RLU/OD₆₀₀ 2 hours after induction of the P_penP Promoter versions (P_penP(long) (Strain 1) and P_penP(short) (Strain 2) in W168 (a) and W168 penP::kan^R(b)) with beta-lactam antibiotics and the controls.

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 beta-lactams reliably by giving a signal way stronger than the basal luminescence.

Furthermore, the analysis of the induction of P_penP by different Beta-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 Beta-lactam antibiotics, which suggests that this enzyme is produced might have other functionalities, too.

2.2 Analyzing the Biosensor`s Behavior on solid Medium via Disk Diffusion assays

To determine whether the results obtained in the plate reader assays using liquid media conditions can be reproduced on solid agar plates, disk diffusion assays with MH-Medium were implemented. This assay would visualize the signal intensity and detection range of the biosensor by a glowing halo at the edge of the inhibition zones, where bacteria are exposed to the antibiotic compounds. Here, we tested the same beta-lactam antibiotics and controls as in the previous experiments, but we chose higher concentrations to cause small inhibition zones around the disks.

Figure7: Photographs of the plates from the disk diffusion assay. The upper row shows a picture of the strains at daylight, while the row beneath shows the plate after detection of chemiluminescence (2 minutes exposure time). At the bottom, the disk layout and the most important remarks of the genotype of all strains are indicated.

We would expect the controls to not cause any luminescence signal at the edge of the inhibition zones, while the beta-lactam antibiotics should lead to a glowing halo around the disks on the plates with the three biosensors. The wildtype strain and control 1 will not show any signal, while the luminescence signal of control 2 should be spread over the whole plate. In Figure 7 on the right, you can see the results of the disk diffusion assay where plates have been photographed after 24 hours of growth.

For the wildtype and control 1, no luminescence signal was detected on the plate, while control 2 displays a strong luminescence signal on the whole plate (see Figure 7). While Biosensor 1 has a similar detection range like biosensor 2 in liquid medium, there is a huge difference in the detection range on agar plates. We could observe a luminescence signal for cefoperazone, cefoxitin and cefalexin on the lawn of Biosensor 1. Though the signal for cefalexin seems weaker, as for the other two compounds. Further, the luminescence halo around the cefoxitin disk is quite broad compared to the others, indicating a far diffusion of the compound into the lawn. Despite biosensor 1 seems to be activated by penicillin G in liquid medium, we could not observe an induction on plate.

Biosensor 2 was activated by all of the beta-lactam compounds tested. 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, all beta-lactams could be detected quite well in the presence of 0.2% xylose. 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 have direct contact with the antibiotic, but rather a bit farer from the inhibition zone on the lawn. Without induction of Biosensor 3 with 0.2% xylose, we could not detect any luminescence signal, despite one very weak signal around the cefoperazone disk (data not shown). Again, these results are comparable to those seen before in the experiments in liquid medium.

Figure8: Results from the Disk Diffusion Assay with the P_penP(long) reporter strains. Below the daylight photographs, luminescence has been detected using an exposure time of 2 minutes. The right schematic shows the disk layout applied in the assay.

Neither bacitracin, nor dH₂O lead to an activation of the biosensor machinery in all strains.

Figure 8 illustrates the results of the disk diffusion assay implemented with the PpenP reporter strains. In comparison to the controls W168 and control 1 shown in figure 6, a slight luminescence signal was detected on these plates, that could not really be related to a specific antibiotic, but rather covered the whole lawn. In this figure, just the results for the long version of the promoter are depicted, as the short version reproduces these findings.

2.3 Examining the Sensitivity of the Biosensor

We carried out Dose Response Assays in order to investigate the detection sensitivity of our Biosensor. In this experiment, 11 different concentrations of the beta-lactam antibiotics and bacitracin (control) have been tested on biosensor 2. This Biosensor was chosen due to its reliable performance in liquid and on solid medium and because there is no need of further induction as there is for biosensor 3.

Figure 9: Dose-Response Curves of the 6 different Beta-Lactam antibiotics showing the sensitivity of biosensor 1 represented by the luminescence signal, when induced with various concentrations.

The graphs in Figure 9 (right) represent the dose-response curves for the different beta-lactams.

These results indicate that biosensor 1 shows the highest sensitivity for cefoperazone and therefore senses very weak concentrations of this compound. An increasing in luminescence signal can be seen for concentrations above 10^-3 for all beta-lactams tested. Biosensor 1 shows the weakest signal in response to cefalexin, which could be observed in the previous experiments, too. The curves cefalexin, ampicillin and penicillin G also demonstrate a decrease in luminescence signal for concentrations higher than 10^-1. The findings of this experiment reproduce the results obtained in the previous experiments described above.

3. Encapsulation of the Biosensor into Peptidosomes – Proofing the Application Potential

After evaluation of the Biosensor we probed its activity when encased into Peptidosomes. An overnight culture was inoculated in FmocFF-Solution with a final OD600=10. Peptidosomes were prepare containing no bacteria (A), W168 (B), Control 2 (C) and Biosensor 1 (D) (see Figure 9 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.

In this experiment, we successfully encapsulated biosensor 1 into Peptidosomes and demonstrated its ability to sense the beta-lactam cefoperazone diffusing into the Peptidosome. Already 2 hours post induction, there is a luminescence signal detectable for control 2 and the encapsulated biosensor 1 (see Figure 9, middle, C and D). Four hours post induction, we could observe an increase in luminescence signal for biosensor 1 in the Peptidosomes (see Figure 9, right, D). The non-induced sample of control 2 shows a breakage of the Peptidosome and thus a spreading of the luminescent bacteria in the well after four hours (Figure 9, right, C).

Figure 10: Encapsulation Experiment with Biosensor 1.The pictures in the upper row show the distribution of the Peptidosomes at the time point of luminescence detection. The plate layout is defined in the legend beneath the photographs. Pink arrows indicate Peptidosomes with a luminescence signal due to the encapsulated biosensor. Upper row of well plate: non-induced samples. Lower row of well plate: induced with cefoperazone (0.2µg µl^-1).

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 1 (D, +AB). No signal is expected for the encapsulated biosensor in absence of cefoperazone (D, -AB).

Conclusion

In this part of EncaBcillus we successfully created and analyzed three biosensors that are able to detect six different Beta-lactam compounds with high sensitivity in liquid and on solid medium. We recommend biosensor 1 as our final Beta-Lactam Biosensor, as its performance was proven in liquid and on solid medium and the results show high reproducibility. In case, an inducible version of the biosensor is of interest, we propose biosensor 3 as the best option to detect beta-lactams. In addition to the evaluation of the different biosensors, we even demonstrated the possibility to encapsulate genetically engineered bacteria into Peptidosomes. The trapping of our biosensor confirmed our hypothesis that the antibiotics can diffuse into the Peptidosome and further are able to activate the biosensor leading to a luminescence output. Finally, our encased biosensor is now ready to be used in a lot of potential field applications.

References

Beam me up!

[1] C. Lee Ventola, MS (2015) The antibiotic resistance crisis: part 2: management strategies and new agents. Pharmacy and Therapeutics 40(5), 344–352

[2] www.aerzteblatt.de, visited 08/23/17 (5:34pm)

[3] www.who.int, visited 09/04/17 (3:21pm)

[4] https://en.wikipedia.org/wiki/Β-lactam_antibiotic, visited 10/27/17 (4:42pm)

[5] Leticia I. Llarrull, Mary Prorok, and Shahriar Mobashery (2010) Binding of the Gene Repressor BlaI to the bla Operon in Methicillin-Resistant Staphylococcus aureus. Biochemistry 49(37), 7975–7977

[6] Radeck, J., Kraft, K., Bartels, J., Cikovic, T., Dürr, F., Emenegger, J., Kelterborn, S., Sauer, C., Fritz, G., Gebhard, S., and Mascher, T. (2013) The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 7(29),

[7] Toth, M., Antunes, N.T., Stewart, N.K., Frase, H., Bhattacharya, M., Smith, C. and Vakulenko, S. (2016) Class D β-lactamases do exist in Gram-positive bacteria. Nature Chemical Biology 12(1),9-14

iGEM-Team TU Dresden
Institut für Mikrobiologie, AG Thorsten Mascher
Zellescher Weg 20b, 01217 Dresden
Germany

@igem.tud2017

@igem_tud_2017

igem2017@mailbox.tu-dresden.de

@@ Line 8: / Line 8: @@
 <div class="contentbox">
 <h1 class="box-heading">Short Description</h1>
-<p>Worldwide, multidrug-resistant germs 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 subproject, we developed a functional and complete heterologous Beta-lactam biosensor in Bacillus subtilis. By the time these specified cells sense a compound of the beta-lactam family, they will respond by producing a measurable luminescence signal.  Thereby, we analyzed the detection range and sensitivity of the biosensor in response to six different Beta-lactam antibiotics from various subclasses. The evaluated Biosensor was then encapsulated into Peptidosomes to proof the concept of our project EncaBcillus. The trapping of engineered bacteria thus will allow for increased control and simplified handling, potentially raising the chances for their application in e.g. sewage treatment plants.</p>
+<p>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 Beta-lactam biosensor in <i>Bacillus subtilis</i>. These specialised cells are capable of sensing a compound of the beta-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 Beta-lactam antibiotics from various subclasses. The evaluated Biosensor was then encapsulated into Peptidosomes to proof the concept of our project EncaBcillus. The encapsulation of engineered bacteria allows an simplified handling and increased biosafety, potentially raising the chances for their application in e.g. sewage treatment plants.</p>
 </div class="contentbox">
@@ Line 27: / Line 27: @@
 <p></p>
 <p>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.<a target="_blank" href ="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422635/">[2]</a><a target="_blank" href ="http://www.who.int/mediacentre/factsheets/fs194/en/">[3]</a></p>
-<p>As Beta-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 beta-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 beta-lactam ring (see Figure1).<a target="_blank" href ="https://en.wikipedia.org/wiki/Β-lactam_antibiotic">[4]</a></p>
+<p>As Beta-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 beta-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 beta-lactam ring (see Figure 1).<a target="_blank" href ="https://en.wikipedia.org/wiki/Β-lactam_antibiotic">[4]</a></p>
 <p>To address the increasing development of multi-drug resistant bacteria our iGEM Team aims at developing a novel beta-lactam biosensor in <i>Bacillus subtilis</i> based on the genetics of the <i>bla</i>-operon found in <i>Staphylococcus aureus</i> (for a detailed description consider our Design section below).
-The genetically engineered Biosensor will help to <b>(I)</b> reliably detect even minimal antibiotic concentrations of compounds from the beta-lactam family in waste and drinking water and <b>(II)</b> unravel producer strains of yet unknown Beta-lactam related antibiotics. After extensive characterization of the detection range and sensitivity, the greater goal is to combine the functional Beta-lactam biosensor with our Peptidosomes. Thereby, we would proof 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 Beta-lactams and two controls (water and bacitracin) to evaluate our Biosensor (table 1).</p></figure>
+The genetically engineered Biosensor will help to <b>(I)</b> reliably detect even minimal antibiotic concentrations of compounds from the beta-lactam family in waste and drinking water and <b>(II)</b> unravel producer strains of yet unknown Beta-lactam related antibiotics. After extensive characterization of the detection range and sensitivity, the greater goal is to combine the functional Beta-lactam biosensor with our Peptidosomes. Thereby, we would proof 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 Beta-lactams and two controls (water and bacitracin) to evaluate our Biosensor (Table 1).</p></figure>
@@ Line 60: / Line 60: @@
 </div>
-<p>For the creation of our biosensor in B. subtilis, the bla-operon from S. aureus was split into three genetic constructs: <b>(A)</b> The Receptor gene blaR1 under control of a strong constitutive promotor (Pveg), <b>(B)</b> the Repressor gene blaI under control moderate strong constitutive promoter (P<sub><i>lepA</i></sub>) and <b>(C)</b> the target promoter region of the <i>bla</i>-operon (P<sub><i>blaZ</i></sub> and P<sub><i>blaR1I</i></sub>) in front of the <i>lux</i>-operon (<i>luxABCDE</i>) (see Figure 3). In addition, an inducible version of the <i>blaR1</i> construct was made by placing the P<sub><i>xylA</i></sub> promoter upstream of the <i>blaR1</i> gene <b>(A)</b>.<a target="_blank" href ="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2942778/">[5]</a></p>
+<p>For the creation of our biosensor in <i>B. subtilis</i>, the <i>bla<i>-operon from <i>S. aureus</i> was split into three genetic constructs: <b>(A)</b> The Receptor gene <i>blaR1</i> under control of a strong constitutive promotor (P<sub><i>veg</i></sub>), <b>(B)</b> the Repressor gene blaI under control moderate strong constitutive promoter (P<sub><i>lepA</i></sub>) and <b>(C)</b> the target promoter region of the <i>bla</i>-operon (P<sub><i>blaZ</i></sub> and P<sub><i>blaR1I</i></sub>) in front of the <i>lux</i>-operon (<i>luxABCDE</i>) (see Figure 3). In addition, an inducible version of the <i>blaR1</i> construct was made by inserting the P<sub><i>xylA</i></sub> promoter upstream of the <i>blaR1</i> gene <b>(A)</b>.<a target="_blank" href ="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2942778/">[5]</a></p>
-<p>All genetic constructs and plasmids have been created using the RFC10 <a target="_blank" href ="http://parts.igem.org/Help:Standards/Assembly/RFC10">RFC10</a> and/or <a target="_blank" href ="http://parts.igem.org/Assembly_standard_25">RFC25</a> cloning standard. Enzymes used were obtained from New England BioLabs&copy;. Cloning procedures were carried out according to the manufacturer`s protocols. </p>
+<p>All genetic constructs and plasmids have been created using the <a target="_blank" href ="http://parts.igem.org/Help:Standards/Assembly/RFC10">RFC10</a> and/or <a target="_blank" href ="http://parts.igem.org/Assembly_standard_25">RFC25</a> cloning standard. Enzymes used were obtained from New England BioLabs<sup><&copy;</sup>. Cloning procedures were carried out according to the manufacturer`s protocols. </p>
-<p>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 <i>Escherichia coli</i> DH10&beta; (see <a target="_blank" href ="https://2017.igem.org/Team:TU_Dresden/Experiments">Experiments and Protocols</a> for details). In this project, we used integrative single-copy B. subtilis specific vectors that stably integrate into the genome at designated loci.<a target="_blank" href ="https://jbioleng.biomedcentral.com/articles/10.1186/1754-1611-7-29">[6]</a></p>
+<p>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 <i>Escherichia coli</i> DH10&beta; (see <a target="_blank" href ="https://2017.igem.org/Team:TU_Dresden/Experiments">Experiments and Protocols</a> for details). In this project, we used integrative single-copy <i>B. subtilis</i> specific vectors that stably integrate into the genome at designated loci.<a target="_blank" href ="https://jbioleng.biomedcentral.com/articles/10.1186/1754-1611-7-29">[6]</a></p>
 <hr>
@@ Line 82: / Line 82: @@
 <h1 class="box-heading">Results</h1>
 <h3><b>1. Determination of Inhibitory Antibiotic Concentrations</b></h3>
-<p>Before starting the actual tests regarding the functionality of the beta-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 beta-lactam antibiotics on the growth of <i>Bacillus subtilis</i> W168 and a strain missing the functional beta-lactamase PenP (W168 <i>penP::kan<sup><i>R</i></sup></i>). The beta-lactams tested in this and the following assays were: Ampicillin, Carbenicillin, Cefoperazone, Cefoxitin, Cefalexin and Penicillin G. As controls we chose water (dH<sub>2</sub>O) and the antibiotic Bacitracin, which does not belong to the group of beta-lactams. </p>
+<p>Before starting the actual tests regarding the functionality of the beta-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 beta-lactam antibiotics on the growth of <i>Bacillus subtilis</i> W168 and a strain lacking the <i>B. subtilis</i> native beta-lactamase PenP (W168 <i>penP::kan<sup><i>R</i></sup></i>). We decided to test the following beta-lactams in our assays: Ampicillin, Carbenicillin, Cefoperazone, Cefoxitin, Cefalexin and Penicillin G. As controls we chose water (dH<sub>2</sub>O) and the peptide antibiotic Bacitracin, which does not belong to the group of beta-lactams. </p>
 <figure>
 <figure class="makeresponsive floatright"  style="width: 60%">
-<figcaption><b>Table 3: Antibiotic concentrations in [µg µl<sup>-1</sup>] (final concentration in well) used in the killing assay carried out in triplicates.
+<figcaption><b>Table 3: Antibiotic concentrations in [µg µl<sup>-1</sup>] (final concentration in well) used in the killing assay.
 </b></figcaption>
        <img src="https://static.igem.org/mediawiki/2017/8/86/T--TU_Dresden--P_Biosensor_Table2_betalactamconcentrations.png"
@@ Line 91: / Line 91: @@
 </figure>
-<p>Several preliminary plate reader experiments have been implemented using a 96 well plate format, in which we tested at least six different concentrations of each antibiotic. These assays were not performed in triplicates. Finally, the concentrations were narrowed down to two concentrations per antibiotic (see Table 3) that would cause a slight growth inhibition, to use in the following assay performed in triplicates (see Figure 4). All tests regarding antibiotic effects were implemented with Mueller Hinton Medium. The strains were induced after 1 hour of growth at 37&#176;C in the plate reader. The growth was followed by measuring the optical density at OD=600nm for 18 hours every 5 minutes. </p>
+<p>In pre-tests, we investigated the growth of <i>B. subtilis</i> wild type and the PenP mutant, upon exposure to different concentrations of each tested beta-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 beta-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 <i>B. subtilis</i> native beta-lactamase (PenP) (see Figure 4). <a href ="https://2017.igem.org/Team:TU_Dresden/Experiments">Plate reader</a> experiments were performed in <a href ="https://2017.igem.org/Team:TU_Dresden/Experiments">Mueller Hinton</a> 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.</p>
 </figure>
-<p>We expected a higher growth inhibition at higher antibiotic concentrations and a higher sensitivity to the antibiotics of the <i>penP</i> mutant. Adding distilled water to the culture should not show any effect on the growth of both tested strains.</p>
+<p>We expected a higher growth inhibition with rising antibiotic concentrations. In the <i>penP</i> mutant we expected an increased sensitivity towards the beta-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 beta-lactam (the peptide antibiotic bacitracin) in all our assays to demonstrate the specificity of the biosensor.</p>
 <figure class="makeresponsive floatright"  style="width: 100%">
@@ Line 105: / Line 105: @@
 <figure>
 <figure class="makeresponsive floatright"  style="width: 40%">
-<figcaption><b>Table4: Antibiotic concentrations in [&#181;g &#181;l<sup>-1</sup>] (final concentration in well) used in all further plate reader experiments.
+<figcaption><b>Table 4: Antibiotic concentrations in [&#181;g &#181;l<sup>-1</sup>] (final concentration in well) used in all further plate reader experiments.
 </b></figcaption>
        <img src="https://static.igem.org/mediawiki/2017/a/ac/T--TU_Dresden--P_Biosensor_Table4_finalconcentrations_correct.png"
@@ Line 135: / Line 135: @@
 <figure>
 <figure class="makeresponsive floatleft"  style="width: 70%">
-<figcaption><b>Table5: Strains of interest with their names and important genotype remarks for differentiation.</b></figcaption>
+<figcaption><b>Table 5: Strains of interest with their names and important genotype remarks for differentiation.</b></figcaption>
        <img src="https://static.igem.org/mediawiki/2017/f/f6/T--TU_Dresden--P_Biosensor_Table5_genotypeBiosensors.png"
             alt="Table 5: Genotype remarks of the Strains" class="zoom">

[1]	C. Lee Ventola, MS (2015) The antibiotic resistance crisis: part 2: management strategies and new agents. Pharmacy and Therapeutics 40(5), 344–352
[2]	www.aerzteblatt.de, visited 08/23/17 (5:34pm)
[3]	www.who.int, visited 09/04/17 (3:21pm)
[4]	https://en.wikipedia.org/wiki/Β-lactam_antibiotic, visited 10/27/17 (4:42pm)
[5]	Leticia I. Llarrull, Mary Prorok, and Shahriar Mobashery (2010) Binding of the Gene Repressor BlaI to the bla Operon in Methicillin-Resistant Staphylococcus aureus. Biochemistry 49(37), 7975–7977
[6]	Radeck, J., Kraft, K., Bartels, J., Cikovic, T., Dürr, F., Emenegger, J., Kelterborn, S., Sauer, C., Fritz, G., Gebhard, S., and Mascher, T. (2013) The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 7(29),
[7]	Toth, M., Antunes, N.T., Stewart, N.K., Frase, H., Bhattacharya, M., Smith, C. and Vakulenko, S. (2016) Class D β-lactamases do exist in Gram-positive bacteria. Nature Chemical Biology 12(1),9-14

Difference between revisions of "Team:TU Dresden/Project/Biosensor"

Revision as of 09:54, 30 October 2017

Short Description

Background

Design

Genetic engineering

Biosensor Characterization

Encapsulation into Peptidosomes

Results

1. Determination of Inhibitory Antibiotic Concentrations

2. Analysis of Detection Range and Sensitivity

2.1 Assessing the Detection Range via Plate Reader Assays

2.2 Analyzing the Biosensor`s Behavior on solid Medium via Disk Diffusion assays

2.3 Examining the Sensitivity of the Biosensor

3. Encapsulation of the Biosensor into Peptidosomes – Proofing the Application Potential

Conclusion

References