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alt="Table 2: β-Lactam concentrations tested all subsequent assays" class="zoom"> | alt="Table 2: β-Lactam concentrations tested all subsequent assays" class="zoom"> | ||
</figure> | </figure> | ||
− | <p>First, we investigated the detection range towards different | + | <p>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 to test our biosensor in liquid conditions. We recorded the luminescence signal and growth behavior (see <a href ="https://2017.igem.org/Team:TU_Dresden/Experiments">Experiments and Protocols</a> 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 (data not shown). |
Furthermore, we analyzed the impact of deleting the <i>Bacillus subtilis</i> gene <i>penP</i> - encoding a β-lactamase (which has not been studied intensively yet) - on the luminescence output. The strain W168 <i>penP::kan<sup>R</sup></i> was created via Long-Flanking Homology PCR (see <a href ="https://2017.igem.org/Team:TU_Dresden/Experiments">Experiments and Protocols</a> for details). </p> | Furthermore, we analyzed the impact of deleting the <i>Bacillus subtilis</i> gene <i>penP</i> - encoding a β-lactamase (which has not been studied intensively yet) - on the luminescence output. The strain W168 <i>penP::kan<sup>R</sup></i> was created via Long-Flanking Homology PCR (see <a href ="https://2017.igem.org/Team:TU_Dresden/Experiments">Experiments and Protocols</a> for details). </p> | ||
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<p>As shown in Figure 3, the wildtype W168 (black with white dots) shows no increase in RLU/OD<sub>600</sub> 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 <i>lux</i> operon resulted in an RLU/OD<sub>600</sub> of over 1.3 million for control 2 (see Figure 3).</p> | <p>As shown in Figure 3, the wildtype W168 (black with white dots) shows no increase in RLU/OD<sub>600</sub> 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 <i>lux</i> operon resulted in an RLU/OD<sub>600</sub> of over 1.3 million for control 2 (see Figure 3).</p> | ||
<p>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/OD<sub>600</sub> (see Figure 3, bar chart with bacitracin and dH<sub>2</sub>O). 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/OD<sub>600</sub>. 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).</p> | <p>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/OD<sub>600</sub> (see Figure 3, bar chart with bacitracin and dH<sub>2</sub>O). 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/OD<sub>600</sub>. 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).</p> | ||
− | <p>For biosensor 2, the detection range and sensitivity is comparable to biosensor 1 | + | <p>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/OD<sub>600</sub>. Even the basal activity of the P<sub><i>blaZ</i></sub> promoter in biosensor 2, as shown in the bar charts with bacitracin and dH<sub>2</sub>O, conforms with the one from biosensor 1.</p> |
</figure> | </figure> | ||
<hr> | <hr> | ||
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<br> | <br> | ||
− | <p>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 <i>lux</i> operon. In the case of control 2 a luminescence signal should be spread over the whole plate, due to the constitutive expression (P<sub><i>veg</i></sub>) 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).</p> | + | <p>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 <i>lux</i> operon. In the case of control 2, a luminescence signal should be spread over the whole plate, due to the constitutive expression (P<sub><i>veg</i></sub>) 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).</p> |
<p>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 dH<sub>2</sub>O 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).</p> | <p>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 dH<sub>2</sub>O 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).</p> | ||
<p>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. | <p>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 dH<sub>2</sub>O were observed.</p> | 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 dH<sub>2</sub>O were observed.</p> | ||
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<figure> | <figure> | ||
<figure class="makeresponsive floatright" style="width: 100%"> | <figure class="makeresponsive floatright" style="width: 100%"> | ||
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<h3 id="peptidosomes">4. Encapsulation of the Biosensor into Peptidosomes – Proving the Application Potential</h3> | <h3 id="peptidosomes">4. Encapsulation of the Biosensor into Peptidosomes – Proving the Application Potential</h3> | ||
<p></p> | <p></p> | ||
− | <p>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), | + | <p>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 0.2 µg µl<sup>-1</sup> cefoperazone happened after 1 hour of growth. |
</p> | </p> | ||
<p>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).</p> | <p>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).</p> | ||
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<img src="https://static.igem.org/mediawiki/2017/9/90/T--TU_Dresden--P_Biosensor_Figure10.png" | <img src="https://static.igem.org/mediawiki/2017/9/90/T--TU_Dresden--P_Biosensor_Figure10.png" | ||
alt="Figure 6: Encapsulation of the biosensor into peptidosomes" class="zoom"> | alt="Figure 6: Encapsulation of the biosensor into peptidosomes" class="zoom"> | ||
− | <figcaption><b>Figure 6: Encapsulation experiment with biosensor 2.</b> 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. The upper row of well plates contains non-induced samples. Lower row of well plates were induced with cefoperazone (0.2µg µl<sup>-1</sup>). | + | <figcaption><b>Figure 6: Encapsulation experiment with biosensor 2.</b> 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. The upper row of well plates contains non-induced samples. Lower row of well plates were induced with cefoperazone (0.2 µg µl<sup>-1</sup>). |
</figcaption> | </figcaption> | ||
</figure> | </figure> |
Latest revision as of 02:35, 2 November 2017