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<img src="https://static.igem.org/mediawiki/2017/4/46/T--TU_Dresden--P_Biosensor_Figure2_mechanismbiosensor.png" | <img src="https://static.igem.org/mediawiki/2017/4/46/T--TU_Dresden--P_Biosensor_Figure2_mechanismbiosensor.png" | ||
alt="Figure 1 Molecular mechanism of the Biosensor"class="makeresponsive zoom"> | alt="Figure 1 Molecular mechanism of the Biosensor"class="makeresponsive zoom"> | ||
− | <figcaption><b>Figure 1: Overall concept showing the components and the molecular mechanism of the biosensor in <i><b>B. subtilis</b></i></b>. Upon binding of a | + | <figcaption><b>Figure 1: Overall concept showing the components and the molecular mechanism of the biosensor in <i><b>B. subtilis</b></i></b>. Upon binding of a β-lactam to the receptor BlaR1 <b>(1)</b>, due to the receptors c-terminal proteolytic activity, the repressor BlaI is degraded and frees the target promoter <b>(2)</b> enabling the expression of an easy detectable reporter <b>(3)</b>. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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</div> | </div> | ||
− | <p>This biosensor project turned out to be successful as our biosensor showed a great performance in all conducted experiments. For this reason, we created this section to apply for “best basic part” with the <a target="_blank" href="http://parts.igem.org/Part:BBa_K2273111">P<sub><i>blaZ</i></sub> promoter [BBa_K2273111]</a>. As this promoter showed high activity and reliability when induced by | + | <p>This biosensor project turned out to be successful as our biosensor showed a great performance in all conducted experiments. For this reason, we created this section to apply for “best basic part” with the <a target="_blank" href="http://parts.igem.org/Part:BBa_K2273111">P<sub><i>blaZ</i></sub> promoter [BBa_K2273111]</a>. As this promoter showed high activity and reliability when induced by β-lactams, a clear differentiation between background and the desired signal was possible. The results demonstrated in the paragraphs below, validate the functionality of the biosensor and thus also the functionality of its composites.</p> |
</div class="contentbox"> | </div class="contentbox"> | ||
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<p>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. As the β-lactamase PenP confers resistance to β-lactam antibiotics in <i>B. subtilis</i>, a rising luminescence signal is estimated for the P<sub><i>penP</i></sub>_<i>lux</i> constructs post induction. Generally, the P<sub><i>xyl</i></sub> 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 <i>blaR1</i>.</p> | <p>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. As the β-lactamase PenP confers resistance to β-lactam antibiotics in <i>B. subtilis</i>, a rising luminescence signal is estimated for the P<sub><i>penP</i></sub>_<i>lux</i> constructs post induction. Generally, the P<sub><i>xyl</i></sub> 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 <i>blaR1</i>.</p> | ||
<p>Further we propose biosensor strains carrying the genotype remark <i>penP::kan<sup>R</sup></i> to give a stronger signal in presence of β-lactam compounds, as they cannot be degraded by the PenP enzyme.</p></figure> | <p>Further we propose biosensor strains carrying the genotype remark <i>penP::kan<sup>R</sup></i> to give a stronger signal in presence of β-lactam compounds, as they cannot be degraded by the PenP enzyme.</p></figure> | ||
− | <p>We could not observe a substantial activation of the P<sub><i>blaR1I</i></sub> promoter by the | + | <p>We could not observe a substantial activation of the P<sub><i>blaR1I</i></sub> 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 3 illustrate the best biosensor constructs identified in the plate reader experiments and compare the RLU/OD<sub>600</sub> values of the strains 2 hours post induction with the antibiotics.</p> |
<figure> | <figure> | ||
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<figure class="makeresponsive floatright" style="width: 60%"> | <figure class="makeresponsive floatright" style="width: 60%"> | ||
<img src="https://static.igem.org/mediawiki/2017/6/65/T--TU_Dresden--P_Biosensor_Figure9.png" | <img src="https://static.igem.org/mediawiki/2017/6/65/T--TU_Dresden--P_Biosensor_Figure9.png" | ||
− | alt="Figure | + | alt="Figure 5: Disk Diffusion Assay showing the Biosensor`s activity on solid agar plates" class="zoom"> |
− | <figcaption><b>Figure | + | <figcaption><b>Figure 5: Dose-Response Curves of the six different β-lactam antibiotics of biosensor 2.</b> 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. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
<p>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).</p> | <p>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).</p> | ||
− | <p>The obtained results 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. The antibiotics cefalexin, ampicillin and penicillin G lead to a decrease in luminescence signal for concentrations higher than 10<sup>-1</sup>, due to the growth inhibition at these concentrations. Over all, a high dynamic range upon antibiotic exposure can be detected for all compounds starting at concentrations above 10<sup>-3</sup>.</p></figure> | + | <p>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<sup>-1</sup>, due to the growth inhibition at these concentrations (see Figure 5). Over all, a high dynamic range upon antibiotic exposure can be detected for all compounds starting at concentrations above 10<sup>-3</sup>.</p></figure> |
<p></p> | <p></p> | ||
<hr> | <hr> | ||
<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 prepare containing no bacteria (A), W168 (B), Control 2 (C) and Biosensor 2 (D) (see Figure | + | <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 prepare 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<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> | ||
+ | <p></p> | ||
<figure class="makeresponsivet" style="width: 100%"> | <figure class="makeresponsivet" style="width: 100%"> | ||
<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 | + | alt="Figure 6: Encapsulation of the biosensor into peptidosomes" class="zoom"> |
− | <figcaption><b>Figure | + | <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. Upper row of well plates contain non-induced samples. Lower row of well plates were induced with cefoperazone (0.2µg µl<sup>-1</sup>). |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
<p></p> | <p></p> | ||
− | <p>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 | + | <p>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. </p> |
− | <p></p><p></p> | + | <p></p> |
+ | </div class="contentbox"> | ||
+ | <div class="contentbox"> | ||
+ | <h1 class="box-heading">Summary</h1> | ||
+ | <p>Taking together all the results obtained in this project, we can conclude that all three biosensors show excellent functionality under various different conditions. All strains are able to detect the six β-lactams, though the biosensors 2 and 3 perform better on solid MH-medium. Generally speaking, the P<sub><i>blaZ</i></sub> promoter, as part of the biosensor strains, generates a high luminescence signal that can be easily detected in liquid and on solid media. Further, our results show high reproducibility of the strong promoter activity in the conducted experiments evaluated in the section above.</p> | ||
+ | <p>Another potential application for the P<sub><i>blaZ</i></sub> promoter other than in the context of a biosensor would be in the framework of an expression system. As already very low concentrations of e.g. cefoperazone are leading to strong activation of the promoter by the BlaR1-BlaI system, you could think of replacing the lux-operon by any gene of interest. This promoter reached even higher activities than the constitutive promoter P<sub><i>veg</i></sub>. For this reason, we also propose this system for the overexpression of proteins of interest. </p> | ||
+ | </div class="contentbox"> | ||
</div> | </div> |
Revision as of 12:54, 1 November 2017