Difference between revisions of "Team:TU Dresden/Basic Part"
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<h1 class="box-heading">Introduction</h1> | <h1 class="box-heading">Introduction</h1> | ||
| − | <p>As part of the EncaBcillus project, we developed a novel and complete heterologous biosensor for | + | <p>As part of the EncaBcillus project, we developed a novel and complete heterologous biosensor for β-lactam antibiotics in <i>Bacillus subtilis</i>. This biosensor is based on a one-component system encoded in the so-called <i>bla-operon</i> naturally found in <i>Staphylococcus aureus</i>. The biosensor is composed of three composites from this operon: The β-lactam receptor BlaR1 receptor and the repressor BlaI which have been codon-adapted for expression in <i>B. subtilis</i> as well as the <a target="_blank" href="http://parts.igem.org/Part:BBa_K2273111">P<sub><i>blaZ</i></sub> promoter [BBa_K2273111]</a>(see Figure 2). This promoter was inserted upstream of the <i>lux</i>-operon, our reporter of choice. Figure 1 displays the molecular mechanism of the established biosensor. In case a β-lactam is bound to BlaR1, the receptor`s proteolytic c-terminal domain degrades the BlaI repressor, thereby releasing the P<sub><i>blaZ</i></sub> promoter. This enables binding of the transcription machinery to the promoter and therefore the expression of the <i>luxABCDE</i> genes, resulting in a luminescence signal produced by the bisosensor. </p> |
| − | + | <div style="display: flex; align-items: flex-start; flex-wrap: wrap;"> | |
| + | <div class="makeresponsive" style="width:50%;"> | ||
| + | <figure class="makeresponsive" style="padding-left:10%; padding-right:10%;"> | ||
<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 | + | 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 beta-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><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 beta-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> | ||
| + | </div> | ||
| + | <div class="makeresponsive" style="width:50%;"> | ||
| + | <figure class="makeresponsive floatleft" style="padding-left:10%; padding-right:10%;"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/3/38/T--TU_Dresden--P_Biosensor_Figure3_biosensorconstructs.png" | ||
| + | alt="Figure 2 Genetic constructs constituting the biosensor." | ||
| + | class="makeresponsive zoom"> | ||
| + | <figcaption><b>Figure 2: Genetic constructs necessary for the functional biosensor strain.</b> <b>(A)</b> <i>blaR1</i> under control of the constitutive promoter P<sub><i>veg</i></sub> or the inducible promoter P<sub><i>xylA</i></sub>, <b>(B)</b> <i>blaI</i> downstream of the promoter P<sub><i>lepA</i></sub>, and <b>(C)</b> the promoters P<sub><i>blaZ</i></sub> or P<sub><i>blaR1I</i></sub> controlling the expression of the <i>luxABCDE</i> operon.</figcaption> | ||
| + | </figure> | ||
| + | </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 P<sub><i>blaZ</i></sub> promoter. As this promoter showed high activity and reliability when induced by the presence of beta-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> | + | <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 the presence of beta-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"> | ||
<div class="contentbox"> | <div class="contentbox"> | ||
| − | <h1 class="box-heading">Proving the functionality of P<sub><i>blaZ</i></sub> [ | + | <h1 class="box-heading">Proving the functionality of P<sub><i>blaZ</i></sub></h1> |
| + | <h3>1. Assessing the activity of P<sub><i>blaZ</i></sub> in liquid medium</h3> | ||
| + | <figure> | ||
| + | <figure class="makeresponsive floatright" style="width: 40%"> | ||
| + | <figcaption><b>Table 1: Antibiotic concentrations in [µg µl<sup>-1</sup>] (final concentration in the 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" | ||
| + | alt="Table 2: β-Lactam concentrations tested all subsequent assays" class="zoom"> | ||
| + | </figure> | ||
| + | <p>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 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> | ||
| + | |||
| + | <figure> | ||
| + | <figure class="makeresponsive floatleft" style="width: 70%"> | ||
| + | <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"> | ||
| + | </figure> | ||
| + | <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>We could not observe a substantial activation of the P<sub><i>blaR1I</i></sub> promoter by the beta-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/OD<sub>600</sub> values of the strains 2 hours post induction with the antibiotics.</p> | ||
| + | |||
| + | <figure> | ||
| + | <figure class="makeresponsive floatleft" style="width: 55%"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/a/ab/T--TU_Dresden--P_Biosensor_Figure5.png" | ||
| + | alt="Figure 5: Bar Charts showing the detection range of the Biosensors" class="zoom"> | ||
| + | <figcaption><b>Figure 5: RLU/OD<sub><i>600</i></sub> values of the different biosensors and the controls are shown 2 hours after induction with the six β-lactams, bacitracin and dH<sub><b>2</b></sub>O</b>. Graphs show the Wild-type (<b>black</b>), control 1 (<b>light gray</b>), control 2 (<b>dark gray</b>), biosensor 1 (<b>pink</b>), biosensor 2 (<b>purple</b>), biosensor 3 (<b>white and black</b>) and biosensor 3 Xylose induced (<b>dark blue</b>). Luminescence (RLU/OD<sub>600</sub>) output is shown two hours after β-lactam antibiotic induction. Mean values and standard deviation are depicted from at least three biological replicates. </figcaption> </figure> | ||
| + | <p></p> | ||
| + | <p>As shown in Figure 5, 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 5).</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 5, 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 5).</p> | ||
| + | <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> | ||
| + | |||
</div> | </div> | ||
</main> | </main> | ||
Revision as of 11:59, 1 November 2017
Introduction
As part of the EncaBcillus project, we developed a novel and complete heterologous biosensor for β-lactam antibiotics in Bacillus subtilis. This biosensor is based on a one-component system encoded in the so-called bla-operon naturally found in Staphylococcus aureus. The biosensor is composed of three composites from this operon: The β-lactam receptor BlaR1 receptor and the repressor BlaI which have been codon-adapted for expression in B. subtilis as well as the PblaZ promoter [BBa_K2273111](see Figure 2). This promoter was inserted upstream of the lux-operon, our reporter of choice. Figure 1 displays the molecular mechanism of the established biosensor. In case a β-lactam is bound to BlaR1, the receptor`s proteolytic c-terminal domain degrades the BlaI repressor, thereby releasing the PblaZ promoter. This enables binding of the transcription machinery to the promoter and therefore the expression of the luxABCDE genes, resulting in a luminescence signal produced by the bisosensor.
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 PblaZ promoter [BBa_K2273111]. As this promoter showed high activity and reliability when induced by the presence of beta-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.
Proving the functionality of PblaZ
1. Assessing the activity of PblaZ in liquid medium
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 to test our biosensor in liquid 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 (data not shown). 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).
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 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 beta-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.
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.
