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 beta-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 beta-lactam receptor BlaR1 receptor and the repressor BlaI which have been codon-adapted for expression in <i>B. subtilis</i> as well as the P<sub><i>blaZ</i></sub> promoter. 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 beta-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 luxABCDE genes, resulting in a luminescence signal produced by the bisosensor. </p> | + | <p>As part of the EncaBcillus project, we developed a novel and complete heterologous biosensor for beta-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 beta-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</a>. 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 beta-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 luxABCDE genes, resulting in a luminescence signal produced by the bisosensor. </p> |
<figure class="makeresponsive floatright" style="width: 60%"> | <figure class="makeresponsive floatright" style="width: 60%"> | ||
<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 2 Molecular mechanism of the Biosensor"class="makeresponsive zoom"> | alt="Figure 2 Molecular mechanism of the Biosensor"class="makeresponsive zoom"> | ||
| − | <figcaption><b>Figure | + | <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> | ||
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| − | <h1 class="box-heading">Proving the functionality of P<sub><i>blaZ</i></sub></h1> | + | <h1 class="box-heading">Proving the functionality of P<sub><i>blaZ</i></sub> [BBa_K2273111]</h1> |
</div> | </div> | ||
</main> | </main> | ||
Revision as of 11:26, 1 November 2017
Introduction
As part of the EncaBcillus project, we developed a novel and complete heterologous biosensor for beta-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 beta-lactam receptor BlaR1 receptor and the repressor BlaI which have been codon-adapted for expression in B. subtilis as well as the PblaZ promoter. 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 beta-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. 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.
