Difference between revisions of "Team:TU Dresden/Project/Biosensor"
| Line 29: | Line 29: | ||
<figure> | <figure> | ||
| − | <figure class="makeresponsive | + | <figure class="makeresponsive floatleft" style="width: 50%;"> |
<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="zoom"> | alt="Figure 2 Molecular mechanism of the Biosensor" class="zoom"> | ||
<figcaption><b>Figure 2: 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>, the signal is transferred to the repressor BlaI, which releases its target promoter <b>(2)</b> enabling the expression of an easy detectable reporter <b>(3)</b>.</figcaption> | <figcaption><b>Figure 2: 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>, the signal is transferred to the repressor BlaI, which releases its target promoter <b>(2)</b> enabling the expression of an easy detectable reporter <b>(3)</b>.</figcaption> | ||
</figure> | </figure> | ||
| − | |||
| − | |||
| − | |||
<figure> | <figure> | ||
| − | <figure class="makeresponsive | + | <figure class="makeresponsive floatright" style="width: 50%;"> |
<img src="https://static.igem.org/mediawiki/2017/3/38/T--TU_Dresden--P_Biosensor_Figure3_biosensorconstructs.png" | <img src="https://static.igem.org/mediawiki/2017/3/38/T--TU_Dresden--P_Biosensor_Figure3_biosensorconstructs.png" | ||
alt="Figure 3 Genetic constructs constituting the Biosensor" class="zoom"> | alt="Figure 3 Genetic constructs constituting the Biosensor" class="zoom"> | ||
| − | <figcaption><b>Figure 3: 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><b>Figure 3: 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><p style="clear: both;"> | ||
| − | </ | + | <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>.</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©. Cloning procedures were carried out according to the manufacturer`s protocols. </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©. Cloning procedures were carried out according to the manufacturer`s protocols. </p> | ||
Revision as of 19:59, 28 October 2017
Background
Antibiotics represent the most effective treatment against bacterial infections. Since the discovery of penicillin by Alexander Fleming in 1928, many new antibiotics have been constantly developed and were successfully applied to treat life-threatening diseases. This significant advancement in medicine saved millions of lives and still does today. However, fighting microorganisms has never been a completed task, but rather an ongoing race between drug discovery and pathogens developing resistances. Thus, multi-drug resistant bacteria still constitute a major threat for humanity, as infectious diseases represent the second leading cause of death worldwide. [1][2]
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.
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).
To address the increasing development of multi-drug resistant bacteria our iGEM Team aims at developing a novel beta-lactam biosensor in Bacillus subtilis based on the genetics of the bla-operon found in Staphylococcus aureus (for a detailed description consider our Design section below). The genetically engineered Biosensor will help to (I) reliably detect even minimal antibiotic concentrations of compounds from the beta-lactam family in waste and drinking water and (II) 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).
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).
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 (Pveg), (B) the Repressor gene blaI under control moderate strong constitutive promoter (PlepA) and (C) the target promoter region of the bla-operon (PblaZ and PblaR1I) in front of the lux-operon (luxABCDE) (see Figure 3). In addition, an inducible version of the blaR1 construct was made by placing the PxylA promoter upstream of the blaR1 gene (A).
All genetic constructs and plasmids have been created using the RFC10 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.[3]
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::kanRwas 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.
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.
