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Biosensing System Design

There are three vital components to our system: the autoinducer, the receptor protein, and the reporter construct. The autoinducer will be supplied by P. aeruginosa. The receptor proteins need to be produced by the E. coli. Finally, the reporter sequence has to be regulated by the inducible promoters (autoinducer/R-protein specific). The three components are depicted below in Figure 1.

**Figure 1:** The three components of the sensing system in *E. coli*: (a) the autoinducer, (b) the receptor R-protein, and (c) the reporter construct.

When combined, the three components of the system facilitate the detection of P. aeruginosa, as depicted in Figure 2. The autoinducer concentration directly influences the level of reporter expression. However, since autoinducers are generally produced at low basal levels by P. aeruginosa, this may complicate the detection of these molecules by our biosensors. Therefore, to enhance the sensitivity of our biosensors to the autoinducers, we chose to express the R-proteins from strong, constitutive promoters. To combine R-protein synthesis and the reporter into a single construct, the inducible promoter and reporter sequences are upstream of the constitutive promoter and R-protein gene to minimize the effects of transcriptional readthrough.

**Figure 2:** The *P. aeruginosa* detecting biosensor system design.

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+<DIV style="font-size:20px">Biosensing System Design</DIV>
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-<DIV style="font-size:20px">The Theory</DIV><DIV style="font:15px Helvetica;">
-Virulence factors produced by Pseudomonas aeruginosa are controlled by a natural regulatory, hierarchical network consisting of two main systems: the las system and the rhl system. The main components of these systems are the receptor proteins lasR and rhlR, and the autoinducers 3-oxo-C12-HSL (PAI-1) and C4-HSL (PAI-2). Upon binding to the induced promoters LasP and RhlP, the LasR/PAI-1 and RhlR/PAI-2 dimers act as transcriptional regulators that enhance the expression of the proceeding genetic sequence.  This transcriptional regulatory mechanism, which known specifically to respond to cell density (via autoinducer/R-proteins) will be employed in our construct for Pseudomonas aeruginosa detection.
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-<DIV style="font-size:20px">Components of the Construct</DIV><DIV style="font:15px Helvetica;">
+There are three vital components to our system: the autoinducer, the receptor protein, and the reporter construct. The autoinducer will be supplied by ''P. aeruginosa''. The receptor proteins need to be produced by the ''E. coli''. Finally, the reporter sequence has to be regulated by the inducible promoters (autoinducer/R-protein specific). The three components are depicted below in Figure 1.
-There are three vital components to our construct, the autoinducer, the receptor protein, and the reporter. The autoinducer will be supplied by P. aeruginosa, which needs to be detected by the system. The receptor proteins need to be produced by the E. coli. Finally, the reporting sequence has to be paired to the induced promoters (autoinducer/r-protein specific). The three components are depicted below in Figure 1.
-</div>
-<br>
 <div align="center"><html><table class="image">
-<caption align="bottom"></html>'''Figure 1:''' The 3 components of the sensing construct in ''E. coli''.<html></caption>
+<caption align="bottom"></html>'''Figure 1:''' The three components of the sensing system in ''E. coli'': (a) the autoinducer, (b) the receptor R-protein, and (c) the reporter construct.<html></caption>
 <tr><td><img src="https://static.igem.org/mediawiki/2011/1/13/General_idea.jpg" style="opacity:1;filter:alpha(opacity=100);" width="700px" height="140px" alt="fig1"/ border="0"></td></tr>
 </table></html></div>
-<br>
-<DIV style="font-size:20px">Construct Design</DIV><DIV style="font:15px Helvetica;">
-When combined, the three components of the system facilitate the detection of P. aeruginosa as depicted in Figure 2. The autoinducers directly influence the level of reporter expression; however, they are traditionally produced at basal levels by P. aeruginosa which would take a while to detect. Therefore, to enhance the sensitivity of the construct to the autoinducers, the R-protein synthase sequence is coupled with a constitutive promoter. Constitutive expression of the R-protein synthase will eventually saturate the cell, and enhance the detection of P. aeruginosa. The induced promoter and reporter sequence is designed upstream of the constitutive promoter and R-protein synthase construct.
-</div>
-<br>
-<br><div align="center"><html><table class="image">
+When combined, the three components of the system facilitate the detection of ''P. aeruginosa'', as depicted in Figure 2. The autoinducer concentration directly influences the level of reporter expression. However, since autoinducers are generally produced at low basal levels by ''P. aeruginosa'', this may complicate the detection of these molecules by our biosensors. Therefore, to enhance the sensitivity of our biosensors to the autoinducers, we chose to express the R-proteins from strong, constitutive promoters.  To combine R-protein synthesis and the reporter into a single construct, the inducible promoter and reporter sequences are upstream of the constitutive promoter and R-protein gene to minimize the effects of transcriptional readthrough.
-<caption align="bottom"></html>'''Figure 2:''' The ''P. aeruginosa''  detecting construct design.<html></caption>
+<div align="center"><html><table class="image">
+<caption align="bottom"></html>'''Figure 2:''' The ''P. aeruginosa''  detecting biosensor system design.<html></caption>
 <tr><td><img src="https://static.igem.org/mediawiki/2011/3/37/Construct_design.jpg" style="opacity:1;filter:alpha(opacity=100);" width="600px" height="400px" alt="fig1"/ border="0"></td></tr>
 </table></html></div>
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