Team:Northwestern/Project/Description

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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.
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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.
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<caption align="bottom"></html>'''Figure 1:''' The three components of the sensing system in ''E. coli'': (a) constitutive expression of LasR, (b) constitutive expression of RhlR, and (c) inducible expression of GFP by receptor/autoinducer complex.<html></caption>
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<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>
<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>
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<DIV style="font-size:20px">Construct Design</DIV>
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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.
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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.
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<caption align="bottom"></html>'''Figure 2:''' The ''P. aeruginosa''  detecting construct design.<html></caption>
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<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>
<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>
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Latest revision as of 02:35, 29 October 2011

RETURN TO IGEM 2010



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.
fig1


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.
fig1




Sponsor northwestern.jpg Sponsor weinberg.jpg Sponsor mccormick.jpg