Team:UT Dallas/pop3 o design

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Revision as of 02:34, 4 October 2012

Three Population Cascade Design

For our three population signal propagation mechanism, we created three different strains using E.coli bacteria. These strains used three different quorum sensing molecules acyl homoserine-lactone (AHL), (autoinducer-1) Ai-1, and (autoinducer-2) Ai-2 coupled with yellow, red, and cyan fluorescent proteins to create a visual oscillating effect.

Population 1:
The sequence begins with the synthesis of LsrK. When LsrK binds with Ai-2 produced by the third population, it creates a phosphorylated dimer, referred to as “Ai-2 +P”, that binds to the PLsrA promoter. This promotes the transcription of both Luxl and the yellow fluorescence protein, YFP. Also, LuxI, when bound with S-adenosylmethionine (SAM), produces AHL.

Population 2:

The second population in the sequence is induced by the AHL produced by Population 1. When AHL is in the presence of the bacteria, it creates a dimer with LuxR that binds to PLuxR. PLuxR then promotes LasI which when bound with SAM produces Ai-1. RFP is attached to the end of the LasI gene, so that when LasI is produced, the bacterium also fluoresces red.

Population 3:

The third population in the sequence is induced by the Ai-1 molecules produced by Population 2. When Ai-1 is in the presence of the bacteria, it creates a dimer with LasR and binds to PLasR. PLasR then promotes LuxS which when bound with SAM produces Ai-2. CFP is attached to the end of the LuxS gene, so that when LuxS is produced, the bacterium fluoresces cyan.

Pseudo-population 3

   This population consists of only a Constitutive promoter and LuxS. LuxS is the gene in Population 2 that produces the quorum sensing molecule Ai-2. This pseudo-population is used in testing whether the output of Population 3 will successfully promoter the production of Ai-2 and the yellow flourescence protein expression level in Population 1

Production and Testing:

       Construct a set of populations that each promote the expression of the next. For example, Population 1 promotes Population 2. Populations 2 promotes Population 3.

       Construct pseudo-populations that only produce the quorum sensing molecule LuxS to mimic the output of Population 3

           Pseudo-population 3: Constitutive promoter-LuxS

       Test the effect of all three basic populations interacting with each other as well as the pseudo population.