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From 2012.igem.org

Three Population Cascade Design

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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

The Perfect Model

Planning and Production

Biobricks we used to construct our three population system:
BBa_C0080: araC
BBa_J23119: Constitutive Promoter
BBa_C0012: LacI
BBa_C0078: LasI
BBa_C0079: LasR
BBa_K091002: LsrK
BBa_C0061: LuxI
BBa_C0062: LuxR
BBa_K091109: LuxS
BBa_I13453: pBad
BBa_K091159: Pbad-LsrK
BBa_R0010: pLac
BBa_R0079: pLasR
BBa_K117002: pLsrA
BBa_R0062: pLuxR
BBa_R0040: pTet
BBa_B0034: RBS
BBa_E0422: RBS-CFP-Term-Term
BBa_K093005: RBS-RFP
BBa_E0432: RBS-YFP-Term-Term
BBa_B0015: Term-Term
BBa_C0040: TetR
BBa_I14015: pLasR-TetO

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.