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From 2012.igem.org

Triple Population Cascade Design

The Three Population System

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Population 1:
· Positive Feedback: The oscillation 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. · Negative Feedback: In population 1, LasR is constitutively promoted. When it binds with AI-1, it creates a dimer that, in turn, binds with PLasR and promotes the transcription of araC. AraA then binds with PBad to repress the production of LsrK, AHL and YFP.

Population 2:
· Positive Feedback: 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.
· Negative Feedback: In population 2, LsrK is constitutively promoted. When it binds with AI-2, it creates a dimer, binds with PLsrA and promotes LacI. LacI then binds with PLac to repress the production of LuxR, AI-1 and RFP. This inhibits the red fluorescence with the populations

Population 3:
· Positive Feedback: 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.
· Negative Feedback: In Population 3, LuxR is constitutively promoted. When it binds with AHL, it creates a dimer which binds with PLuxR and promotes TetR. TetR then binds with PTetR to repress the production of LasR, LuxS, and CFP.

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

In planning the production of our three population system we decided to take a two phase approach.

Phase 1: Promotion

       Construct a basic 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 molecules of their respective population counterparts.

           Pseudo-population 1: Constitutive promoter-LuxI

           Pseudo-population 2: Constitutive promoter-LasI

           Pseudo-population 3: Constitutive promoter-LuxS


       Test each population individually using the pseudo-population that produces the quorum sensing molecules to promote their expression.

           Basic Population 1 with pseudo-population 3

           Basic Population 2 with pseudo-population 1

           Basic Population 3 with pseudo-population 2


       Test the effects of each population with its neighboring populations and pseudo-populations to see if one basic population can successfully promote another population instead of its respective pseudo-population.

           Basic Population 1 and Basic Population 2 with pseudo-population 3

           Basic Population 2 and Basic Population 3 with pseudo-population 1

           Basic Population 3 and Basic Population 1 with pseudo-population 2

       Test the effect of all three basic populations interacting with each other without any pseudo-populations.

Phase 2: Promotion and Repression

       Reconstruct the three populations from step one, but add the necessary parts that will cause each system to be repressed by the proper quorum sensing molecule.

       Test each population again using the pseudo-population that produces the quorum sensing molecules to promote its fluorescent protein expression and another time with the molecules that will repress its fluorescent protein expression.

           Population 1 with pseudo-population 3

           Population 1 with pseudo-population 2

           Population 2 with pseudo-population 1

           Population 2 with pseudo-population 3

           Population 3 with pseudo-population 1

           Population 3 with pseudo-population 2


       Test the effects of each population with its neighboring populations and pseudo-populations to see if one basic population can successfully promote another population instead of its respective pseudo-population.

           Population 1 and Population 2 with pseudo-population 3

           Population 1 and Population 3 with pseudo-population 3

           Population 2 and Population 1 with pseudo-population 1

           Population 2 and Population 3 with pseudo-population 1

           Population 3 and Population 1 with pseudo-population 2

           Population 3 and Population 2 with pseudo-population 2


       Finally test the effect of all three populations interacting with each other without any pseudo-populations and watch the magic happen.