SELF-DIFFERENTIATED BACTERIAL ASSEMBLY LINEPEKING 2007 IGEMAll ideas and images from Peking 2007 IGEM team unless otherwise notedAllen Lin*

IssueProteins whose production interferes with each otherNeed for seperationSeperation from homogenous conditions in division of laborSolutions:Temporal DifferentiationSpatial Differentiation*

Push-on-Push-off SwitchTemporal SeperationSame input at different times -> different outputSimple finite state machine (has a current state and cyclic) binary

*

Binary SwitchBistable switch (stabilizes over time)NOR gate to reset to initial conditions*

Binary Switch*

Details of Binary SwitchAt first, Prm on -> CI and lacI produced, red colorC1 represses Pr, lacI represses PkuAfter UV light, CI degraded; Pr stronger promoter -> CI434 produced, green colorPr represses Prm, lacI production falls (mRNA degraded), so only LexA represss PkuAfter 2nd UV light, LexA degraded, Pku on -> Prm on, red color, back to startcI(ind-) repressions Pr*

Theoretical Computation (with ODE) Allens comments:NOR gates can be used to build AND, OR, INV gatesNote the time scaleGFP and RFP can be replaced by two different production systems*

Choosing the right parts*

Confirmation of Bistable SwitchTop figure: Each colony has a stable colorBottom figure: Two states exist*

How to Select Best PartsGraphical display of effectiveness (NOR gate)Blue graph desired; others not so much*

Allens ThoughtsHow was the circuit designed?The circuit is a 2-state, cyclic system can it be expanded? What components are specific to this implementation?Needed a NOR gate to reset from 2nd state to 1st stateUse of computation beforehand Identical input to produce varied output based on previous history -> counter?*

Hop CountSpatial Seperation need for different production systems that interfere with each other to happen in fixed proportions*

PlasmidsMain idea: Plasmid vector length as counterIn conjugation, DNA between two OriC sites deleted*

Conjugation*

oriTtraITraI

Isolating Components in Separate PlasmidFirst delete oriT and traI and place it signaling plasmidCounter will be in signaling plasmidOriginal Plasmid*

Theoretical Model*

DetailsImportant that the different oriT/traI systems do not interfere (crosstalk) with each otherThings to note:One promoter only traI nearest promoter is transcribed, terminator ends that transcriptionDNA replication occurs in opposite direction of promoterThus lose traI + terminator + functional oriT every step*

Experimental ResultAfter conjugationBefore conjugationBefore conjugation, promoter stops at terminatorsConjugation step removes two terminators between oriT sitesThus, after conjugation, GFP expressed*

Experimental Result IIShows that absence of TraI prevents conjugation completelyPlasmid F with traI deletedWild type plasmid F*

Allens ThoughtsShows two steps needed for hop count system to work once Place assembly line specific genes in between oriT sites before terminatorEnsures that one cell does each step; other cells in assembly line not far awayWhat would happen if we combined temporal and seperation? Get bistable neighboring cells? Checkerboard configuration?*

SourcesPeking 2007 iGEM presentation and powerpointhttp://parts.mit.edu/igem07/index.php/PresentationsPeking 2007 Wiki Project Noteshttp://parts.mit.edu/igem07/index.php/Peking_The_Projects*

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