A synthetic gene-metabolic oscillatorFung et al. 2005
A synthetic genemetabolic oscillator Fung et al. 2005
influxConceptualdesign ofMoscillatorycircuitEt2InfluxInfluxM,OutfluxFlux1>Flux2+Influx→M,decreasesE,HFlux1>Flux2+Outflux-+Mz increasesCFlux<1Flux2+Infaux→M,increasesFlux1Flux2+Outflux-→+M,decreasesMaInfluxOutfluxOutfluxMEHEM2Outfiux
Conceptual design of oscillatory circuit
Realization of the oscillatory circuit using the acetatepathway in E. coliSugersAcP:acetylphosphateFattyacidsGlycerulOAc:acetateEthanolHOAcprotonated formTCAcycleAcetyi-CoAof acetatePPPta: phosphatePtarACSATPLeclacetyltransferaseAck:acetatekinaseAcPAcs: acetyl-CoAAckAIPsynthetaseOACTheinductionofAcs bytheHOACpresenceof acetateis undercataboliterepression byglucosein wild-type strainso as to avoid futilecyclingHOACOAC
Realization of the oscillatory circuit using the acetate pathway in E. coli AcP: acetyl phosphate OAc- : acetate HOAc protonated form of acetate Pta: phosphate acetyltransferase Ack: acetate kinase Acs: acetyl-CoA synthetase The induction of Acs by the presence of acetate is under catabolite repression by glucose in wild-type strain so as to avoid futile cycling
E.coli strain and plasmid constructsAcPChromosomeOriRaKykanginAp2lacluAA品att,att,catOriRp15Atacgfpmut3.1AAVLaclAcsPEF3ReporterptaAAaCSLAAOriRpBR322blalacO-1ginAp2PtaAGFPisligateddownstreamofaplasmid-borneLacl-repressibletacpromoter, to be used as a readoutAcetylphosphate in the M2poolactivates acsexpression,andatthe same time, represses ptaexpressionindirectly
E. coli strain and plasmid constructs AcP Acs LacI Pta Acetyl phosphate in the M2 pool activates acs expression, and at the same time, represses pta expression indirectly. A GFP is ligated downstream of a plasmid-borne LacI-repressible tac promoter, to be used as a readout
Representative3.000single cell(ne)dynamicsooso2.0001,000060120180240Time (min)3.000(cne)coeoso2,0001,000601201802400Time (min)About 60% of the initial colonies oscillatewithvarying amplitudeand aperiod of 45± 10 min (fora total of 85 cells in 10 experiments),for a totaldurationofatleast4h
Representative single cell dynamics About 60% of the initial colonies oscillate with varying amplitude and a period of 45 ±10 min (for a total of 85 cells in 10 experiments), for a total duration of at least 4 h