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Constitutive Promoter
Key for Diagrams “AND” Gate- both inputs are necessary for an output. Constitutive Promoter Inducible Promoter Repressible Promoter Ribosome Binding Site Open Reading Frame Terminator
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Device 1- Oxidative Stress Induced RA Generator
NO Input 8 3 4 2 SoxS Promoter BC-RA Converter Generator Retinoic Acid Const. SoxR Generator 5 Metabolite 7 Beta-Carotene 6 Const. BC Generator
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Crt Cluster- BC Producer
Device 1- Oxidative Stress Induced RA Generator SoxR NO SoxR gene SoxS Promoter Blh- BC to Retinal RALDH II- Retinal to RA NO Sensor BC-RA Converter Retinoic Acid Crt Cluster- BC Producer Beta-Carotene Generator
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Device 1- Oxidative Stress Induced RA Generator
NO SoxR NO Sensor BC to RA Converter J23100 Const Prom. B0034 RBS k SoxR gene B0015 TT k SoxS Prom. B0034 RBS k223035 Blh gene B0034 RBS k223036 Raldh II gene B0015 TT BC to Retinal Retinal to RA Beta-Carotene Generator Retinoic Acid J23100 Const Prom. B0034 RBS ? Crt Cluster B0015 TT
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Device 2- Il-6 Secretion System
Il-6 Generator Constitutive Export System Generator POPS Invertor 5 MT 5 4 3 2 1
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Repressible 5MT Operator
Device 2 5MT Level Sensitive Sensor Il-6 Generator Repressible 5MT Operator Il-6 gene HlyA Linker Il-6 Secreted Export System TolC gene Hly R,B,D,C
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Device 2 5MT Level Sensitive Il-6 Generator Sensor Il-6 Secreted
k223023 Trp Operator B0034 RBS B0015 TT k223022 Il-6 + HlyA linker Allows Il-6 to leave through the export channel Il-6 Secreted Export System J23100 Const Prom. B0034 RBS k223025 Hly R,B,D,C B0015 TT k223024 TolC gene Create an Export Channel and Chaperone
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Fourth Round of Cloning
Device 2 Fourth Round of Cloning 2.4a Trp Operator, RBS,Il-6+HlyA,TT Rbs, TrpR, TT (Cloned from step 2.3a) (Cloned from step 2.1e) + J #? (Const. Promoter) (Synthesized de novo as 1part = RBS + trpR + TT) #k B k B0015 (Trp operator-) (RBS) (Il-6 and HlyAs linker) (TT) synthesized) Cut w/ EcoR1/Spe1 Cut w/ EcoR1/Xbal
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Device 2 + Fourth Round of Cloning
2.4b Trp Operator Rbs, TolC, HlyR,B,D,C, TT (will be registered) (Cloned from step 2.3b) + #k223023 (Trp operator - synthesized) B k k B0015 (RBS) (TolC ) (HlyR,B,D,C) (TT) Cut w/ ? Cut w/ ?
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