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Editor's Notes

• #3: Instead of having circuits with switches and oscillators, you are building a circuit made of biological counter-parts. Goal: Whole-cell biosensors: made with e.coli. Focus of talk: variety of different networks and switches that are used and how they are constructed
• #4: Control at: Signal Repressor Translation Structure of Operon Activation of proteins
• #5: Zinc finger domain: bind DNA Proteins are modular
• #6: Zinc finger domain: bind DNA Proteins are modular
• #7: Memory elements and genetic counters Go to board to explain recombinatiom ~100 natural recombinases are known Recombinases recognize a short DNA sequence motif, and they do so by contact at a few a.a. residues Easily genetically engineered for greater diversity and sequence specificity
• #8: Ribosome-binding Site A sequence that is complementary to the ribosome binding site (cis-repressor sequence) is placed upstream of it, so that transcription of this RNA leads to hairpin This secondary structure blocks 30S ribosomal subunit binding to RBS->inhibit translation However, if you express a short trans-activating non-coding RNA, called a taRNA, that contains a sequence that is complementary to the cis-regulatory sequence Then this sequence will bind to the cis-repressor sequence, disrupting the hairpin structure and relieving the ribosome binding site cis-acting  generally means "acting from the same  molecule " ( i.e. , intramolecular). It may be considered the opposite of trans-acting which generally means "acting from a different molecule" ( i.e. , intermolecular).
• #11: This is the motivating factor for the toggle switch The toggle switch is composed of two repressors and two promoters. Each promoter is inhibited by the repressor that is transcribed by the opposing promoter If both promoters have then same strength, then this results in a network with bistable behaviour
• #12: The toggle switch is composed of two repressors and two promoters. Each promoter is inhibited by the repressor that is transcribed by the opposing promoter If both promoters have then same strength, then this results in a network with bistable behaviour
• #13: Adaptive learning networks Natural networks in bacteria can exhibit anticipatory behaviour for related environmental simuli If we design this into our circuit, we could code the network to “learn” Synaptic interconnections between neurons Activator A and B are expressed in response to a different stimulus Suppose both transcriptional activators drive expression of effector proteins (Effector A and Effector B) which contorl distinct genetic pathways … explain circuit This creates associative memory
• #14: In another example of a learning network We could design a “winner take all” behaviour in detecting stimuli In the future, will respond with an output only in the presence of the single type of stimuli This system could be adapted to create chenmotactic bacteria that remember a particular location or landmark and only respond to gradient of one chemoattractant
• #15: Protein based circuits Advantage: react with shorter time scales (transcription takes time, so does translation and folding; activation is just conformational change) Advantage: target synthetic activities to subcellular locations Here we have a protein circuit that exhibits Amyloid-based memory (CONST is a constitutive promoter, meaning that it’s always on. ON and OFF are inducible) Prionogenic domain (PD) fused to an effector gene, such as a transcriptional activator Prions propagate by transmitting a mis-folded protein state induces pre-existing normal forms of the protein to convert into the rogue form. this triggers a chain reaction that produces large amounts of the prion form Prions are the cause of a number of diseases in a variety of mammals, including bovine spongiform encephalopathy(BSE, also known as "mad cow disease") Chaperones disaggregate amyloids
• #16: Light responsive elements Bacteria engineered to seek out hazardous chemicals or heavy metals in the environment, perform cleanup and return to origin to report hazardous sites encountered via analysis by microfluidic devices Production of chemoattractant receptors aka chemotaxis network
• #17: One example of this is used to Engineered circuits for biological containment For ensuring genetically modified orgnaisms do not spread throughout natural environment Call active containment: Cells engineered to express toxic compounds when located out of their target environments, for programmed cell death
• #18: Here we have a two-counter: P-BAD = arabinose promoter Both genes T7RNAP and T3RNAP are regulated by riboregulators. Recall that these contain a cis-repressor sequence (cr) upstrem of the ribosome binding site Cr is placed between transcription start site and ribosome binding site. The first arabinose pulse translates taRNA, which relieves RBS and allows translation of T7 RNAP Remove arabinose from cell environment What happens if you don’t remove the arabinose? ALSO: Notice basal signal –> Why do you think this is? (arabinose)
• #19: Three-counter Notice that the peak of GFP expression is at 150 minutes. Why do you think this takes so long? (Counter limited by rate of transcription and protein translation)
• #20: Mathematical model predictions. Used model to analyze the effects of pulse frequency and pulse length on performance of the RTC 2-counter and 3-counter Color scale = expression level/fluorescence Maximum expression occurs with pulse lengths of 20-30min and intervals of 10-40min
• #21: DNA Invertase Cascade (DIC) counter Cre FLPe ssrA tag causes rapid protein degradation Term – transcription terminus
• #22: DNA Invertase Cascade (DIC) counter Cre FLPe
• #23: DNA Invertase Cascade (DIC) counter Cre FLPe
• #24: Multiple-inducible DIC three-counter
• #25: Notice basal level again: why do you think this is? (In this case, not clearing of chemical inducers. All inducible promoters are a little leaky aka constitutive transcription)
• #27: Analog-to-digital converters could translate external analog inputs, such as inducer concentrations or exposure times, into internal digital representations for biological processing Depending on the level of analog inputs, different genetic pathways could be activated Cells with these type of converters could be used as biosensors in medical and environmental settings. For example, whole-cell biosensors in the cut could be engineered to generate different reporter molecules based on the level of gastrointestinal bleeding, and this could be measured in stool Not binary
• #28: Alternatively, we could have digital to analog to translate digital representations into analog outputs Eg. Instead of fine-tuning transcriptional activity with the perfect amount of chemical inducers, cell uptake, etc, we could have a digital to analog converter Which in this case takes in 3 different signals So you would have three genetic switches, each sensitive to a different inducer Allow you to generate a gradient of responses Here, we have a bank of recombinase-based switches, known as single invertase memory modules (SIMMs) Recall: recombinase is an enzyme that will recombine homologous regions, recognize specific motif Explain recombinase circuit If each promoter is of a different strength, then digital combinations of inducers can be used to program defined levels of transcriptional activities Use for reliable expression of different pathways