What sets the switching threshold of a toggle?

• The size of the culture flask
• Promoter strength, repressor levels, and binding affinity
• The color of the fluorescent reporter

What makes a two repressor toggle bistable?

• Two repressors blocking each other, so one side wins and stays on
• One repressor working alone at high speed
• Two activators firing at fixed intervals

Attaching a downstream module can change the behavior of the module driving it.

A circuit works on day one and fades after many generations of growth. What is the most likely cause?

• The threshold was calculated with the wrong units
• Mutations breaking circuit genes, with freeloaders outgrowing producers
• The cells learned to ignore all promoters

You attach a downstream reporter and the driver signal sags. What happened?

• The reporter mutated within minutes
• The inducer evaporated from the flask
• The downstream part consumed the shared signal, dragging the driver down

Which wiring holds states and which wiring oscillates?

• Positive feedback holds states, negative feedback oscillates
• Negative feedback holds states, positive feedback oscillates
• Both wirings always oscillate

Your toggle flips at a tiny unwanted signal. Which change raises its switching threshold?

• Strengthen the promoter driving the repressor
• Delete one of the two repressor genes
• Dilute the culture with fresh medium

A student blames a faded 50 generation culture on the toggle threshold drifting, while non producers dominate the flask. What is the likelier cause?

• Threshold drift in every cell at once
• Mutation plus selection: broken freeloaders outgrew the producers
• Retroactivity from a reporter added years ago