1. How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.

ERNs find a matching site on a piece of RNA and chops up the RNA there, which prevents the RNA from being translated.

So ERNs chop up RNA, whereas proteases chop up protein. And ERNs perform their work before translation, whereas proteases do their work after translation.

2. How does lipofectamine 3000 work? How does DNA get into human cells and how is it expressed?

Lipofectamine 3000 is a lipid nanoparticle used to get DNA into cells. It serves the same functional purpose as, e.g., heat shocking bacteria, except that you can’t heat-shock mammalian cells much. You take the DNA that you want to put into the cell, wrap it in lipid nanoparticles, dump the lipid nanoparticles onto your cells, and the lipid nanoparticles melt into the cell membrane, and release the payload DNA into the cell.

3. Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.

Poly-transfection means you have several tubes of lipid nanoparticles each with just one DNA sequence. This lets you pour different amounts of each DNA you want into the cells.

4. Genetic Toggle Switches:

Provide a detailed explanation of the mechanism behind genetic toggle switches, including how bi-stability is established and maintained.

You have 2 genes. Gene A produces something that suppresses the expression of Gene B, and Gene B produces something that suppresses the expression of Gene A.

So there are 2 states where this is stable: Gene A can win, and it’s expressed a lot while Gene B is suppressed; or vice versa.

Describe at least one induction method used to switch states, including molecular signals or environmental factors involved.

Courtesy of https://en.wikipedia.org/wiki/Promoter_(genetics):

“1: RNA Polymerase. 2: Repressor. 3: Promoter. 4: Operator. 5: Lactose. 6: lacZ. 7: lacY. 8: lacA. Top: The transcription of the gene is turned off. There is no lactose to inhibit the repressor, so the repressor binds to the operator, which obstructs the RNA polymerase from binding to the promoter and making the mRNA encoding the lactase gene. Bottom: The gene is turned on. Lactose is inhibiting the repressor, allowing the RNA polymerase to bind with the promoter and express the genes, which synthesize lactase. Eventually, the lactase will digest all of the lactose, until there is none to bind to the repressor. The repressor will then bind to the operator, stopping the manufacture of lactase.”

So one way is, you can normally have a repressor that binds to the DNA, blocking the gene from being transcribed. But the repressor acts as a sensor: when lactose attaches to the repressor, it falls off the DNA, allowing the DNA to be transcribed.

Are there any limitations? How many ‘switches’ can we potentially chain? Is there a metabolic cost?

I think there are a limited number of known repressors that you can play with that don’t affect critical cell functionality?

The cost is that each item you add to the chain requires transcription and producing proteins, which costs the cell energy.

5. Natural Genetic Circuit Example: