Questions 1-3 are mandatory for all students.

1. How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.
   1. ERSs are a type of enzyme that cleaves RNA. When an ERS cleaves an mRNA, it results in decreased protein levels.
   2. One difference between how ERNs work and how proteases work is that ERNs decrease protein levels at the RNA level while proteases decrease protein levels at the protein level
   3. ERNs are single turnover proteins, so it can only be used once. Proteases can degrade protein after protein after protein.
2. How does lipofectamine 3000 work? How does DNA get into human cells and how is it expressed?
   1. It is the name of a specific region that allows us to put DNA into humans. We use a chemical transformation that contains a lipid nanopartycle with the DNA of interest. When the cell interacts with the lipid particle, it will take it up, and the DNA will make its way to the nucleus.
3. Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.
   1. A transfection is using lipofectamine 3000 to put DNA into cells. A poly-transfection is when you mix n different nanoparitcals that each contain a distinct DNA combinations. this is useful when building neuromorphic circuits because it allows for combinatorial variation between cells. This results in continuous data coverage across the circuit design space.

Questions 4-6 are optional for but highly encouraged for MIT/Harvard Students and mandatory for **Committed Listeners.

• *Responses should be no more than 1 paragraph each, and hand drawn diagrams (iPad, digital or paper) are HIGHLY encouraged

1. Genetic Toggle Switches:

   • Provide a detailed explanation of the mechanism behind genetic toggle switches, including how bi-stability is established and maintained.
   • Describe at least one induction method used to switch states, including molecular signals or environmental factors involved.
   • Are there any limitations? How many ‘switches’ can we potentially chain? Is there a metabolic cost?

   The traditional mechanism behind a genetic toggle switch are two promoters that code for each other’s repressor. The system has two stable states: a) Gene 1 is on, Gene 2 is off b) Gene 1 is off, Gene 2 is on. There is also equilibrium point which is inherently unstable. The system will fall into one of the two stable states. One induction method used to switch states is to use molecules that bind to each of the promoters. Depending on which molecule you introduce to the system, either promoter 1 or promoter 2 shuts off, allowing you to switch between states. Theoretically, we could chain as many ‘switches’ together as we want, but this is not realistic. Because the switches are all floating around together, there is the potential for crosstalk where proteins from one switch may interfere with another. In addition, protein expression is metabolically costly, requiring ATP, amino-acids, as well as taking up precious ribosomes.

   

2. Natural Genetic Circuit Example:

   • Identify and describe in detail a naturally occurring genetic circuit, emphasizing its biological function, components, and regulatory interactions.

   An example of a naturally occurring genetic circuit found in nature is the lambda phage circuit,. There are two primary promoters involved in the circuit. P_R regulates gene cro and P_RM regulates gene cI. When P_R is on, the virus goes into lytic mode. When P_RM is on, the virus goes into lysogenic mode. when P_RM is activated, cI is expressed, whcih codes for a molecule that preferentially binds to Operator Sites 1 and 2 over site 3. When cI2 binds to these operator sites, it represses P_R. Alternatively, when P_R is activated, cro is expressed. cro has highest binding affinity to OR3, shutting PRM off. The result is a bistable switch, with either P_R or P_RM being on at a given time, but never both.

   

   

   

3. Synthetic Genetic Circuit:

• Select and critically analyze a synthetic genetic circuit previously engineered by researchers (e.g., pDAWN). Provide details about its construction, components, intended function, and performance.
• Discuss potential limitations or improvements suggested in subsequent literature or experimental data.

One of the most famous genetic circuits is the Repressilator. In the example below, TetR represseslamda_cl which represses Lacl which represses TetR. Like an infinite game of rock-paper-scissors, this circuit results in individual genes being turned on and off over time. TetR in turn represses a GFP gene. The result of this is that GFP content in the cell oscillates over time. The Repressilator circuit is on a separate plasmid from the reporter circuit. This is by design. The repressilator is on a low-copy plasmid, which limits toxicity from over-expressed repressors. The reporter circuit is on a high-copy plasmid, which helps maximize readout.