1. How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.
   1. They degrade speciifc mRNA in the cells, which are the templates to make proteins. This in turn reduces the protein produced by the cell.
   2. While ERN work on mRNA, proteases directly cut and degrade proteins. Also, ERN stop protein production, while proteases destroy proteins after they’ve already been made.
2. How does lipofectamine 3000 work? How does DNA get into human cells and how is it expressed?
   1. It forms oily bubbles called liposomes which surround the DNA and help it travel to the cell. They attach to the cell membrane which is also made of lipids, and this allows the DNA to enter the cell.
   2. The DNA reaches the nucleus, is transcribed to mRNA, which is translated by ribosomes into proteins.
3. Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.
   1. This is where multiple DNA plasmids are introduced into a cell in a specific ratio. This is useful because neuromorphic circuits are complex, and you can quickly test how different ratios of DNA can affect and be tuned to receive the desired outcome. This way, the circuits also mimic neural networks, where different signals and weights are included.
4. Genetic Toggle Switches:
   • Provide a detailed explanation of the mechanism behind genetic toggle switches, including how bi-stability is established and maintained.
     • Its a synthetic gene circuit that can flip between two states (0 and 1). It consists of two genes that mutually inhibit each other’s expression. That is, Gene A produces a repressor protein that inhibits Gene B, and Gene B produces a repressor that inhibits Gene A. This way, when one protein is on (1) the other is off (0) due to production of the repressor.
     • This mutual repression creates bi-stability, where the system has two stable equilibrium states and resists small fluctuations.
   • Describe at least one induction method used to switch states, including molecular signals or environmental factors involved.
     • An inducer is introduced that temporarily inhibits one repressor (by binding to it), allowing the other gene to be active. Otherwise, when both repressors are active then the circuit would not work.
   • Are there any limitations? How many ‘switches’ can we potentially chain? Is there a metabolic cost?
     • Yes, there can be noise or variability in gene expression leading to instability. There needs to be careful tuning of promotor and repressor strength/levels.
     • Also, while multiple toggle switches can be chained to create logic circuits, the complexity grows quickly due to cross talk between switches, and limited resources in the cell (polymerase, ribosomes, etc.) to scale effectively.
5. Natural Genetic Circuit Example:
   • Identify and describe in detail a naturally occurring genetic circuit, emphasizing its biological function, components, and regulatory interactions.
     • lac operon: controls the ability of E. coli bacteria to metabolize lactose. It enables the bacteria to switch between using glucose (the preferred energy source) and lactose, depending on which sugar is available in the environment. This allows it to survive and conserve energy.
     • The lacl repressor binds to the operator to block RNA polymerase from transcribing operon genes. The CAP (Catabolite Activator Protein) allows transcription when glucose levels are low, binding near the promoter to help RNA polymerase. [Used the help of ChatGPT]
6. 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.
     • pDAWN uses a blue-light-responsive histidine kinase (YF1) and response regulator (FixJ) to control gene expression in E. coli. It provides reversible, precise blue-light control of gene expression with a strong on/off ratio.
   • Discuss potential limitations or improvements suggested in subsequent literature or experimental data.
     • It has limited light penetration in dense cultures, metabolic burden on cells, and dependency on E. coli as a host. We can do protein engineering to reduce background expression, promoter optimization, signal amplification via feedback, and improved light delivery systems. [Used the help of ChatGPT]