——— Jenny Wang

1. How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.
2. How does lipofectamine 3000 work? How does DNA get into human cells and how is it expressed?
3. Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.
4. 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?
5. Natural Genetic Circuit Example:
   • Identify and describe in detail a naturally occurring genetic circuit, emphasizing its biological function, components, and regulatory interactions.
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.
   • Discuss potential limitations or improvements suggested in subsequent literature or experimental data.

1. Endoribonucleases (ERNs) and Protein Level Reduction

Endoribonucleases (ERNs) are enzymes that cleave RNA molecules internally at specific sequences or structural motifs, leading to RNA degradation. By targeting messenger RNA (mRNA), ERNs prevent its translation into protein, effectively reducing protein synthesis. For example, RNase E in E. coli initiates mRNA decay by binding to the 5’-monophosphate group of RNA, while human endoribonucleases like XRN1 process RNA during stress responses (Lodish et al. 512; Schoenberg and Maquat 890).

Differences Between ERNs and Proteases:

1. Substrate Specificity: ERNs degrade RNA (e.g., mRNA, rRNA), while proteases hydrolyze peptide bonds in proteins (Lodish et al. 78).
2. Mechanistic Role: ERNs act pre-translationally by destroying RNA templates, whereas proteases act post-translationally by breaking down existing proteins (Schoenberg and Maquat 891).

2. Lipofectamine 3000 Mechanism and DNA Expression

Lipofectamine 3000 is a cationic lipid-based transfection reagent. Its positively charged lipid nanoparticles complex with negatively charged DNA, forming lipoplexes that fuse with the cell membrane. These lipoplexes enter cells via endocytosis, followed by endosomal escape triggered by the “proton sponge effect,” where the lipoplexes buffer endosomal pH, causing osmotic swelling and membrane rupture (Thermo Fisher Scientific).

DNA Entry and Expression:

1. Cellular Uptake: DNA-lipid complexes enter the cytoplasm via endocytosis.
2. Nuclear Entry: DNA passively enters the nucleus during mitosis when the nuclear envelope disassembles or via nuclear pore complexes in non-dividing cells (Mellott et al. 12).
3. Expression: Nuclear DNA is transcribed into mRNA, which is exported to the cytoplasm and translated into protein by ribosomes (Lodish et al. 105).