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

Endoribonucleases (ERNs) decrease protein levels by cleaving the mRNA encoding the protein, thereby preventing its translation. By cutting the mRNA internally, ERNs promote its rapid degradation, reducing the availability of the transcript for protein synthesis.

key differences:

1. Target Molecule:
   • ERNs cleave RNA (specifically mRNA), preventing translation into protein.
   • Proteases cleave proteins directly, breaking them down into peptides or amino acids.
2. Stage of Intervention:
   • ERNs act before translation, reducing protein levels by degrading the mRNA template.
   • Proteases act after translation, degrading already synthesized proteins.

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

Lipofectamine 3000 is a widely used lipid-based transfection reagent that delivers DNA (or RNA) into human cells. It works through the following steps:

1. Formation of Lipoplexes
   • Lipofectamine 3000 contains cationic lipids that electrostatically bind to the negatively charged DNA, forming lipid-DNA complexes (lipoplexes).
   • A helper lipid (often neutral, like DOPE) enhances membrane fusion and endosomal escape.
2. Cellular Uptake via Endocytosis
   • The positively charged lipoplexes interact with the negatively charged cell membrane and are internalized via endocytosis.
3. Endosomal Escape
   • Inside the cell, lipoplexes are trapped in endosomes (acidic vesicles).
   • The cationic and helper lipids disrupt the endosomal membrane, releasing the DNA into the cytoplasm (endosomal escape).
4. Nuclear Entry & Expression
   • The DNA must reach the nucleus for expression (in dividing cells, nuclear membrane breakdown during mitosis helps).
   • Once inside the nucleus, the DNA is transcribed into mRNA, which is exported to the cytoplasm and translated into protein.

How DNA Gets into Human Cells & Is Expressed

1. Delivery Methods

   • Chemical (Lipofectamine, Calcium Phosphate, Polymers) – Forms complexes with DNA for endocytosis.
   • Physical (Electroporation, Microinjection, Gene Gun) – Uses force or electricity to introduce DNA.
   • Viral (Lentivirus, AAV, Adenovirus) – Engineered viruses deliver DNA efficiently.

2. Cellular Expression Process

   • Transcription: DNA is transcribed into mRNA in the nucleus.
   • Nuclear Export: mRNA exits the nucleus via nuclear pores.
   • Translation: Ribosomes synthesize the encoded protein.
   • Post-Translational Modifications: The protein may be folded, modified, or localized to its functional site.

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

A genetic toggle switch is a synthetic gene regulatory circuit that exhibits bi-stability, allowing it to stably exist in one of two distinct states (e.g., high GFP vs. high RFP) without intermediate equilibria—functioning much like an electrical flip-flop. This behavior arises from mutual repression between two transcriptional regulators, forming a positive feedback loop that locks the system into one state until an external signal triggers a switch. As a foundational concept in synthetic biology, toggle switches enable cellular memory, decision-making, and programmable logic operations by maintaining their state until deliberately flipped by specific inputs.

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

Genetic toggle switches can be flipped between stable states using specific molecular inducers like IPTG and aTc. In the classic LacI-TetR system, IPTG inactivates the LacI repressor, allowing TetR expression to dominate, while aTc inhibits TetR to restore LacI dominance. This switching persists due to mutual repression creating hysteresis - the system "remembers" its state even after inducers are removed. The process depends on precise, orthogonal molecular interactions: IPTG specifically targets LacI, while aTc only affects TetR. Alternative induction methods include temperature-sensitive repressors, light-activated systems, or metabolite-responsive regulators. All these approaches work by temporarily relieving one arm of the toggle's mutually repressive feedback loop, triggering a complete and stable state transition. This predictable, tunable switching behavior makes genetic toggle switches valuable for engineering cellular memory and decision-making in synthetic biology applications. The system's robustness comes from its nonlinear response to inducer concentration, ensuring clear transitions between discrete states rather than intermediate expression levels.