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

Endoribonucleases (ERNs) are enzymes that cleave RNA at internal phosphodiester bonds, generating smaller RNA fragments. By degrading or processing RNA, ERNs can reduce protein levels by either directly degrading mRNA, preventing its translation into protein, or modifying or processing specific RNA sequences, which can alter their stability or translational efficiency.

Main differences:

(a) Target substrate:

ERNs act on RNA by cleaving phosphodiester bonds. Proteases act on proteins, hydrolyzing peptide bonds. (b) Mode of regulation:

ERNs regulate protein levels indirectly by preventing or altering mRNA translation, which can lead to gradual or variable effects on protein expression. Proteases regulate protein levels directly by degrading the protein itself, resulting in an immediate and binary effect (present vs. degraded).

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

Lipofectamine 3000 is a transfection reagent composed of (1) Cationic lipids (DOSPA and DOPE) that bind and encapsulate nucleic acids through electrostatic interactions. (2) Neutral co-lipid (P3000), which enhances the efficiency of DNA delivery and transfection

When Lipofectamine 3000 is mixed with target DNA, it forms lipid-DNA complexes. The cationic lipids bind to the negatively charged DNA through ionic interactions. The lipid-DNA complexes interact with the negatively charged cell membrane. This promotes endocytosis, allowing the complexes to enter the cell in endocytic vesicles. Lipofectamine 3000 facilitates the release of DNA from the endosomes into the cytoplasm, preventing its degradation. The DNA is transported into the nucleus, where it undergoes transcription into mRNA. The mRNA is then translated into protein by the ribosomes, resulting in gene expression.

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

Poly-transfection refers to the simultaneous introduction of multiple DNA constructs into a single cell or cell population. This method allows for co-expression of multiple genes, enabling complex genetic manipulations.

Poly-transfection Is Useful in Neuromorphic Circuit Building since it allows for the simultaneous expression of multiple proteins. The proportional delivery of multiple DNA constructs allows for fine-tuning of gene expression levels, which is essential for creating circuits with specific and controlled activity patterns. This is crucial for neuromorphic systems, which require the coordinated expression of multiple genes to replicate neuronal processing and memory functions

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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?

2. Natural Genetic Circuit Example:

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