Homework#7

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

• 📕 Definition

Endoribonucleases (ERNs) are highly specific enzymes that cut or cleave double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA). This prevents translation and consequently reduces protein levels. This, in turn, promotes their degradation by ERNs via various pathways.

• ⛔ Differences between ERNs and proteases:

  • Objective: RNAs are responsible for RNA degradation, while proteases target proteins.
  • Mechanism and timing of action: RNAs interfere with protein synthesis by altering mRNA before the translation process. Proteases, on the other hand, are responsible for the degradation of proteins that have already been synthesized. RNAs primarily target phosphodiester bonds, while proteases primarily target peptide bonds.

• Li, W. M., Barnes, T., & Lee, C. H. (2010). Endoribonucleases--enzymes gaining spotlight in mRNA metabolism. The FEBS journal, 277(3), 627–641. https://doi.org/10.1111/j.1742-4658.2009.07488.x

• Schoenberg, D., Maquat, L. Regulation of cytoplasmic mRNA decay. Nat Rev Genet 13, 246–259 (2012). https://doi.org/10.1038/nrg3160 Houseley, J., & Tollervey, D. (2009). The many pathways of RNA degradation. Cell, 136(4), 763-776. https://doi.org/10.1016/j.cell.2009.01.019

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

https://theory.labster.com/mechanism-lipofectamine-3000/

• 📕 Definition:

L3000 is a lipid nanoparticle-based transfection reagent that facilitates the entry of nucleic acids (such as DNA or RNA) into eukaryotic cells --> Transfection.

• 👷‍♀️ Mechanism:

1. First, there is complex formation, which occurs by the binding of the cationic lipids of Lipofectamine to the DNA. These protect the DNA and facilitate its entry into the cell.
2. Second, the complexes are internalized by the cell through endocytosis, entering endosomes.

<<(under normal circumstances, the endosome matures to a lysosome and degrades the DNA)>>

1. Third, Lipofectamine 3000 helps the release of DNA into the cytoplasm, preventing its degradation.

2. Finally, the DNA will be transported to the cell nucleus where it will follow the normal process for gene expression.

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

A polytransfection (poly-TX) refers to a transfection where each plasmid is mixed with the transfection reagent separately, forming transfection complexes containing only one plasmid species. The transfected cells take up different combinations of the complexes, resulting in cells containing none, both or only one plasmid species, and all in varying total amounts of plasmid.

Neuromorphic circuits mimic the structure and function of the human brain through neural networks. Therefore, these tools would be useful as they would allow the simultaneous expression of multiple genes involved in neuronal activity, synapses, and synaptic networks to be studied. As well as the construction of synthetic networks that allow understanding of the expression of certain proteins.

https://pmc.ncbi.nlm.nih.gov/articles/PMC6765116/

1. Genetic Toggle Switches:

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

   📕 Definition:

   A genetic switch is a type of synthetic genetic circuit capable of alternating between two states, this is known bistability. This allows it to regulate gene expression in a controlled manner. It involves the mutual inhibition of two repressor genes, each suppressing the expression of the other.

   That is, two repressor genes (1 and 2) are interconnected in a negative feedback loop.

   Therefore, when 1 is expressed, the expression of 2 is inhibited, and vice versa. Therefore, stability is maintained because the system has two stable states that operate continuously.

   Gardner, T. S., Cantor, C. R., & Collins, J. J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature, 403(6767), 339–342. doi:10.1038/35002131

   

   The toggle switch consists of two repressors and two constitutive promoters (Fig. 1). Each promoter is inhibited by the repressor that transcribes the opposite promoter.

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

   One of the most common ways to induce state changes in a genetic toggle switch is the use of inducing molecules that interfere with repression. Such is the case of IPTG (Isopropyl-β-D-thiogalactoside), which binds to LacI, reducing its ability to inhibit transcription of its target gene.

   • Are there any limitations? How many ‘switches’ can we potentially chain? Is there a metabolic cost?

In theory, multiple switches can be designed in series or in parallel, but the biological noise and genetic instability involved must be considered. In addition, the overload of repressors and additional components can overload the cellular metabolism, affecting the stability of the circuit.

1. Natural Genetic Circuit Example:

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

     

https://es.khanacademy.org/science/ap-biology/gene-expression-and-regulation/regulation-of-gene-expression-and-cell-specialization/a/the-lac-operon

In E. coli, there is a well-studied gene regulation system known as the lactose operon (lac operon), responsible for regulating the use of lactose as a carbon source.

Key Components

• Structural Genes:
  • lacZ: encodes β-galactosidase, responsible for the hydrolytic breakdown of lactose into glucose and galactose.
  • lacY: encodes lactose permease, responsible for transporting lactose into the cell.
  • lacA: encodes transacetylase, which does not play such a central role in the system.
• Regulators:
  • lacI: encodes the LacI repressor protein. This protein binds to the operator, inhibiting transcription of the operon in the absence of lactose.
  • Operator (O): binding site for the LacI repressor, which prevents transcription in the absence of lactose. - Promoter (P): region where RNA polymerase binds to initiate transcription.