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

Endoribonucleases are enzymes that degrade RNA molecules, particularly messenger RNA (mRNA), to regulate protein synthesis. By cleaving mRNA, these enzymes prevent translation, effectively reducing protein levels in cells. Two key differences between endoribonucleases and proteases are their substrates and mechanisms. Endoribonucleases target RNA, cleaving phosphodiester bonds to halt translation, while proteases degrade proteins directly by hydrolyzing peptide bonds. Additionally, endoribonucleases often require specific RNA sequences or structures for activity, whereas proteases recognize amino acid motifs or protein conformations.

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

Lipofectamine 3000 is a lipid-based transfection reagent that facilitates DNA delivery into human cells. It forms cationic lipid-DNA complexes that fuse with the cell membrane, enabling endocytosis. Once inside, the complexes escape endosomes via pH-dependent membrane disruption and release DNA into the cytoplasm. The DNA then enters the nucleus during cell division or through nuclear pore complexes, where it is transcribed into mRNA and translated into protein. This process bypasses lysosomal degradation, enhancing transfection efficiency compared to older reagents.

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

Poly-transfection involves simultaneous delivery of multiple plasmids into cells, each encoding distinct circuit components. This method is critical for constructing neuromorphic circuits, which require precise ratios of neuronal receptors, ion channels, and signaling molecules. By transfecting multiple genes at once, researchers can emulate the complexity of neural networks and study emergent behaviors like signal integration and plasticity. Poly-transfection also accelerates prototyping of synthetic circuits by testing numerous genetic configurations in parallel.

Genetic Toggle Switches:

Genetic toggle switches are bistable circuits where two repressors mutually inhibit each other’s expression. For example, LacI represses TetR expression, while TetR represses LacI. Bistability arises from nonlinear feedback, creating two stable states: high LacI/low TetR or low LacI/high TetR. Induction methods include adding small molecules like IPTG (which inhibits LacI) or anhydrotetracycline (which inhibits TetR) to flip the switch. Limitations include metabolic burden from constitutive repressor production and crosstalk between components, which restricts the number of switches that can be chained. Theoretical models suggest a practical limit of 3–5 switches due to noise accumulation.

Natural Genetic Circuit Example:

The lac operon in E. coli is a classic genetic circuit regulating lactose metabolism. It consists of three structural genes (lacZ, lacY, lacA) controlled by a promoter, operator, and the lacI repressor. In the absence of lactose, LacI binds the operator, blocking transcription. Allolactose, a lactose metabolite, binds LacI, causing its release from the operator and enabling gene expression. The operon also integrates catabolite repression via cAMP-CRP, which enhances transcription when glucose is scarce. This dual regulation ensures efficient resource allocation, prioritizing glucose metabolism while retaining lactose utilization capacity.

Synthetic Genetic Circuit:

The pDAWN system is a light-inducible genetic circuit using a blue light-sensitive protein (YF1) to control gene expression. In darkness, YF1 inhibits transcription by binding the promoter. Blue light exposure causes conformational changes in YF1, freeing the promoter and enabling RNA polymerase to initiate transcription. The system’s modular design allows insertion of target genes, making it versatile for applications like optogenetic control of neuronal activity. However, limitations include basal leakage in the dark and reduced efficiency in thick tissues due to light scattering. Recent improvements involve engineering red light-responsive variants for deeper tissue penetration.