How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.
ERNs target specific sequences within mRNA and cleave RNA molecules at specific sites, which leads to mRNA degradation. This results in the reduced production of proteins, since the mRNA cannot effectively translate into proteins.
Proteases break down proteins by cleaving peptide bonds, directly degrading the protein itself. They recognize specific sequences in the target protein and clean them into smaller peptides or amino acids. They target the protein itself, not the translation layer like ERNs.
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 used to deliver DNA or RNA into mammalian cells. It forms lipid-DNA complexes (lipoplexes) by encapsulating the DNA in lipid layers. These complexes are positively charged, allowing them to interact with the negatively charged cell membrane. The lipoplex fuses with the cell membrane via endocytosis, a process where the cell engulfs the complex in vesicles. The DNA is then released inside the cell, often into the cytoplasm or nucleus for transcription.
Lipofectamine facilitates endocytosis, where the DNA enters the cell within a vesicle. The vesicle (a lipid-bilayer enclosed structure, like a viral vector) containing the DNA will destabilize, triggering an escape. This destabilization releases DNA into the cytoplasm. For transcription to occur, the DNA (typically a plasmid) must enter the nucleus. This happens naturally via nuclear pores. Once inside the nucleus, the DNA is transcribed into mRNA. The mRNA is then translated by ribosomes in the cytoplasm into a protein.
Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.
Neuromorphic circuits are electronic circuits designed to mimic the structure and functioning of the human brain. They emulate biological neural networks, like neurons and synapses, to process information in a brain-like way. The goal is to create systems that are more efficient and capable of learning from sensory inputs.
Poly-transfection is the process of introducing multiple genetic materials simultaneously into a single cell (DNA, RNA, plasmids, genes).
When building neuromorphic circuits , poly-transfection is used to introduce multiple components, like receptors, signaling proteins, or ion channels, into cells to build a more complex, interactive network that mimics biological neural circuits. It allows the engineering of cells with multiple properties, which is crucial for creating more sophisticated neuromorphic circuits. These all lead to more accurate models of biology.
Genetic Toggle Switches:
Provide a detailed explanation of the mechanism behind genetic toggle switches, including how bi-stability is established and maintained.
I mean they are environmental-dependent. Like in the Lambda Phage, the decision between lysogeny vs lysis is a switch in the valence difference of the environment. CI and Cro fight over OR1, OR2, and OR3, but the fight is influenced by the overall cell health- should the virus proliferate now, while the cell is healthy, or before it is about to die, so that the manufacturing of the viruses can take advantage of the environment?
Describe at least one induction method used to switch states, including molecular signals or environmental factors involved.
Induction, in the Lambda Phage, example, is driven by UV light, at least experimentally. This triggers RecA to cleave the repressor, CI, which allows Cro to bind to OR3 and start manufacturing. Naturally it is driven by the cell’s state of health.
Are there any limitations? How many ‘switches’ can we potentially chain? Is there a metabolic cost?
Theoretically, there is an approximate carrying capacity- the balance between CI and Cro production that is keeping the cell in balance is bound by the available materials in the cell/what it can attract through its membrane. There is a metabolic cost- the balance of CI vs Cro production is driven by maintaining a balance of production. They are both being produced from the infected DNA, which actually uses RNA + whatever other materials are needed in order to produce the proteins, that then balance the environment.
Natural Genetic Circuit Example:
Identify and describe in detail a naturally occurring genetic circuit, emphasizing its biological function, components, and regulatory interactions.
One naturally occurring genetic circuit is the lac operon in E. coli, which regulates the metabolism of lactose. The operon allows E. coli to metabolize lactose when glucose is scarce (needs to reach a specific system level). It turns on the genes needed to break down lactose (lacZ, lacY, lacA) only when lactose is present.
Components
Promoter: binds RNAP to DNA to initiate transcription (enables transcription)
Operator: DNA sequence where the repressor protein binds to inhibit transcription when lactose is absent
Repressor (lacI): inhibits transcription by binding to the operator in the absence of lactose
Inducer (Allolactose): metabolite of lactose that binds to LacI, preventing it from binding to the operator and enabling transcription
Structural genes
lacZ: encodes β-galactosidase, which breaks down lactose into glucose and galactose
lacY: encodes lactose permease, which transports lactose into the cell
lacA: encodes thiogalactoside transacetylase, which detoxifies byproducts
Regulatory interactions
The LacI repressor binds to the operator to block RNAP from transcribing the genes. When lactose or allolactose is present, it binds to LacI, causing a conformational change that prevents LacI from binding to the operator, allowing transcription. In the
cAMP-CAP complex: In the absence of glucose, cyclic AMP (cAMP) binds to the Catabolite Activator Protein (CAP), which then binds to the promoter, enhancing RNAP binding and increases expression of the operon.
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.
pDAWN is a circuit designed for light-responsive gene expression. It uses a light-inducible promoter (PpsbA2) that activates the transcription of downstream genes in response to a specific range of light waves. The circuit integrates a light-sensitive protein (BphS or PhyB), which senses light and activates the promoter for gene expression, which is a fluorescent protein or antibody. The performance is measured by machines that read the light emitted, most likely from the protein that is expressed, via fluorescence assays.
Discuss potential limitations or improvements suggested in subsequent literature or experimental data.
One of the main constraints, as everything in synthetic biology, is capturing complete systematic information. Only a range of waves trigger this, when in reality, the body is a harmonic city of vibes balancing out the entire population of cells. There is emergent behavior from the system we are not yet able to capture.
Prolonged light exposure may increase the resistance over time, or may burn out the cell by stretching the circuits feedback loop to the asymptote.
The circuit could also impose a metabolic burden on the cell, where it pulls away resources from other functions, especially in the feedforward, continuous-until-complete function above.
All the improvements are fine-tunes or deterministic formal proof-like systems that nullify the original system from experience unintended behavior. Again, we lose information, but gain predictability, which helps lower the cost of engineering for that boundedness. We would then be able to reinvest in expanding that boundary.
