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 the delivery of nucleic acids (DNA, RNA) into human cells. It forms lipid-DNA complexes that fuse with the cell membrane, enabling endocytosis. Once inside, the DNA escapes the endosome and reaches the nucleus, where it can be transcribed into mRNA. The mRNA is then translated into protein by ribosomes in the cytoplasm.

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

Poly-transfection is a technique that allows the simultaneous introduction of multiple DNA sequences into cells. It is useful in building neuromorphic circuits because it enables the controlled expression of multiple genes in synthetic neural networks. This is essential for replicating brain functions, enhancing artificial synaptic connectivity, and modeling neurological disorders.

Genetic Toggle Switches:

Mechanism and Bistability

Genetic toggle switches work by using two mutually repressing genes. Each gene suppresses the expression of the other, creating a bistable system. Positive feedback loops ensure that the system stays in one state until an external signal triggers a switch.

Induction Methods

The states of a toggle switch can be flipped using various methods:

• Chemical inducers like IPTG (inhibits LacI) or aTc (inhibits TetR).
• Temperature shifts with heat-sensitive repressors.
• Optogenetics, which uses light to control gene expression.
• CRISPRi for precise gene repression.

Limitations and Constraints

• How many switches can be chained?

  Multiple switches can be linked, but genetic instability and cross-talk between genes limit scalability. Too many switches can lead to complexity and noise, making the system harder to control.

• Is there a metabolic cost?

  Yes, constant production of repressors and reporters adds a metabolic burden to the cell, potentially slowing growth and reducing efficiency. Larger circuits increase this burden.

Natural Genetic Circuit Example:

Example: pMUT2-pLac-mCherry

• Biological function: This plasmid contains the Lac promoter, which regulates the expression of the mCherry fluorescent protein. It functions as a reporter system, allowing fluorescence-based monitoring of gene expression.
• Components:
  • pLac (Lac promoter): Inducible by IPTG or lactose.
  • mCherry: A red fluorescent protein used as a reporter.
  • Backbone elements: Selection markers and replication origin.
• Regulatory interactions: When lactose or IPTG is absent, the lacI repressor binds to the operator, preventing mCherry expression. When IPTG or lactose is added, the repressor is inactivated, allowing transcription and fluorescence detection.

Synthetic Genetic Circuit:

Example: pHR_PGK_SNIPR_synRobo/Notch

Construction and Components:

The pHR_PGK_SNIPR_synRobo/muesca plasmid encodes a synthetic Notch receptor (synNotch) fused with the Robo signaling domain. Under the PGK promoter, the synNotch receptor is expressed in mammalian cells. Upon ligand binding, the receptor undergoes proteolytic cleavage, releasing an intracellular domain that acts as a transcription factor to activate specific gene expression.

Function and Importance:

• Cell-to-Cell Communication: The system enables precise cell-to-cell communication through designed receptors that respond to specific ligands, triggering a customized genetic response.
• Immunotherapy Applications: It is commonly used to program T cells for targeted immunotherapy, allowing recognition of tumor antigens while minimizing off-target effects.
• Genetic Control: The system is highly modular, providing controlled expression of target genes in response to external stimuli. It allows for the fine-tuned regulation of gene activation.

Limitations and Improvements:

• Ligand Dependency: The system’s effectiveness is dependent on the availability and specificity of ligands, and there may be challenges in identifying ligands that are both highly specific and efficient.
• Cleavage Efficiency: The proteolytic cleavage may not always be 100% efficient, leading to variability in gene expression.
• Off-Target Effects: Despite the advancements in specificity, there is still a potential risk for unintended activation of genes or immune responses.
• Safety Concerns: The use of synthetic receptors and genetic manipulation could lead to unforeseen immune responses or off-target effects, requiring rigorous testing.
• Development Time and Cost: The process of creating and optimizing these systems can be time-consuming and costly. Additionally, large-scale production remains challenging.
• Human Applications: While promising in preclinical models, the transition to clinical human therapies still faces hurdles related to safety, dosage, and long-term efficacy.