Lecture (03/18)

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<aside> <img src="/icons/video-camera_yellow.svg" alt="/icons/video-camera_yellow.svg" width="40px" /> Class Recording

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Recitation (03/19)

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Homework

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> This homework is based off of the Week 7 Lab. This is a good week to start honing in final projects and focusing on developing / researching protocol.

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<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links: https://docs.google.com/document/d/1DwQ7I2By4BIKbnY48m6iQ_081TFI-C4xmoVOrPgcNVQ/edit?tab=t.0

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Questions 1-3 are mandatory for all students.

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1. How do endoribonucleases (ERNs) work to decrease protein levels? Name 2 differences between how ERNs work and how proteases work.
2. How does lipofectamine 3000 work? How does DNA get into human cells and how is it expressed?
3. Explain what poly-transfection is and why it’s useful when building neuromorphic circuits.

• How do endoribonucleases (ERNs) work to decrease protein levels?

  Endoribonucleases (ERNs) cleave specific sites within mRNA molecules, leading to their degradation. This prevents translation, thereby reducing protein levels.

  Two differences between how ERNs and proteases work:

  • Target specificity: ERNs degrade mRNA, preventing protein synthesis, whereas proteases degrade existing proteins.
  • Effect timing: ERNs act at the transcriptional level, reducing future protein production, while proteases act post-translationally to degrade already synthesized proteins.

• How does Lipofectamine 3000 work?

  Lipofectamine 3000 is a lipid-based transfection reagent that forms lipoplexes (lipid-DNA complexes), which facilitate the delivery of nucleic acids into cells via endocytosis.

  How DNA gets into human cells and is expressed:

  • The Lipofectamine-DNA complex is taken up by cells through endocytosis.
  • The complex escapes the endosome, releasing DNA into the cytoplasm.
  • If plasmid DNA is used, it enters the nucleus where it is transcribed into mRNA.
  • The mRNA is translated into protein by ribosomes, leading to gene expression.

• What is poly-transfection and why is it useful when building neuromorphic circuits?

  Poly-transfection refers to the simultaneous transfection of multiple genes or genetic elements into cells.

  Why it’s useful for neuromorphic circuits:

  • Neuromorphic circuits often require the expression of multiple ion channels, receptors, or signaling proteins to mimic neuronal behavior.
  • Poly-transfection allows precise control over multiple genetic components, enabling the design of artificial networks that replicate biological neural processing.

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Questions 4-6 have been added on March 19.

Questions 4-6 are optional for but highly encouraged for MIT/Harvard Students and mandatory for **Committed Listeners.

**Responses should be no more than 1 paragraph each, and hand drawn diagrams (iPad, digital or paper) are HIGHLY encouraged

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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.

3. 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.
   • Discuss potential limitations or improvements suggested in subsequent literature or experimental data.

   1. Genetic Toggle Switches

   Mechanism and Bi-Stability

   A genetic toggle switch is a synthetic biological system that enables cells to stably maintain one of two gene expression states (bi-stability) and switch between them in response to an external stimulus. The classic genetic toggle switch consists of two mutually repressive transcription factors (TFs) that inhibit each other’s expression.

   • Bi-Stability Establishment:
     • Two genes, A and B, encode transcriptional repressors that inhibit each other’s promoter activity.
     • If A is dominant, it suppresses B, reinforcing its own expression and maintaining a stable state. Conversely, if B dominates, it inhibits A, creating the alternative stable state.
   • Bi-Stability Maintenance:
     • The system remains locked in a state unless an external signal disrupts the equilibrium.
     • Positive feedback loops strengthen stability, preventing spontaneous flipping due to molecular noise.

   Induction Methods to Switch States

   A common method to induce switching is using an external small molecule or environmental factor to transiently disrupt repression.

   • Example: IPTG and aTc Induction
     • In a LacI/TetR-based toggle switch, LacI represses Gene B, while TetR represses Gene A.
     • Adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) inhibits LacI, allowing Gene B expression, which then represses Gene A.
     • Alternatively, adding anhydrotetracycline (aTc) inhibits TetR, favoring Gene A expression and repressing Gene B.

   Other methods include temperature shifts, light-inducible systems (e.g., optogenetics), and pH or ion-based regulation.

   Limitations and Scalability

   • Metabolic Cost: Continuous expression of repressors and associated regulatory proteins imposes an energy burden on cells, affecting growth and stability.
   • Leakiness: Imperfect repression can lead to unwanted state flipping or mixed expression.
   • Chaining Multiple Switches: Theoretically, multiple switches can be chained in a cascade, but each additional layer increases noise and metabolic load, limiting practical scalability. The number of switches feasible depends on factors like plasmid copy number, cellular resource availability, and protein stability.

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   2. Natural Genetic Circuit Example

   Quorum Sensing in Vibrio fischeri

   Quorum sensing (QS) is a regulatory genetic circuit that enables bacterial populations to coordinate gene expression based on cell density.

   • Key Components:
     • LuxI: Synthesizes the autoinducer molecule (AHL - acyl-homoserine lactone).
     • LuxR: A transcriptional activator that binds AHL when it reaches a critical threshold.
     • Lux Operon: Includes genes encoding luciferase enzymes responsible for bioluminescence.
   • Regulatory Interactions:
     • At low cell densities, AHL diffuses away, and LuxR remains inactive.
     • At high cell densities, accumulated AHL binds LuxR, activating the lux operon, leading to light production.
     • Positive feedback reinforces the response, ensuring a robust, synchronized population-wide switch.

   This circuit exemplifies natural bistability and collective behavior regulation, critical in bacterial communication and pathogenicity.

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   3. Synthetic Genetic Circuit: pDAWN

   Overview

   pDAWN is a light-controlled genetic switch engineered for spatial and temporal control of gene expression using blue light.

   • Key Components:
     • YF1-FixJ System: A blue light-sensitive two-component system where YF1 (a photoreceptor) regulates FixJ, a response regulator.
     • FixK2 Promoter (P_FixK2): Activated by phosphorylated FixJ, controlling gene expression.
     • Circuit Logic: In darkness, YF1 phosphorylates FixJ, inducing expression from P_FixK2. Blue light inhibits phosphorylation, turning off expression.
   • Performance:
     • Enables reversible and precise light-induced gene expression.
     • Fast response time (~30 min) with high dynamic range.

   Limitations & Potential Improvements

   • Leakiness: Residual expression in the "off" state due to incomplete repression.
   • Metabolic Burden: Continuous protein expression of YF1 and FixJ affects cell fitness.
   • Improvement Ideas:

   This synthetic circuit demonstrates how optogenetics can be applied for fine-tuned, non-invasive gene regulation.