<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.

   Ans: Endoribonucleases (ERNs) decrease protein levels by cleaving RNA molecules internally, especially messenger RNAs (mRNAs). This cleavage initiates mRNA degradation, preventing the mRNA from being translated into protein, and thereby reducing the overall protein output in the cell For example, the coronavirus endoribonuclease nsp15 targets host and viral RNAs, leading to their degradation and a subsequent drop in protein synthesis

   Two differences between ERNs and proteases:

   • Substrate: ERNs act on RNA (mainly mRNA), while proteases act on proteins.
   • Stage of regulation: ERNs prevent protein synthesis by degrading mRNA before translation, whereas proteases degrade proteins after they have already been synthesized

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

   Ans: Lipofectamine 3000 is a lipid-based transfection reagent that forms complexes with DNA (or RNA). These complexes interact with the cell membrane and enter the cell via endocytosis. Once inside, Lipofectamine 3000 helps the DNA escape from the endosome, preventing its degradation in lysosomes. The DNA is then transported to the nucleus, where it can be transcribed and expressed as protein. In dividing cells, DNA can enter the nucleus during mitosis; in non-dividing cells, Lipofectamine 3000 also facilitates nuclear entry

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

   Ans: Poly-transfection is a method where each plasmid DNA is separately complexed with transfection reagent before being added to cells, rather than mixing all plasmids together as in co-transfection. This results in cells receiving different combinations and ratios of the DNA constructs, allowing researchers to sample a wide range of genetic circuit stoichiometries within a single experiment. This is especially useful for building neuromorphic circuits, which require precise tuning of multiple components, because poly-transfection enables rapid, high-throughput optimization of circuit performance by testing many configurations in parallel

<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?

   Ans: Mechanism and Bi-stability:

   A genetic toggle switch is a synthetic circuit typically composed of two genes that mutually repress each other. Each gene encodes a repressor protein that inhibits the expression of the other gene. This mutual inhibition creates two stable states (bi-stability):

   • State 1: Gene A is ON, repressing gene B (OFF)

   • State 2: Gene B is ON, repressing gene A (OFF)

     Once the system is in one state, it remains there until an external signal induces a switch

   Induction Methods:

   Switching states can be achieved by adding small molecules that inhibit the activity of one repressor. For example, isopropyl-β-D-thiogalactopyranoside (IPTG) can inhibit LacI, and anhydrotetracycline (aTc) can inhibit TetR in the classic toggle switch, flipping the system from one state to the other.

   Limitations and Scalability:

   • Limitations: The number of toggle switches that can be chained is limited by circuit complexity, genetic part orthogonality, and cellular burden.
   • Metabolic Cost: Each additional switch increases metabolic load, potentially reducing cell viability and circuit stability
   • Chaining: While theoretically possible to chain multiple switches, practical constraints (such as crosstalk, noise, and resource competition) limit the number to a handful in most systems

2. Natural Genetic Circuit Example:

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

   Ans: A classic example is the lac operon in E. coli.

   • Function: Allows bacteria to metabolize lactose only when glucose is absent and lactose is present.
   • Components:
     • lacZYA structural genes (encode enzymes for lactose metabolism)
     • lacI repressor (inhibits operon in absence of lactose)
     • Operator and promoter regions (control transcription)
   • Regulatory Interactions:
     • In absence of lactose, LacI binds the operator, blocking transcription.
     • When lactose is present, it binds LacI, causing it to release from the operator, allowing transcription and lactose metabolism

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.

   Ans:

   Construction and Components:

   • pDAWN is a light-inducible genetic circuit designed for precise control of gene expression in bacteria.
   • It uses a blue-light responsive transcription factor (YF1) fused to a response regulator (FixJ), which controls a promoter driving target gene expression.

   Intended Function:

   • Enables gene expression to be turned ON or OFF in response to blue light, allowing spatial and temporal control.

   Performance and Limitations:

   • pDAWN enables robust, reversible, and tunable gene expression with high dynamic range in response to light.
   • Limitations:
     • Light penetration can be a challenge in dense cultures or tissues.
     • Potential for leaky expression in the dark.
   • Improvements:
     • Subsequent studies have sought to reduce background activity, increase sensitivity, and adapt the system for use in other organisms or with different wavelengths of light.