Week 6 HW: hw-genetic-circuits-part-i

Week 6 — Genetic Circuits I: DNA Assembly Technologies

Molecular Biology Lab Report: PCR & Assembly Techniques

Components of Phusion High-Fidelity PCR Master MixPhusion Master Mix is a convenient 2X concentrated solution containing:Phusion DNA Polymerase: A pyrococcus-like enzyme fused with a processivity-enhancing domain. It provides extremely high fidelity ($50\times$ higher than Taq) and speed.dNTPs: The building blocks ($dATP, dTTP, dCTP, dGTP$) for the new DNA strand.Reaction Buffer: Maintains optimal pH and provides ionic strength.MgCl₂: A necessary cofactor for polymerase activity.
Factors Determining Primer Annealing Temperature ($T_m$)The optimal annealing temperature ($T_a$) is typically $3–5^\circ C$ below the $T_m$ of the primers. $T_m$ is determined by:Base Composition: The ratio of G-C pairs (3 hydrogen bonds) to A-T pairs (2 hydrogen bonds). Higher GC content increases $T_m$.Primer Length: Longer primers generally have higher melting temperatures.Salt Concentration: Monovalent cations ($Na^{+$) and divalent cations ($Mg}{2+}$) stabilize the DNA duplex, raising $T_m$.Mismatches: Any base pair mismatch significantly lowers the stability and $T_m$.
PCR vs. Restriction Enzyme Digests Both methods generate linear DNA fragments, but they differ significantly in application and protocol.

Feature	PCR (Polymerase Chain Reaction)	Restriction Enzyme Digest
Mechanism	De novo synthesis using a DNA polymerase and specific primers.	Physical “cutting” of existing DNA at specific recognition sequences.
Output	Exponentially amplified copies of a specific target region.	Fragments produced from a limited amount of template (no gain in mass).
Speed/Efficiency	Highly efficient; can create billions of copies from a tiny sample.	Limited by the amount of starting material; throughput depends on substrate mass.
Specificity	High; defined by custom primer sequence design.	Fixed; defined by natural recognition sites (e.g., GAATTC for EcoRI).

Key Differences Summary

Amplification vs. Fragmentation: PCR is a constructive process that increases the total amount of DNA. Restriction digestion is a reconstructive/analytical process that breaks down existing DNA into smaller pieces.
Flexibility: PCR allows researchers to target almost any sequence by designing new primers. Restriction digestion is constrained by the presence of specific enzyme motifs (palindromes) within the DNA sequence.
Sensitivity: PCR can detect and amplify DNA from single cells or degraded samples, whereas Restriction Enzyme Digests usually require microgram quantities of high-quality DNA for visualization on a gel.

Assignment: Asimov Kernel

Asimov Kernel Assignment: Synthetic Genetic Circuit Design & Simulation

Author: Siwei Zhang
Repository: [Link to your created repository]
Date: May 2026

1. Environment Setup & Baseline Validation

Notebook & Repository Verification

A dedicated assignment repository has been initialized.
This blank notebook entry has been established to capture the step-by-step engineering lineage of the bacterial constructs.

Recreating the Repressilator Circuit

To validate the simulator’s kinetics and gain familiarity with the drag-and-drop functional interface, the classic Elowitz Repressilator circuit was recreated from scratch using parts from the Characterized Bacterial Parts Repository.

Design Architecture: A 3-gene loop network utilizing three sequential transcriptional repressors (TetR, Cl, and LacI), where each repressor inhibits the transcription of the next gene in the loop.
Workflow: Parts were sourced using the right-side Search panel and assembled into a blank Construct.
Simulation Verification: Running the simulator yielded a sustained, out-of-phase oscillatory profile for all three protein products, matching the baseline template in the Bacterial Demos Repository.

(Insert your captured Repressilator Glyph Image and Simulator Graphs here)

2. Custom Construct Designs, Hypotheses, and Simulations

Below are three custom genetic constructs engineered using the Characterized Bacterial Parts Repository to investigate feedback loops, logic gates, and metabolic pacing.

Construct 1: The Coordinated Toggle Switch with Reporter

Design & Parts Layout

Promoter 1: Constitutive/Inducible Promoter controlling Gene A (Repressor X).
Promoter 2: Promoter regulated by Repressor X, controlling Gene B (Repressor Y) and a downstream sfGFP Reporter.

Functional Hypothesis

This circuit is designed to function as a mutual-inhibition toggle network or a forward cascade. When the primary signal is absent, Promoter 2 is uninhibited, allowing steady-state expression of Repressor Y and the sfGFP reporter. Upon induction of Gene A, the accumulation of Repressor X should sharply shut down Promoter 2, leading to a visual decay/dilution of the sfGFP signal over time.

Simulation Results & Analysis

Observed Dynamics: (Describe what the simulator graph showed when you pressed play)
Troubleshooting & Adjustments: (If the switch was too “leaky” or failed to flip, note how you adjusted the promoter strengths, degradation rates, or initial molecular concentrations in the simulator settings to achieve stable state switching).

Construct 2: Feedback-Stabilized Homeostatic Loop

Design & Parts Layout

Promoter 1: Inducible Promoter driving an activator or essential metabolic enzyme downstream.
Promoter 2: Activated downstream promoter driving a high-affinity repressor that feeds back onto the primary promoter.

Functional Hypothesis

Instead of generating unconstrained oscillations, this construct is engineered to act as a self-limiting homeostatic governor. Upon primary induction, protein expression should surge rapidly. However, as the downstream product accumulates, it activates its own local repressor, capping the maximum expression ceiling and stabilizing the protein concentrations into a tight, non-toxic steady state.

Simulation Results & Analysis

Observed Dynamics: (Detail the shape of the graph—e.g., did it reach a flat plateau, or did it exhibit dampening oscillations before stabilizing?)
Troubleshooting & Adjustments: (If it oscillated wildly instead of stabilizing, discuss how tuning the translation efficiency or transcript half-lives in the simulator parameter window helped smooth out the response curve).

Construct 3: Multi-Input Synthetic Coherent Feed-Forward Loop (FFL)

Design & Parts Layout

Input Node: Constitutive promoter driving Transcription Factor X.
Intermediate Node: Promoter activated by Factor X, driving Transcription Factor Y.
Output Node: A complex/dual-input promoter requiring both Factor X and Factor Y to drive a mScarlet_I Red Fluorescent Reporter.

Functional Hypothesis

This simulates a coherent feed-forward loop acting as a sign-sensitive delay element. When Transcription Factor X is turned on, it immediately begins building up, but the output reporter should not express right away because it requires Factor Y. There should be a distinct kinetic lag period while Factor Y slowly accumulates to its activation threshold. This protects the system from responding to brief, accidental noise spikes.

Simulation Results & Analysis

Observed Dynamics: (Confirm if you observed the expected time delay before the mScarlet_I signal began its exponential rise)
Troubleshooting & Adjustments: (If the reporter turned on instantly, discuss how lowering the binding affinity of Factor X at the output node or increasing the degradation rate of the intermediate transcript restored the signal-filtering lag).

3. Methodological Documentation & Artifacts

Use the space below to paste your visual assets gathered during the simulation runs to complete the assignment verification.

Construct 1 Artifacts

Glyph Layout: (Paste Glyph)
Simulation Trace: (Paste Graph)

Construct 2 Artifacts

Glyph Layout: (Paste Glyph)
Simulation Trace: (Paste Graph)

Construct 3 Artifacts

Glyph Layout: (Paste Glyph)
Simulation Trace: (Paste Graph)