Week 06: Genetic Circuits Part I

Assignment 1 — DNA Assembly Questions

Q1. Components of Phusion High-Fidelity PCR Master Mix

Components

Phusion DNA Polymerase
Thermostable, high-fidelity enzyme with proofreading activity that synthesizes new DNA strands.
dNTPs
The four nucleotide building blocks (A, T, G, and C) used to construct DNA.
Reaction Buffer
Maintains optimal pH and ionic conditions. Also contains MgCl₂, an essential cofactor for polymerase activity.

Q2. Factors Determining Primer Annealing Temperature

The annealing temperature mainly depends on:

Primer length
GC content

Longer primers and primers with higher GC content generally require higher annealing temperatures because GC base pairs form three hydrogen bonds, compared to two hydrogen bonds in AT pairs.

Q3. PCR vs Restriction Enzyme Digest

Feature	PCR	Restriction Digest
Equipment	Thermocycler	Heat block / incubator
Inputs	Template DNA + primers	DNA with restriction sites + enzymes
DNA Ends Produced	Usually blunt ends	Sticky or blunt ends
Best Use Case	Amplifying or modifying DNA; adding overhangs	Removing inserts; directional cloning
Purification Required	Yes	Yes

Q4. Ensuring Fragments are Appropriate for Gibson Assembly

To ensure fragments are suitable for Gibson cloning:

Design and verify overlaps in silico using the Benchling Assembly Wizard.
Run PCR or digest products on an agarose gel to confirm expected fragment sizes.
Purify the fragments before assembly.
For maximum confidence, sequence the purified products before Gibson assembly.

Q5. How Plasmid DNA Enters E. coli During Transformation

During heat-shock transformation:

Cells are treated with CaCl₂, which helps neutralize the negative charges on DNA and the bacterial membrane.
Cells are first kept on ice and then briefly exposed to 42°C.
This sudden temperature shift temporarily creates pores in the membrane, allowing plasmid DNA to enter the cell.

Q6. Golden Gate Assembly

Golden Gate Assembly uses Type IIS restriction enzymes such as BsaI or AarI.

Key Features

These enzymes cut outside their recognition sequence.
Custom 4-base sticky ends can be designed for directional assembly.
Multiple DNA fragments can be assembled in a single reaction.

Why It Works Efficiently

Correctly assembled fragments lose the recognition sites, preventing further cutting.
Incorrect assemblies retain recognition sites and are cut again by the enzyme.

This creates a highly efficient, seamless, and scarless DNA assembly method.

Assignment 2 — Asimov Kernel

Tasks

Create a repository for your work.
Create a blank notebook entry and save it to the repository.
Explore devices in the Bacterial Demos Repository.
Run simulations using the Simulator and follow the instructions provided in the Info Panel (i icon).
Reconstruct the Repressilator construct.
Create Original constructs.