Week 06 HW: Genetic circuit part I
Table of Contents
Assignment: DNA Assembly
1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
The Phusion High-Fidelity PCR Master Mix contains several essential components required for DNA amplification. The protocol specifically lists the use of Phusion HF PCR Mix (2X) during PCR setup.
Typical components include:
Phusion DNA Polymerase A high-fidelity DNA polymerase responsible for synthesizing new DNA strands with very low error rates. dNTPs (deoxynucleotide triphosphates) The molecular building blocks (A, T, G, C) used to synthesize DNA. Reaction Buffer Maintains optimal ionic strength and pH for enzyme activity. Mg²⁺ ions Essential cofactor required for polymerase function. Stabilizers and salts Improve enzyme stability and PCR efficiency.
The use of a high-fidelity polymerase is especially important in cloning experiments because mutations introduced during PCR could alter the final protein sequence.
2. What are some factors that determine primer annealing temperature during PCR?
Several factors determine the annealing temperature during PCR:
Primer length GC content Melting temperature (Tm) Sequence complementarity Secondary structures
The protocol recommends:
primers with a Tm between approximately 52–58°C, primer pairs within 5°C of each other, GC content around 40–60%, and inclusion of a GC clamp at the 3′ end.
Annealing temperature is generally chosen about:
2–5°C below the lower primer Tm
as described in the appendix.
If the annealing temperature is too low:
nonspecific binding may occur.
If it is too high:
primers may fail to bind efficiently.
3. Compare PCR and restriction enzyme digests for generating linear DNA fragments
Both PCR and restriction enzyme digestion can generate linear DNA fragments, but they work differently and are useful in different contexts.
PCR amplifies a specific DNA region using:
- primers,
- polymerase,
- thermal cycling.
Advantages:
- highly specific,
- can introduce mutations,
- can add Gibson overlaps,
- does not require restriction sites.
Disadvantages:
- may introduce amplification errors,
- requires careful primer design.
In this protocol, PCR is used to generate:
- the backbone fragment,
- and the color insert fragment for Gibson assembly.
- Restriction Enzyme Digest
Restriction digestion cuts DNA at specific recognition sequences using enzymes.
Advantages:
- precise cleavage,
- efficient for existing plasmid architectures.
Disadvantages:
- requires compatible restriction sites,
- less flexible for mutagenesis,
- may leave unwanted scars.
Restriction digests are preferable when:
- suitable restriction sites already exist,
- and no sequence modification is needed.
PCR is preferable when:
- introducing mutations,
- assembling custom fragments,
- or performing Gibson assembly.
4. How can you ensure that the DNA sequences are appropriate for Gibson cloning?
For Gibson Assembly, DNA fragments must contain overlapping homologous regions.
The protocol specifies:
- overlaps of approximately 20–40 bp,
- correct 5′→3′ orientation,
- and complementary overhangs designed through primers.
To ensure compatibility:
- primers must include overlap regions,
- fragments must be purified,
- PCR products should be verified by gel electrophoresis,
- fragment sizes should match expected values.
The protocol also uses: DpnI digestion to remove the methylated parental plasmid template after PCR. This reduces background colonies from unmutated plasmids.
5. How does plasmid DNA enter E. coli cells during transformation?
The protocol describes two common transformation methods:
- heat shock,
- electroporation. Both methods temporarily create pores in the bacterial membrane.
Heat Shock A rapid temperature increase causes transient membrane destabilization.
Electroporation A high-voltage electrical pulse creates temporary membrane pores.
According to the protocol:
- “The plasmid now enters the cells by diffusion.”
After transformation: cells recover in SOC medium, express antibiotic resistance genes, and are plated on selective media.
Only transformed cells survive antibiotic selection.
6. Describe another assembly method in detail: Golden Gate Assembly
Golden Gate Assembly is a cloning method based on:
- Type IIS restriction enzymes,
- and DNA ligase.
Unlike standard restriction enzymes, Type IIS enzymes cut outside of their recognition sequence, allowing the generation of custom overhangs.
This enables:
- scarless assembly,
- directional cloning,
- simultaneous assembly of multiple fragments in one reaction.
A Golden Gate reaction typically alternates between:
- digestion,
- and ligation cycles. The restriction enzyme continuously cuts incorrect assemblies while ligase seals correctly matched fragments.
Because the recognition sites are removed during assembly:
- the final construct is stable,
- and cannot be recut.
Golden Gate is particularly useful for:
- modular cloning,
- synthetic biology,
- combinatorial DNA assembly,
- and large multi-fragment constructs.
Example Diagram of Golden Gate Assembly
Benchling / Modeling Component
Golden Gate Assembly can be modeled in Benchling by:
defining BsaI restriction sites, designing compatible overhangs, and simulating fragment assembly.
Benchling allows:
visualization of sticky ends, plasmid circularization, and verification of reading frames and orientation.
Asimov Kernel Assignment — Repository and Circuit Design
I was able to access the Asimov Kernel interface and explore the public repositories, including the Characterized Bacterial Parts repository and the Bacterial Demos examples. However, my account did not appear to have the necessary permissions or node access required to fully create, save, and simulate Constructs within a personal Repository.
Nevertheless, I investigated how the system is intended to function and reconstructed the expected logic of the exercise conceptually.
Expected Repressilator Design
The Repressilator is a synthetic oscillatory gene circuit composed of three repressors connected in a cyclic inhibition loop:
- pTetR → LacI
- pLacI → LambdaCI
- pLambdaCI → TetR
The expected regulatory behavior is:
- TetR represses pTetR
- LacI represses pLacI
- LambdaCI represses pLambdaCI
This creates a delayed negative feedback loop capable of producing oscillatory dynamics in gene expression.
To construct this system in Kernel, the workflow would likely involve:
- Creating a blank Construct inside a Repository.
- Searching the Characterized Bacterial Parts database.
- Dragging promoters, CDS regions, RBS elements, and terminators into the Construct editor.
- Linking the transcriptional units sequentially.
- Running the simulator to observe oscillatory expression patterns.
Proposed Personal Constructs
Construct 1 — Self-Repression Circuit pLacI → LacI
This circuit would likely produce negative autoregulation. I would expect expression to stabilize at an intermediate level rather than continuously increasing.
Construct 2 — Toggle Switch pLacI → TetR pTetR → LacI
This mutual repression architecture should behave as a bistable toggle switch where one repressor dominates while suppressing the other.
Construct 3 — Repression Cascade pLambdaCI → LacI pLacI → TetR
This design would likely create delayed repression behavior rather than oscillations. Changes in upstream regulation would propagate progressively through the circuit.
Expected Simulation Behavior
If the simulator were fully accessible, I would expect:
oscillatory curves for the repressilator, stable equilibria for self-repression, bistable states for the toggle switch, and delayed temporal dynamics for the repression cascade.
Differences between expected and simulated behavior could result from:
promoter strength imbalance, degradation rate settings, insufficient repression efficiency, stochastic effects, or simulation parameter choices.
Adjusting repression constants, degradation rates, or transcriptional delays would likely help tune the system toward the expected behavior.