Week 6 HW: Genetic Circuits Pt 1

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Part 1 — Concept Questions

1. Components of Phusion High-Fidelity PCR Master Mix

Phusion PCR master mix typically contains:

High-fidelity DNA polymerase • enzyme that synthesizes new DNA strands • has proofreading ability, reducing mutation errors
dNTPs (deoxynucleotide triphosphates) • building blocks used to create new DNA strands
Reaction buffer • maintains proper pH and salt conditions for enzyme activity
Mg²⁺ ions (magnesium) • essential cofactor for polymerase activity
Stabilizers and enhancers • improve enzyme stability and reaction efficiency

Purpose: To allow accurate amplification of DNA during PCR.

2. Factors that determine primer annealing temperature

Primer annealing temperature depends mainly on:

Primer length • longer primers bind more strongly

GC content • G-C pairs have stronger hydrogen bonding

Primer sequence complementarity • mismatches lower binding strength

Melting temperature (Tm) • annealing temperature is typically 3–5°C below Tm

Salt concentration in buffer • affects DNA duplex stability

3. PCR vs Restriction Enzyme Digests Feature PCR Restriction Digest Purpose Amplify DNA Cut DNA at specific sites Enzyme DNA polymerase Restriction endonuclease Output Many copies of a DNA fragment Linear fragments from cutting Flexibility Primers allow custom fragments Limited to existing restriction sites Use case Creating new DNA fragments Preparing plasmids or inserts

PCR is preferable when you need to generate or modify a DNA fragment.

Restriction digestion is useful when cutting DNA at known sequences for cloning.

4. Ensuring fragments work for Gibson Assembly

For Gibson assembly, DNA fragments must have:

overlapping homologous regions (20–40 bp) so fragments can anneal to each other.

These overlaps can be created by:

• designing primers with overlap sequences • cutting plasmids at appropriate sites

DNA fragments must also be:

• clean and correctly sized • free of incompatible ends

5. How plasmid DNA enters E. coli during transformation

Plasmids enter bacteria through competent cell transformation.

Common methods:

Heat shock transformation

cold competent cells + plasmid DNA ↓ heat shock (~42°C) ↓ pores form in membrane ↓ DNA enters cell

Electroporation

electric pulse ↓ temporary membrane pores ↓ DNA enters cell

After transformation, cells replicate the plasmid.

6. Another Assembly Method: Golden Gate Assembly

Golden Gate Assembly is a cloning method that uses Type IIS restriction enzymes and DNA ligase in the same reaction.

Type IIS enzymes cut DNA outside their recognition site, creating custom overhangs. These overhangs allow multiple DNA fragments to assemble in a predefined order.

The process cycles between digestion and ligation, allowing fragments to be cut and joined repeatedly until the correct construct forms.

Golden Gate is highly efficient and allows the assembly of many DNA fragments in a single reaction. It is commonly used in synthetic biology to build complex genetic circuits.

Simple diagram Fragment A Fragment B Fragment C | | |

Type IIS enzyme cuts → custom overhangs

A – B – C

DNA ligase seals the backbone

PART 2 - Create a Repository for your work

Asimov Kernel is the computational synthetic biology environment. 1️ Create a repository 2️ Create a notebook entry 3️ Rebuild the Repressilator (The repressilator is a famous synthetic gene circuit.)

Repressilator idea:

Gene A represses Gene B
Gene B represses Gene C
Gene C represses Gene A

This results in an oscillating gene expression A ─| B B ─| C C ─| A

Design three synthetic circuits This assignment is teaching you how genetic circuits are engineered This is basically electrical engineering for cells.

Promoters = switches
Genes = components
Repressors = logic gates

#1 TOGGLE SWITCH

Gene A represses Gene B Gene B represses Gene A

Result: stable on/off state.

#2 Inducible gene

Promoter → gene expression when signal present

#3 Oscillator variant Add feedback loops to control amplitude.