Week 6 HW: Genetic Circuits I
DNA Assembly
Answer these questions about the protocol in this week’s lab:
What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
I didn’t find anything pertaining the matter in the protocol itself, though a quick websearch (Link 1, Link 2), revealed that the PCR Master Mix contains 4 main ingreadients.
- Phusion DNA Polymerase
- Copying the Gene
- Deoxynucleotide Triphosphates (dNTPs)
- Building blocks (ATCG) used by the polymerase to synthesize the new strand of DNA
- Reaction Buffer
- Maintaining the ideal environment for the polymerase to function
- Magnesium Chloride
- Cofactor for the polymerase reaction
What are some factors that determine primer annealing temperature during PCR?
The annealing temperature Ta is determined by the melting temperature Tm, around 3-5°C lower. The melting temperature is determined by the primer length and the GC content.
- Primer length and GC Content: The longer the primer length, the and the higher the GC content, the higher the annealing temperature can become. GC have three hydrogen bonds, that needs more energy (temperature) to break.
- Salt and Ion Concentration: DNA backbone is negatively charged and two strand would repel each other. The salt provides cations to cancel the negative charge.
- Primer Concentration: Similar logic as 1). The higher the concentration of the primer the higher the annealing temperature
There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other.
In short PCR builds DNA up from scratch, while restriction digests cut existing DNA down. PCR binds to single stranded DNA and complements the strand with free flowting dtNs, effectively “negative copying” the DNA. It needs little input DNA. The polymerase docks at custom primers. It can output billions of strands of DNA. The PCR Protocol is thermocyclic and consists of denaturation (95°C), annealing (55-65°C) and extension (72°C). This process is then repeated 30-40 times. Restriction Enzyme Digests cuts DNA fragments by enzymatic cleavage. It needs large amounts of clean DNA fragments. The enzyme binds to restriction sites and cuts them. Protocol for restriction enzyme digest is isothermal. The main steps include mixing DNA, restriction enzyme and buffer, incubation at 37°C for 30 min, then inactivation of the enzyme (and the reation) through heat (around 65-85°C).
The two methods are very different, but from the description it gets clear when to pick each method. PCR should be used when you have little samples, when you want to add sequences, like tags and the sequence is known (essential for primer design) Restriction enzymes should be used when you are cloning (as you need sticky sites, checking the validity of a plasmid, or you’re working with unknown sequences.
How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
Gibson Assembly operates with homologous overlapping ends, and doesn’t require specific restriction sites or leave behind “scars” like traditional restriction cloning. Therefore the main requirement for a successful Gibson Assembly is the correct design of the 20-40bp long overlap.
How does the plasmid DNA enter the E. coli cells during transformation?
There are several transformation methods, like heat shock, electroporation or conjugation, etc. The lab went into detail with heat shock, therefore this will be the focus of the answer. The cells and plasmids are suspended in a cold calcium chlorid solution (mostly in an ice bucket), then heated up in a waterbath or PCR machine (42°C) for 30-60 seconds.
Here is a detailed instruction video
Describe another assembly method in detail (such as Golden Gate Assembly)
I choose Golden Gate Assembly.
Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).
Golden Gate Assembly is a molecular cloning method that uses Type IIS restriction enzymes to assemble multiple DNA fragments into a single vector in one reaction. Type IIS enzymes (like BsaI or BbsI) cut at a specific distance away, leaving behind 4-base “overhangs” that are not resticted by the enzyme’s binding sequence, unlike traditional enzymes that cut within their recognition site. Plainly they have a recognition site, where they dock, but then instead of cutting in their recognition site, they reach over and cut a few basepairs away. By thoughtfully designing these overhangs, you can ensure that multiple fragments anneal only in a specific, predetermined order. Because the recognition sites are oriented to be “cut out” during the reaction, the final assembled product lacks the original enzyme sites, making the process irreversible . This “one-pot” reaction combines digestion and ligation in a single tube, allowing for the seamless assembly of multiple fragments simultaneously.
Model this assembly method with Benchling or Asimov Kernel!
Asimov Kernel
NOTICE 2026-03-17: As of the deadline, we have not received access to the Asimov Kernel