Week 6 Homework: Genetic Circuits Part I
Week 6 — Genetic Circuits Part I: Assembly Technologies
Assignment: DNA Assembly
1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
Some components that are found in the Phusion High-Fidelity PCR Master Mix are:
- Phusion DNA Polymerase: a enzyme that assembles the new DNA strand and proofreads for errors.
- Nucleotide bases (A, T, C, G) used as building blocks for the new DNA.
- Optimized reaction buffer including MgCl2 :maintains the optimal pH and ionic strength to keep the enzyme stable during thermal cycling.
2. What are some factors that determine primer annealing temperature during PCR?
Some factors That determine the primer annealing temperature during PCR are:
- Base Composition: higher G-C content increases the melting temperature
- Primer Length: longer primers generally have higher Tm values.
- Primer Concentration: higher concentrations can shift the kinetics of annealing.
3. 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.
| PCR | Restriction Enzyme Digest |
|---|---|
| Protocol | |
| Thermal Cycling: You move through specific temperatures (98°C, ~60°C, 72°C) to unzip, prime, and build DNA. | Isothermal Incubation: You hold the sample at a steady temperature (usually 37°C) to let the enzymes “chew” the DNA. |
| Input DNA | |
| Needs very little “template” DNA; it creates millions of copies from almost nothing. | Needs a high concentration of DNA because you are only cutting what is already there. |
| Customization | |
| High: You can add “overhangs” or mutations to the ends of your DNA. | Low: You can only cut where a specific “recognition site” (like GAATTC) already exists. |
- You can use PCR when you need to copy-paste a gene out of a genome or prepare fragments for Gibson Assembly by adding specific overlapping ends.
- You can use Digest for diagnostic purposes (to check if your plasmid is correct) or for traditional cloning if the gene already has the right cut sides at the ends.
4. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
To ensure that the DNA sequences are appropriate:
- each fragment must share a 20–40 bp sequence with the fragment next to it
- purity: must “clean" your DNA (using a kit or gel extraction)
- If there are leftover Polymerase or Restriction Enzymes from the previous steps, they will destroy the Gibson reaction
- If you use a digest, ensure the restriction site isn’t leaving behind DNA that will ruin the protein’s code
5. How does the plasmid DNA enter the E. coli cells during transformation?
Plasmid DNA enters E. coli cells during transformation through temporary pores formed in the cell membrane. These pores are created artificially using methods like: heat shock and electroporation
6. Describe another assembly method in detail (such as Golden Gate Assembly)
a.Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).
b.Model this assembly method with Benchling or Asimov Kernel!
Golden Gate Assembly (GGA) allows you to snap multiple pieces of DNA together in a specific order, all in one go. Most restriction enzymes (like EcoRI) cut DNA right inside their recognition sequence. Golden Gate uses Type IIS restriction enzymes (like BsaI or BbsI), which are unique because they bind to a specific DNA sequence but cut the DNA a few base pairs away from that site. Because the enzyme cuts outside its designated sequence, you can design the sticky ends to be whatever 4 letters you want.By designing the end of Fragment A to match the beginning of Fragment B, you ensure they can only stick to each other.This allows for directional assembly, meaning the parts will never move into the wrong position.In GGA, you put your DNA fragments, the Type IIS enzyme, and DNA Ligase into a single tube. You then cycle the temperature:
- cooler (~37°C): the enzyme cuts the DNA
- warmer (16°C or 25°C): the ligase pastes the matching ends together
In GGA the recognition sites are designed to be “thrown away.” When the enzyme cuts the fragment, the recognition site is on the piece of DNA that gets discarded. Once the fragments are ligated together into the final circular plasmid, the recognition site is gone.