Week 6 HW: Genetic Circuits Part I

Assignment: DNA Assembly

  1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

Some components in the Phusion High-Fidelity PCR Master Mix are:

➜ Phusion DNA Polymerase - This enzyme copies the DNA with very high accuracy.

➜ dNTPs (deoxynucleotide triphosphates) - These are the building blocks that the polymerase uses to synthesize new DNA.

➜ Reaction Buffer - This keeps the pH and salt conditions so the enzyme works properly.

➜ Mg²⁺ ions (Magnesium ions) - Magnesium is required for the DNA polymerase to function during DNA synthesis.

➜ Stabilizers and additives - These help keep the enzyme stable and improve the efficency of the PCR reaction.

  1. What are some factors that determine primer annealing temperature during PCR?

➜ One factor that determines primer annealing during PCR is the primer length, because longer primers usually have a higher annealing temperature because they bind more strongly to the DNA.

➜ Furthermore, the GC content of the primer is important because primers with more G and C bases have a higher annealing temperature since G–C pairs form stronger bonds than A–T pairs.

➜ Another factor is the primer sequence, because the exact order of bases can affect how strongly the primer binds to the DNA template.

➜ Salt and magnesium concentration is another important factor because higher concentrations can stabilize primer binding and influence the optimal annealing temperature.

  1. 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.

Restriction Enzyme DigestThis method uses restriction enzymes to cut DNA at specific sequences. It is commonly used to analyze DNA fragments or to prepare DNA for cloning.
PCR (Polymerase Chain Reaction)PCR is used to amplify a specific DNA sequence using DNA polymerase and primers.
The experiment first uses PCR to amplify DNA fragments and introduce mutations into the amilCP gene and after PCR, the samples are treated with the DpnI restriction enzyme, which digests the original methylated plasmid template so that only the newly amplified PCR DNA remains. Then the PCR fragments are combined using Gibson Assembly and later transformed into E. coli. When you want to make many copies of a DNA region, PCR is required, but when you need to cut DNA at specific sites, the restriction enzyme is preferable over PCR.

  1. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

To ensure the DNA fragments work for Gibson cloning, you need to design them carefully so they can join together correctly. The DNA fragments must have matching overlapping ends (usually ~20–40 base pairs). These overlaps allow the fragments to stick together during Gibson Assembly. Furthermore, when doing PCR, primers should be designed to add these overlapping regions to the ends of the DNA fragments and you also need to make sure the overlaps match the correct neighboring fragment so everything assembles in the right order. After PCR or digestion, the DNA should be pure and free of contaminants so the assembly reaction works efficiently. Unlike restriction cloning, you don’t need specific restriction sites, but the ends must be designed to be complementary.

  1. How does the plasmid DNA enter the E. coli cells during transformation?

Plasmid DNA enters E. coli cells during transformation by making the cells temporarily permeable so the DNA can pass through the membrane. A common method is the heat shock transformation, where the cells are first treated with calcium chloride to make their membranes more permeable. Then a sudden increase in temperature (heat shock) creates a temporary opening, allowing the plasmid DNA to enter the cells. Antoher method electroporation, where a short electrical pulse is applied to the cells, which creates tiny pores in the membrane. The plasmid DNA can then pass through these pores into the cell.

  1. Describe another assembly method in detail (such as Golden Gate Assembly).

I found another assembly method on the website Addgene.org. This assembly method is called SLIC (Sequence and Ligation Independent Cloning). SLIC is a method that joins DNA fragments using short homologous (matching) sequences, similar to Gibson, but with fewer enzymes. SLIC joins DNA fragments by creating matching overhangs that anneal, and the final DNA is repaired inside the bacteria. How does SLIC work?

1. Each DNA fragment (for example, your insert and plasmid) is designed so that their ends share 15–25 base pairs of identical sequence. These overlaps are essential because they will allow the fragments to recognize and bind to each other.

2. The DNA is treated with an enzyme such as T4 DNA polymerase. This enzyme “chews back” the ends of the DNA, removing nucleotides from the 3′ ends and creating single-stranded overhangs.

3. When the treated DNA fragments are mixed together, the complementary single-stranded overhangs base-pair (anneal) with each other. This brings the fragments together in the correct order based on their matching sequences.

4. At this stage, the DNA fragments are joined, but there may still be missing bonds in the backbone. So the DNA is not fully complete yet.

5. The partially assembled DNA is introduced into E. coli cells.

6. The bacteria’s natural DNA repair systems fill in missing nucleotides and seal the nicks by using ligase enzymes. This results in a fully intact plasmid.

SLIC Method Diagram
DNA Fragments with Overlaps
→
Exonuclease Treatment
(creates overhangs)
→
Annealing
(fragments stick together)
→
Transformation into E. coli
→
DNA Repair in Cell