Week 6 HW: Genetic Circuits Part I: Assembly Technologies
Answers to Questions About This Week’s Lab Protocol
1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
Phusion High-Fidelity PCR Master Mix typically contains several important components: Phusion DNA polymerase, dNTPs, reaction buffer, and MgCl2. The polymerase synthesizes new DNA strands and has proofreading activity, which lowers the error rate compared with standard Taq polymerase. The dNTPs provide the nucleotide building blocks needed to make the new DNA strands. The buffer maintains the proper chemical environment, including pH and salt concentration, for the enzyme to work efficiently. MgCl2 is an essential cofactor that allows the polymerase to function properly. In this lab, the master mix is provided as a 2X mix, so only template DNA, primers, and water are added separately. (neb.com)
2. What are some factors that determine primer annealing temperature during PCR?
Primer annealing temperature is mainly determined by the melting temperature (Tm) of the primer binding region. According to the course page, a good primer binding region is usually about 18–22 base pairs, with a Tm around 52–58 °C, and the Tm values of the two primers should ideally be within about 5 °C of each other. GC content affects annealing temperature because GC base pairs are more stable than AT base pairs, so primers with higher GC content tend to have higher Tm values. A GC clamp at the 3′ end can improve binding stability, while hairpins or primer dimers can reduce efficiency. In this lab, the backbone PCR uses an annealing temperature of 57 °C, while the insert PCR uses 53 °C, showing that different primers may require different annealing conditions. (docs.google.com)
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 creates a linear DNA fragment by amplifying a selected region from a DNA template using primers, polymerase, dNTPs, and a thermocycler. In this week’s lab, PCR is used both to generate the backbone fragment and the color fragment, and it also introduces mutations through the primer sequence. Restriction enzyme digestion, by contrast, cuts DNA at specific recognition sites already present in the sequence. This means restriction digestion is limited by whether suitable restriction sites exist in the right positions, while PCR allows much more flexibility in choosing the exact boundaries of the fragment. (docs.google.com)
PCR is often preferable when you want to customize the fragment, such as adding Gibson overlaps, introducing mutations, or amplifying a region that does not have convenient restriction sites. Restriction enzyme digestion is often preferable when a plasmid already has well-placed restriction sites and you want a straightforward way to cut out a fragment without designing long primers. PCR is more versatile but can introduce amplification errors or require more optimization. Restriction digests are simpler in some cases, but they are less flexible because they depend on the DNA sequence already containing the right enzyme recognition sites. In this lab, PCR is the better choice because the experiment depends on introducing specific mutations into the color sequence and creating Gibson-compatible overlaps at the same time. (docs.google.com)
4. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
To make DNA fragments appropriate for Gibson cloning, the adjacent fragments must contain the correct overlapping homologous sequences. The course page states that Gibson Assembly usually requires 20–40 bp overlaps between neighboring fragments, and these overlaps are designed directly into the primers. You also need to verify that the fragments are in the correct orientation and that their ends match exactly with the intended assembly junctions. In practice, this means checking the fragment design in a sequence editor before running the experiment, and then confirming fragment size by gel electrophoresis after PCR. (docs.google.com)
Another important step in this lab is DpnI digestion. DpnI digests the original methylated plasmid template but leaves the new unmethylated PCR products intact. This reduces background from the original plasmid and helps ensure that the fragments going into Gibson Assembly are the newly generated ones rather than leftover parental template. After that, the DNA is purified and quantified before assembly, which helps confirm that the samples are clean and present at usable concentrations. (docs.google.com)
5. How does the plasmid DNA enter the E. coli cells during transformation?
In this lab, plasmid DNA enters E. coli cells during transformation after the cells are made permeable by heat shock. The course page explains that heat shock or electroporation causes the bacterial membrane to temporarily open up, allowing plasmid DNA to enter the cell by diffusion. After the DNA enters, the cells are placed in SOC medium to recover and start expressing the antibiotic resistance gene carried by the plasmid. The cells are then plated on selective media containing chloramphenicol, so only cells that successfully took up the plasmid can grow. (docs.google.com)
6. Describe another assembly method in detail (such as Golden Gate Assembly).
Another widely used DNA assembly method is Golden Gate Assembly. Golden Gate uses a Type IIS restriction enzyme such as BsaI or BsmBI together with T4 DNA ligase in a one-pot reaction. Unlike standard restriction enzymes, Type IIS enzymes cut outside of their recognition sequence, which allows the user to design custom sticky ends. These sticky ends determine the order in which fragments assemble, so multiple fragments can be assembled directionally in one reaction. During repeated digestion-ligation cycles, incorrectly assembled products are recut, while correctly assembled products are ligated and preserved because the recognition sites are removed in the final construct. This makes Golden Gate highly efficient for assembling multiple modular parts in a predefined order, often without leaving scars between fragments. (neb.com)
7. Explain the other method in 5–7 sentences plus diagrams.
Golden Gate Assembly is a one-pot DNA assembly method that uses a Type IIS restriction enzyme and DNA ligase. The Type IIS enzyme cuts outside its recognition site, creating user-defined sticky ends instead of fixed ones. Those sticky ends determine which DNA fragments can join to each other, allowing multiple fragments to assemble in a chosen order. During thermal cycling, the enzyme cuts unassembled or misassembled molecules, while ligase seals the correct junctions. Because the recognition sites are typically removed during assembly, the final product is often scarless and is no longer recut in the same reaction. Compared with Gibson Assembly, Golden Gate uses short sticky ends rather than long homologous overlaps. This makes it especially useful for modular cloning systems with many interchangeable parts. (neb.com)