Week 6: Genetic Circuits Part I

Week 6: Genetic Circuits Part I

Homework — DUE BY START OF MAR 17 LECTURE

Assignment: DNA Assembly

Answer these questions about the protocol in this week’s lab:

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

Phusion High-Fidelity PCR Master Mix is a chemical environment designed to ensure the most accurate duplication of DNA possible. At its core is the Phusion DNA Polymerase, which is enzyme created by fusing a Pyrococcus-like proofreading polymerase to a double-stranded DNA-binding domain. This structural modification allows the enzyme to remain attached to the DNA template for much longer than standard enzymes, resulting in high processivity and a significantly lower error rate. To support this activity, the mix contains a balanced concentration of dNTPs, which serve as the raw material for the new DNA strands, and a specialized reaction buffer. This buffer includes magnesium chloride, a vital cofactor that helps the polymerase coordinate with the phosphate groups of the incoming nucleotides, as well as various stabilizers that prevent the enzyme from denaturing during the intense heat of the thermal cycling process.

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

Determining the correct annealing temperature for a PCR reaction requires balancing several molecular factors. The primary driver is the melting temperature of the primers, which is largely dictated by their length and their GC content. Because Guanine-Cytosine pairs are held together by three hydrogen bonds compared to the two bonds in Adenine-Thymine pairs, primers with more G and C bases require more energy—and thus a higher temperature—to separate and re-anneal. Furthermore, the concentration of salts and ions in the master mix, such as potassium and magnesium, can stabilize the negative charges on the DNA backbone, effectively raising the required annealing temperature. If the temperature is set too low, the primers may bind non-specifically to the wrong parts of the template, while setting it too high may prevent the primers from binding at all, resulting in no amplification.

  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.

PCR:PCR is a synthetic process that builds billions of new copies of a specific DNA segment using thermal cycling and a polymerase enzyme, making it the preferred choice when starting with a tiny amount of DNA or when you need to add custom sequences, like Gibson tails, to the ends of a fragment. Restriction enzyme digests: restriction digest is an analytical or preparatory process that uses specialized proteins to cut an existing, purified piece of DNA at specific recognition sequences. While PCR is ideal for generating large quantities of modified DNA, restriction digests are often preferred for simpler tasks like subcloning between classic plasmids or performing a diagnostic check to see if a plasmid contains the correct insert.

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

Careful attention must be paid to the design of the fragment ends. Unlike traditional cloning, Gibson Assembly relies on an exonuclease enzyme that chews back the ends of DNA to reveal single-stranded overlaps. Therefore, you must ensure that each PCR-generated fragment has a “tail” that is identical to the end of the adjacent fragment, typically between 20 and 40 base pairs in length. It is also critical to treat your PCR products with an enzyme like DpnI to destroy any original template DNA and to purify the final product through a column or a gel. This prevents leftover primers or incorrect DNA templates from interfering with the assembly process, ensuring that only the intended overlapping fragments are available for the final reaction.

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

circular plasmid is introduced into a bacterial cell, and it relies on making the E. coli “competent” to receive foreign DNA. In a typical chemical transformation, the cells are treated with a calcium chloride solution that helps neutralize the repulsive negative charges between the DNA and the cell membrane. By applying a sudden heat shock at 42°C, a pressure difference is created between the inside and outside of the cell, which momentarily opens up small pores or “adhesion zones” in the lipid bilayer. This allows the plasmid to be pulled into the cytoplasm, where the cell can then begin to express the genes carried on the plasmid, such as antibiotic resistance.

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

    • Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).

    Golden Gate Assembly a method that uses Type IIS restriction enzymes, such as BsaI, to assemble multiple parts simultaneously. These specific enzymes are unique because they recognize a DNA sequence but cut the DNA several bases away from that site, allowing for the creation of custom four-base overhangs. Because the recognition site is actually removed during the cutting process, the final assembled product no longer contains the enzyme’s “handle” and cannot be cut again. This creates a “one-pot” reaction where digestion and ligation happen in a continuous cycle, eventually driving the reaction toward the fully assembled, stable circular plasmid. This method is exceptionally modular and is frequently used in synthetic biology toolkits to mix and match different promoters, genes, and terminators with nearly 100% efficiency.

    • Model this assembly method with Benchling or Asimov Kernel!

Assignment: Asimov Kernel

no asimov account - on pause

  1. Create a Repository for your work
  2. Create a blank Notebook entry to document the homework and save it to that Repository
  3. Explore the devices in the Bacterial Demos Repo to understand how the parts work together by running the Simulator on various examples, following the instructions for the simulator found in the “Info” panel (click the “i” icon on the right to open the Info panel)
  4. Create a blank Construct and save it to your Repository:
    • Recreate the Repressilator in that empty Construct by using parts from the Characterized Bacterial Parts repository
    • Search the parts using the Search function in the right menu
    • Drag and drop the parts into the Construct
    • Confirm it works as expected by running the Simulator (“play” button) and compare your results with the Repressilator Construct found in the Bacterial Demos repository
    • Document all of this work in your Notebook entry - you can copy the glyph image and the simulator graphs, and paste them into your Notebook
  5. Build three of your own Constructs using the parts in the Characterized Bacterials Parts Repo:
    • Explain in the Notebook Entry how you think each of the Constructs should function
    • Run the simulator and share your results in the Notebook Entry
    • If the results don’t match your expectations, speculate on why and see if you can adjust the simulator settings to get the expected outcome

Reading & Resources