Week 6 HW: Genetic Circuits Part I: Assembly Technologies

Part A. DNA Assembly

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

Phusion High-Fidelity DNA Polymerase: A Pfu-like enzyme fused to a dsDNA-binding domain (Sso7d). This increases processivity and ensures an error rate 50 times lower than Taq polymerase. 5X Phusion HF Buffer (including $MgCl_2$): Maintains optimal pH and provides Magnesium ions, which act as essential cofactors for the polymerase to catalyze the addition of dNTPs. dNTPs (Deoxynucleotide Triphosphates): The molecular “bricks” (dATP, dTTP, dCTP, dGTP) used to synthesize the new DNA strand. Stabilizers: Often including glycerol or mild detergents to maintain enzyme stability through repeated thermal cycling.

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

The annealing temperature is usually calculated as $T_m - 5^\circ\text{C}$. Key factors include:GC Content: G-C pairs have three hydrogen bonds (compared to two in A-T), requiring more thermal energy to denature. Higher GC content increases $T_m$.Primer Length: Longer primers have more total hydrogen bonds, leading to a higher melting temperature.Salt Concentration (Cations): $Na+$ and $Mg{2+}$ ions in the buffer stabilize the DNA double helix by neutralizing the negative charges of the phosphate backbone.Primer Concentration: Higher concentrations can slightly shift the kinetics of hybridization.

  • 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.

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

To ensure success in a Gibson Cloning reaction, you must verify the following:

  1. Overlapping Ends: Adjacent fragments must share 15–40 bp of identical sequences at their ends. This is achieved by designing PCR primers with “overhangs” that match the neighboring fragment.
  2. DNA Purity: You must remove the original template (via DpnI digestion) and residual primers to prevent non-specific products.
  3. Correct Concentration: Fragments should be added in specific molar ratios (e.g., 1:2 or 1:3 vector-to-insert) to optimize assembly efficiency.
  • How does the plasmid DNA enter the E. coli cells during transformation?

During Chemical Transformation (using $CaCl_2$):Neutralization: Calcium ions ($Ca^{2+}$) neutralize the negative charges of both the DNA phosphate backbone and the cell membrane’s phospholipids, allowing them to come into close contact.Heat Shock: A sudden increase to 42°C creates a thermal gradient that generates temporary pores in the plasma membrane.Entry: The pressure difference and thermal motion “push” the DNA into the cytoplasm before the cells are moved to a recovery medium to heal the membrane.

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

  1. Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online). Golden Gate Assembly is a molecular cloning method that utilizes Type IIS Restriction Enzymes (such as BsaI or BsmBI) and T4 DNA Ligase. Unlike standard enzymes, Type IIS enzymes cut outside of their recognition sites, allowing for the creation of custom, non-palindromic “sticky ends” (overhangs). The reaction is performed in a “one-pot” format, where digestion and ligation occur simultaneously; because the ligation product lacks the original restriction site, it cannot be re-digested, driving the reaction toward the final assembly. This method is scarless and highly efficient, enabling the directional assembly of 10+ fragments in a single step. It is the gold standard for creating complex genetic circuits and modular libraries in Synthetic Biology.

  2. Model this assembly method with Benchling or Asimov Kernel!

Part B. Asimov Kernel