Week 6 HW: Genetic Circuits Part I

Part 1: 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?

The master mix can be found here and contains “Phusion DNA Polymerase, nucleotides, and optimized reaction buffer including MgCl2”. The polymerase is an essential part of PCR, nucleotides are the base materials needed to form new DNA sequences, and reaction buffer lowers the energy needed to start the process.

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

The master mix can be found here and contains “Phusion DNA Polymerase, nucleotides, and optimized reaction buffer including MgCl2”. The polymerase is an essential part of PCR, nucleotides are the base materials needed to form new DNA sequences, and reaction buffer lowers the energy needed to start the process.

  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.
MethodPCRRestriction Enzyme
ThroughputHigh (2^n)Low (n)
ProtocolRequires 3 step process of varying temperature, 1 pot reactionRequires plasmid preparation to form recognition sites in DNA
Protein InvolvedPrimer, PolymeraseEndonuclease
  1. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

You can verify experimentally how well the DNA is assembled. Plasmids are usually built with an antibiotic resistance gene, and in the test you put an antibiotic on your plasmids to see which survives. The ones that survive should be fully assembled.

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

The heat shock method puts the bacteria and plasmid at a high temperature for about a minute, which “shocks” the bacteria into forming pores that the plasmid can enter through.

  1. 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 also a form of molecular cloning, but uses restriction enzymes unlike Gibson Assembly. It uses recognition sites instead of overlapping sequences, and the enzymes are responsible for stripping the DNA (in place of exonuclease) and removing recognition sites to create the final construct (in place of ligase). This allows Golden Gate Assembly to accommodate multiple fragments in a one-pot reaction to create a single long strand of DNA. On the other hand, it needs more specific design and choosing of enzymes, including making sure recognition sites are unique within the sequence and do not show up accidentally.

    1. Model this assembly method with Benchling or Asimov Kernel! Further modeling in Asimov in link below.

Part 2: Asimov Kernel

  1. 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)
  2. Recreate the Repressilator in that empty Construct by using parts from the Characterized Bacterial Parts repository
  3. Build three of your own Constructs using the parts in the Characterized Bacterials Parts Repo
    1. Explain in the Notebook Entry how you think each of the Constructs should function
    2. Run the simulator and share your results in the Notebook Entry
    3. 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

My folder, along with the constructs and notebook documentation, can be found here.