Week 6 - Genetic Circuits Part I: Assembly Technologies

This week we learn core molecular biology tools and techniques for processing and assembling DNA, including PCR and Gibson Assembly.

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Assignment: DNA Assembly

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

Phusion High-Fidelity PCR Master Mix usually contains the DNA polymerase, dNTPs, buffer, and MgCl2. Its purpose is to copy DNA accurately and efficiently during PCR.

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

Primer annealing temperature depends on the primer melting temperature, primer length, GC content, salt concentration, and additives like DMSO. Primers with higher Tm usually need a higher 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.

PCR and restriction digest both make linear DNA fragments, but PCR amplifies a chosen region using primers, while restriction enzymes cut DNA at specific recognition sites. PCR is best when you want a precise fragment without depending on restriction sites, and restriction digestion is best when the cut sites already exist in the DNA.

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

To make DNA suitable for Gibson cloning, the insert and vector need matching overlapping ends, usually about 20–30 bp of homology. The PCR product should also be clean and in the correct orientation and size

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

During transformation, plasmid DNA enters E. coli cells through the membrane after the cells are made competent, usually by heat shock or electroporation. The treatment helps the DNA cross into the cell.

  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).
  • Model this assembly method with Benchling or Asimov Kernel!

Golden Gate Assembly uses type IIS restriction enzymes that cut outside their recognition sites, creating custom overhangs. You design primers so each DNA fragment has recognition sites at the ends and matching overhangs between fragments.In one reaction, the enzymes cut all fragments and DNA ligase joins the compatible overhangs in the correct order.The final product has no restriction sites left, so you can repeat the process for hierarchical assembly. This method is great for assembling 5+ fragments quickly and precisely without needing specific restriction sites.

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Assignment: Asimov Kernel

  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
  1. 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

I’m late and I was finishing the other HM before doing this one so far, i’m doing it soon.