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.

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?
2. What are some factors that determine primer annealing temperature during PCR?
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.
4. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
5. How does the plasmid DNA enter the E. coli cells during transformation?
6. 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).
   2. Model this assembly method with Benchling or Asimov Kernel!

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
   1. Recreate the Repressilator in that empty Construct by using parts from the Characterized Bacterial Parts repository
   2. Search the parts using the Search function in the right menu
   3. Drag and drop the parts into the Construct
   4. 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
   5. 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
   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