Week 6 — Genetic Circuits Part I: Assembly Technologies

DNA Assembly

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

What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The Phusion High-Fidelity PCR Master Mix contains several components:

Phusion DNA polymerase → a high-fidelity enzyme that synthesizes DNA with very low error rates (With a failure rate 50 times lower than Taq and 6 times lower than Pfu, these polymerases are an excellent choice for cloning and other applications requiring high fidelity), which is critical when amplifying fragments of the amilCP gene.
dNTPs (deoxynucleotide triphosphates) → building blocks for new DNA strands
MgCl₂ → cofactor necessary for polymerase activity
Buffer system → maintains optimal pH and ionic conditions These components work together to ensure accurate and efficient DNA amplification, also Phusion DNA polymerases offer robust performance with short protocol times, even in the presence of PCR inhibitors. They generate higher yields with less enzyme than other DNA polymerases. In this protocol, the master mix is used to amplify amilCP fragments that will later be assembled using Gibson Assembly.

What are some factors that determine primer annealing temperature during PCR? Primer annealing temperature depends on:

Primer length → longer primers have higher melting temperatures,
GC content → higher GC increases stability and raises Tm. Higher melting temperatures are caused due to stronger hydrogen bonding. In this protocol, primers include additional overhangs (20–22 bp) for Gibson Assembly, but only the binding region determines the annealing temperature. The annealing temperature is typically set a few degrees below the melting temperature (Tm) to ensure specific binding.

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. In this protocol, PCR amplify specific regions of the amilCP gene, including mutated regions in the chromophore, allowing precise control over sequence design In contrast, restriction digestion (using PvuII) is used to linearize the pUC19 plasmid backbone. PCR is more flexible and allows introduction of mutations and overlaps, while restriction digestion relies on specific enzyme recognition sites. PCR is preferable for designing new constructs, whereas digestion is useful for preparing existing plasmid backbones.
How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? To ensure compatibility with Gibson Assembly, DNA fragments must have overlapping homologous regions of ～20–22 base pairs. In this protocol, these overlaps are introduced through primer design during PCR amplification of the amilCP fragments. The pUC19 backbone generated by restriction digestion also contains compatible ends. These overlaps allow fragments to anneal and be joined seamlessly during the Gibson Assembly reaction.
How does the plasmid DNA enter the E. coli cells during transformation? Plasmid DNA enters E. coli cells during transformation through heat shock or electroporation. In heat shock, cells are chemically treated (for example with CaCl₂) and briefly heated, creating pores in the membrane In electroporation, an electric pulse temporarily disrupts the membrane These methods allow DNA to pass into the cell, where it can replicate. Once inside, the plasmid replicates and expresses the amilCP gene, allowing colonies to be visually identified by color.
Describe another assembly method in detail (such as Golden Gate Assembly) a) Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online). Golden Gate Assembly is a molecular cloning method that uses Type IIS restriction enzymes (such as BsaI) and DNA ligase in a single reaction. These enzymes cut DNA outside their recognition site, generating customizable overhangs. This allows multiple DNA fragments to be assembled in a specific order without leaving unwanted sequences (scarless assembly). The reaction cycles between digestion and ligation, increasing efficiency. Because of its precision, Golden Gate is ideal for assembling multiple fragments simultaneously. It is widely used in synthetic biology for modular cloning. Compared to Gibson Assembly, it relies more on restriction sites rather than homologous overlaps. ![Gibson Assembly]( b) Model this assembly method with Benchling or Asimov Kernel!

Assignment: Asimov Kernel

Create a Repository for your work
Create a blank Notebook entry to document the homework and save it to that Repository
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)
Create a blank Construct and save it to your Repository a) Recreate the Repressilator in that empty Construct by using parts from the Characterized Bacterial Parts repository b) Search the parts using the Search function in the right menu c) Drag and drop the parts into the Construct d) Confirm it works as expected by running the Simulator (“play” button) and compare your results with the Repressilator Construct found in the Bacterial e) Demos repository f)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
Build three of your own Constructs using the parts in the Characterized Bacterials Parts Repo a) Explain in the Notebook Entry how you think each of the Constructs should function b) Run the simulator and share your results in the Notebook Entry c) 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