Week 6 HW: Genetic Circuits Part I

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?

The Phusion High-Fidelity PCR Master Mix contains several key components necessary for efficient and accurate DNA amplification. First, it includes Phusion DNA polymerase, a high-fidelity enzyme with proofreading activity (3’ → 5’ exonuclease), which reduces errors during DNA replication. It also contains dNTPs (deoxynucleotide triphosphates), which are the building blocks used to synthesize new DNA strands. The mix includes a reaction buffer, optimized with the correct pH and salt concentrations to ensure proper enzyme activity. Additionally, it contains Mg²⁺ ions, which act as essential cofactors for the polymerase. Some mixes may also include stabilizers to maintain enzyme activity during thermal cycling.

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

Primer annealing temperature depends mainly on the melting temperature (Tm) of the primers. Tm is influenced by primer length, GC content (since G-C pairs have stronger bonding than A-T), and sequence composition. Typically, the annealing temperature is set about 5°C below the Tm. Moreover, other factors include primer specificity, as mismatches lower effective binding, and salt concentration, which affects DNA duplex stability. If the temperature is too low, nonspecific binding may occur; if too high, primers may not bind efficiently.

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.

PCR amplifies a specific DNA region using primers and DNA polymerase, allowing you to generate large amounts of a defined fragment and even introducing mutations or overlaps. It is highly flexible and does not require specific restriction sites. In contrast, restriction enzyme digestion cuts DNA at specific recognition sequences using restriction enzymes. This method is precise but limited by the presence of those recognition sites in the DNA.

PCR is preferable when you need to amplify DNA, modify sequences or add overlaps for cloning. On the other hand, restriction digestion is preferable when working with existing plasmids and known restriction sites, especially for traditional cloning methods.

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

DNA fragments must have overlapping homologous regions (typically 20–40 base pairs) at their ends. These overlaps can be designed into PCR primers or generated through careful restriction digestion. It is also important to verify that sequences are correct (no mutations) and in the proper orientation. DNA fragments should be clean and free of contamination. Finally, checking sequences using software (like Benchling) ensures that overlaps align correctly for seamless assembly.

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

Plasmid DNA enters E. coli induced by chemical treatment or electroporation. In chemical transformation, cells are treated with calcium chloride, which neutralizes the negative charges on DNA and the cell membrane. A heat shock step creates a temporary imbalance in the membrane, allowing DNA to enter the cell. Alternatively, in electroporation, an electrical pulse creates transient pores in the membrane, through which DNA can pass. Once inside, the plasmid replicates independently if it has an origin of replication.

6. 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).

Golden Gate Assembly relies on Type IIS restriction enzymes that cut DNA outside their recognition sites, generating unique overhangs. These overhangs are designed to be complementary between adjacent fragments, ensuring correct assembly order. During the reaction, the enzyme cuts the DNA and DNA ligase joins the fragments together. Because the recognition sites are removed after cutting, the assembled DNA cannot be re-cut, making the process highly efficient. Multiple fragments can be assembled in a single reaction tube in a predefined sequence.

Diagram:

- Model this assembly method with Benchling or Asimov Kernel!

The Golden Gate Assembly was modeled in Benchling by inserting a pre-designed genetic circuit I previously had, into the pXTK058 backbone. The circuit consisted of a constitutive promoter, a ribosome binding site (RBS), the coding sequence for butyryl-CoA dehydrogenase and a terminator, forming a complete expression cassette.

Type IIS restriction enzyme sites (BbsI) were used to generate compatible overhangs for directional assembly. The Assembly Wizard was used to simulate the process by treating these overhangs as overlaps. The final construct was verified to ensure correct insertion, orientation, and sequence integrity.

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 repository4

  • 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

HTGAA WEEK #6: IAN SEBASTIAN TERAN GARCIA’S HOMEWORK

1. Exploring the devices in the Bacterial Demos Repo:

Found a construct called “Circuit 3” in the Bacterial Demos Repore and I could observe it corresponds to a plasmid backbone containing the AmeR resistance gene, which allows bacterial selection under antibiotic conditions. The promoter pAmeR drives the expression of this resistance gene.

2. Recreating the Repressilator:

After recreating the repressilator, no noticeable differences were observed between the original circuit obtained from the repository and the one recreated in the simulation environment. Both produced nearly identical graphical outputs, indicating that the reconstruction was accurate and functionally equivalent.

Graphs interpretation:

The graph of RNA concentrations over time shows clear periodic oscillations for the three transcripts. Each gene’s mRNA level rises and falls in a regular pattern, with a noticeable phase shift between them. When one gene is highly expressed, it represses the next gene, causing its expression to decrease, while the third gene begins to increase. This cyclical pattern confirms that the circuit is functioning as an oscillator, with coordinated and repeating changes in gene expression.

Similarly, the protein concentration graph also displays oscillatory behavior, although the fluctuations are smoother and slightly delayed compared to the RNA levels. This delay occurs because protein production depends on the prior synthesis of mRNA, and proteins generally have longer degradation times. Therefore, protein dynamics tend to be more stable and less abrupt than RNA dynamics, which is consistent with biological expectations.

The RNAP flux graph represents the transcriptional activity of each gene at a specific moment in time. Higher values indicate stronger promoter activity, meaning that more RNA polymerase is actively transcribing that gene. In contrast, lower values suggest that the gene is being repressed. This snapshot reflects the regulatory interactions within the circuit at that particular time point.

Finally, the ribosome flux graph shows the rate of protein synthesis for each gene. Similar to the RNAP flux, higher values correspond to increased translation activity. The patterns observed here are consistent with the RNA levels but may show slight delays due to the time required for translation. Overall, these flux measurements provide additional confirmation of the dynamic regulation and oscillatory behavior of the repressilator system.

2.2. Repressilator simulation recreated by me:

3. My constructs:

3.1. Construct 1.

The first genetic circuit consists of the TetR gene under the control of the inducible pBad promoter. This represents a simple gene expression system without regulatory feedback. The RNA concentration graph shows a rapid increase followed by a stable plateau, indicating that transcription is activated and reaches a steady state where production and degradation are balanced.

The protein concentration shows a delayed increase compared to RNA levels, which is expected due to the time required for translation. Eventually, protein levels stabilize, indicating equilibrium.

The RNAP and ribosome flux graphs show relatively constant activity, suggesting sustained transcription and translation under the simulated conditions. Overall, this circuit behaves as a simple inducible expression system, producing a stable amount of TetR without dynamic regulation.

3.2. Construct 2.

For the second construct, the genetic circuit expresses QacR under the control of the pSrpR promoter. Similar to the first construct, this system lacks explicit feedback regulation and behaves as a simple expression module. The RNA concentration rapidly increases and stabilizes, indicating steady transcriptional activity. Compared to Construct 1, the higher RNA levels suggest that the pSrpR promoter is stronger under the simulation conditions.

Moreover, the protein concentration follows the expected delayed increase and reaches a higher steady-state level. RNAP and ribosome fluxes confirm sustained transcription and translation activity. Overall, this construct demonstrates stable gene expression and highlights how promoter strength affects system output.

3.3. Construct 3.

The third construct includes two genes which are QacR, under the inducible pBad promoter and LitR under the pLitR promoter, introducing regulatory interactions and feedback into the system as the simulation results show a strong dominance of LitR expression, while QacR remains near zero. This suggests that the inducible promoter pBad is not sufficiently activated under the simulation conditions, resulting in minimal QacR production.

As a consequence, repression of pLitR by QacR is ineffective, allowing LitR to accumulate. Additionally, LitR negatively regulates its own promoter, creating a feedback loop that stabilizes its expression level.

The protein concentration reflects this behavior, with LitR reaching a high steady-state level and QacR remaining negligible. RNAP and ribosome fluxes confirm strong transcription and translation for LitR and minimal activity for QacR.