Week 6 — Genetic Circuits Part I Protocol: DNA Assembly
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 required for efficient and accurate DNA amplification. The key components are:
Phusion DNA Polymerase: A high-fidelity thermostable polymerase derived from Pyrococcus. It has proofreading (3’→5’ exonuclease) activity, which reduces the error rate during DNA synthesis
Subsections of Labs
Week 1 Lab: Pipetting
Week 6 Lab: Gibson assembly
Week 6 — Genetic Circuits Part I
Protocol: DNA Assembly
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 required for efficient and accurate DNA amplification. The key components are:
Phusion DNA Polymerase: A high-fidelity thermostable polymerase derived from Pyrococcus. It has proofreading (3’→5’ exonuclease) activity, which reduces the error rate during DNA synthesis
dNTPs (deoxynucleotide triphosphates): These are the building blocks used by the polymerase to synthesize the new DNA strand
Reaction buffer: Provides optimal ionic strength and pH for polymerase activity. It usually contains Mg²⁺, which is required as a cofactor for DNA polymerase
MgCl₂: Magnesium ions stabilize primer-template interactions and are essential for the catalytic activity of the polymerase
Stabilizers and additives: These help maintain enzyme stability and improve amplification efficiency
The master mix in PCR is prepared to simplify the reaction setup and ensure consistency across multiple reactions. Instead of adding each component separately, the master mix contains the essential reagents required for DNA amplification, such as the polymerase, buffer, Mg²⁺ ions, and dNTPs.
In practice, the total master mix volume is calculated based on the number of reactions to be performed, and it is recommended to prepare additional volume (usually enough for one or two extra reactions) to account for a negative control and possible pipetting errors.
In general, using a master mix also helps reduce variability between reactions and ensures high specificity and low mutation rates during amplification when using high-fidelity enzymes such as Phusion polymerase.
What are some factors that determine primer annealing temperature during PCR?
The primer annealing temperature (Ta) is critical for PCR specificity and efficiency. Some factors that determine the annealing temperature include:
Primer melting temperature (Tm): The annealing temperature is typically 3–5°C below the primer Tm
Primer length: Longer primers generally have higher melting temperatures
GC content: Primers with higher GC content have stronger hydrogen bonding and therefore higher Tm
Primer sequence composition: Runs of GC or secondary structures can affect annealing behavior
Salt concentration in the buffer: Ionic conditions influence primer-template hybridization
Template complexity: Complex templates or repetitive regions may require optimized annealing temperatures
(Rychlik et al., 1990)
The important part is choosing the correct annealing temperature, which helps ensure that primers bind specifically to the intended target sequence. As well, remember the principal steps of PCR in Figure 1.
Figure 1. MiniPCR graphic ‘Depiction of one PCR cycle.’
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.
Both PCR amplification and restriction enzyme digestion can generate linear DNA fragments, but they differ significantly in methodology and application. In Table 1, the differences between PCR and restriction enzyme digest are summarized.
Table 1. Comparison of PCR vs. Restriction enzyme digest
Category
PCR
Restriction Enzyme Digest
Principle
DNA amplification using primers and polymerase
Cutting DNA using sequence-specific enzymes
DNA requirement
Small amount of template DNA
Requires plasmid or DNA containing restriction sites
Specificity
Determined by primer design
Determined by enzyme recognition sequences
Output
Amplified fragment of specific length
Linearized DNA or defined fragments
Flexibility
Can add overhangs or modifications through primers
Limited to existing restriction sites
When is PCR preferable?
When amplifying a specific gene or fragment
When introducing mutations or overhangs
When restriction sites are not present
When are restriction enzymes preferable?
When cutting plasmids or large DNA constructs
When working with known restriction maps
When cloning using classical restriction-ligation methods
PCR offers greater flexibility, while restriction enzyme digestion provides precise cleavage at defined sequences. Also, PCR amplifies many copy of the DNA or genetic material, while restriction enzymes cut a specific region as mentioned before.
How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
To ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson Assembly, DNA fragments must contain overlapping homologous regions.
Table 2. Requirements for Gibson Assembly
To ensure compatibility:
Design PCR primers with 20–40 bp overlaps matching adjacent fragments.
Verify sequences in silico using tools such as Benchling.
Ensure fragments are linear and free of secondary structures.
Confirm correct fragment size via gel electrophoresis.
Remove template plasmid contamination if necessary (e.g., DpnI digestion).
Then, those overlapping sequences allow the Gibson reaction enzymes to:
Generate single-stranded overlaps
Anneal complementary regions
Fill gaps and ligate the fragments.
Extra: Thermofisher graphic:
Figure 2. Gibson Assembly 101: Expert Cloning Tips You Need to Know (thermofisher)
How does the plasmid DNA enter the E. coli cells during transformation?
During transformation, plasmid DNA enters E. coli cells that have been made competent, the most common methods are: Chemical transformation & Electroporation.
Table 3. Chemical transformation vs. Electroporation
Chemical transformation
Electroporation
Cells are treated with CaCl₂, which neutralizes negative charges on DNA and the cell membrane
Cells are washed to remove salts
DNA is added to the competent cells
A high-voltage electric pulse creates temporary pores in the membrane
A heat shock (~42°C) briefly disrupts the membrane
After transformation, cells are plated on selective media containing antibiotics to identify successful transformants.
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 technique that allows the simultaneous assembly of multiple DNA fragments in a single reaction. The method uses Type IIS restriction enzymes, such as BsaI, which cut DNA outside of their recognition sequence to generate customizable overhangs. These overhangs allow DNA fragments to ligate together in a specific and predetermined order. During the reaction, the restriction enzyme first digests the DNA fragments, generating compatible sticky ends, and DNA ligase then joins the fragments together. Because the recognition sites are removed during digestion, the final construct does not contain unwanted restriction sequences, resulting in scarless cloning. This technique is widely used in synthetic biology because it enables efficient multi-fragment assembly in a single tube (Bird et al., 2018).
Diagram:
flowchart LR
A[Fragment A] --> D[BsaI digestion]
B[Fragment B] --> D
C[Fragment C] --> D
D --> E[Sticky overhangs]
E --> F[Ligation]
F --> G[Final plasmid<br>A-B-C]
b. Model this assembly method with Benchling or Asimov Kernel!
Assembly Method with Benchling
For this activity, the Golden Gate Assembly method was modeled using Benchling. The project folder used for this exercise can be accessed through the following link:
To perform the assembly, the plasmid pUC19 was selected as the backbone sequence. The full plasmid sequence was obtained from Addgene, which provides verified plasmid maps and DNA sequences commonly used in molecular biology. Sequence
This fragment includes recognition sites for the Type IIS restriction enzyme BsaI, which is required for Golden Gate Assembly. And its structure has a short alanine-rich peptide used as a synthetic example insert. Golden Gate Assembly uses Type IIS restriction enzymes that cut outside their recognition sequence, allowing the creation of custom overhangs. These overhangs guide the directional ligation of DNA fragments, enabling multiple pieces of DNA to be assembled in a single reaction.
In Benchling, the pUC19 backbone and the synthetic insert were added to the assembly workspace. The Golden Gate cloning method was selected, and BsaI was defined as the restriction enzyme. The insert fragment was generated using a primer pair to simulate PCR amplification, which allows the introduction of compatible overhangs for the assembly.
After defining the fragments, Benchling automatically generated compatible sticky ends between the backbone and the insert. Once the fragments were validated, the assembly was finalized to generate the construct pUC19_backbone–small_peptide_insert.
Final Golden Gate Assembly plasmid:
Figure 3. pUC19_backbone–small_peptide_insert by Golden Gate Assembly method
Tutorial for assembly method with Benchling
Additionally, I created a document that shows the process I used to create the plasmid.
Next part of the homework is at: Homework- Week 6 section
References & sources:
Asif, A., Mohsin, H., Tanvir, R., & Rehman, Y. (2017). Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation. Frontiers in microbiology, 8, 2169. https://doi.org/10.3389/fmicb.2017.02169Open Access
Bird, J. E., Marles-Wright, J., & Giachino, A. (2022). A User’s Guide to Golden Gate Cloning Methods and Standards. ACS synthetic biology, 11(11), 3551–3563. https://doi.org/10.1021/acssynbio.2c00355Open Access
Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd, & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6(5), 343–345. https://doi.org/10.1038/nmeth.1318Open Access
Liu, J., Chang, W., Pan, L., Liu, X., Su, L., Zhang, W., Li, Q., & Zheng, Y. (2018). An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments. Indian journal of microbiology, 58(4), 448–456. https://doi.org/10.1007/s12088-018-0743-zOpen Access
Rychlik, W., Spencer, W. J., & Rhoads, R. E. (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic acids research, 18(21), 6409–6412. https://doi.org/10.1093/nar/18.21.6409Open Access
