Week 6 HW: Genetic circuits I

Part A

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

The Phusion HF PCR Master Mix is a ready-to-use mixture designed for high accuracy DNA amplification. It contains several component that make PCR efficient, like dNTPs (the building blocks of DNA which are incorporated by the polymerase in the newly synthesized DNA strand), Reaction buffer which can either help maintain the optimal chemical environment or a GC buffer that can amplify GC-rich DNA templates, and Mg²⁺ ions that are an essesntial cofactor for the DNA polymerase activity. The most notable component is the Phusion High-Fidelity DNA polymerase, which has proofreading activity between 3’-> 5’ exonuclease which corrects mistakes during replication, having a lower rate than the standard Taq polymerase. The Phusion HF PCR Master Mix can also contain Tracking Dye, allowing direct loading of PCR product on agarose gels without adding loading buffer.

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

The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. The primer annealing temperature (Ta) in PCR is the temperature at which primers bind to complementary DNA template. Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield. Too low Ta may possibly lead to non-specific products caused by a high number of base pair mismatches.

Some factors that influence the Ta are the primer melting temperature (Ta = 0.3 x Tm(primer) + 0.7 Tm (product) – 14.9, best primers have melting temperature at 52-58 °C), the primer length (Longer primers form more hydrogen bonds => higher Tm => higher Ta, typical primers are between 18-22 nucleotides long), GC content (since G-C base pairs have 3 hydrogen bonds, higher GC content mean higher Tm, best primers have around 40-60% GC content), GC clamps (more than 3 G’s or C’s should be avoided in the last 5 bases at the 3’ end of the primer because they can help promote specific binding at the 3’ end) and primer secondary structures (strong secondary structures can reduce effective binding to the template => adjustments to Ta)

  1. Compare and contrast PCR and Restriction Enzymes Digest

PCR

  • amplifies a specific DNA segment using primers and DNA polymerase;
  • produces many copies of the target DNA region;
  • They need a DNA template, forwards and reverse primers, dNTPs, reactions buffer and DNA polymerase;
  • Require thermal cycling;
  • Produces copies of a specific sequence (with variable size given by the primers positions);
  • Preferred for studying a specific gene.

Restriction Enzyme Digest

  • uses restriction endonucleases to cut DNA at specific recognition sequences;
  • Produce DNA fragments with defined ends;
  • They need DNA substrate, restriction enzymes, restriction buffer and stabilizers;
  • Require constant temperature;
  • Produce predictable fragments depending on the location of restrictions sites in the DNA;
  • Preferred for cutting DNA for cloning into vectors.
  1. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

Gibson Assembly requires that all fragments share designed overlapping ends, without relying on restriction sites.

We can ensure that the DNA is suitable following the next steps:

1. Design overlapping sequences

Each adjacent DNA fragment must share ~20–40 bp of identical sequence at their ends. Typically, the PCR primer design add at the 5’ end sequences homologous to the neighboring fragment or vector. These overlaps determine the order and orientation of assembly. If the fragments don’t overlap, then they will not assemble in Gibson.

2. PCR product design consideration

Standard Taq polymerase may cause occasional errors. To prevent that we can use high-fidelity polymerase to minimize mutations and ensure correct overlapping and clean single-band amplification. Like this we can avoid unwanted sequences between overlaps and inserts.

3. Preparing Restriction-Digested DNA

For Gibson, restriction enzymes are not required, but we can still use them to linearize a vector and ensure the cut region exposed ends match the designed overlaps.

4. End Compatibility

Gibson Assembly requires double-stranded DNA with overlapping ends so we must ensure that the Gibson mix overlaps are exact matches (no mismatches or frame shifts). The Gibson mix uses exonuclease to chew back 5’ ends, allow complementary overlaps to anneal, fill gaps and anneal.

5. Fragment quality

DNA needs to be purified of primers, enzymes and nonspecific products.

6. Avoid internal problems

We need to check for secondary structures disruptions, mis-annealing caused by repetitive sequences and unintended homology elsewhere.

7. Sequence verification

In silico design tools we can confirm overlaps.

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

Plasmid DNA doesn’t normally pass through the membrane of E.coli, so we need to “shock” the membrane, making it temporarily permeable. The most common methods are:

Heat Shock Transformation

Cells are first made “competent” using cold salts like CaCl₂, to help neutralize negative charges on both DNA and the cell membrane. DNA is added and cells are kept on ice, the a short heat shock (~42°C for ~ 30-60 seconds) is applied. Like this we can cause temporary pores or increased fluidity in the membrane, pulling the DNA into the cell.

Electroporation

It acts with a similar purpose: to create pores in the membrane of the cells. Cells are washed to remove salts and placed in a special buffer where DNA is added and a brief high-voltage pulse is applied, causing transient pores in the cells membrane.

  1. Describe another assembly method in detail

Another interesting method that I’ve learned about is the traditional restriction-ligation cloning.

Restriction–ligation cloning is a molecular biology method used to insert a specific DNA fragment into a plasmid vector. It begins by cutting both the vector and the DNA insert with restriction enzymes such as EcoRI or XhoI, which recognize specific DNA sequences. These enzymes often generate “sticky ends,” short single-stranded overhangs that can base-pair with complementary sequences. When the vector and insert have compatible ends, they can anneal through hydrogen bonding. The enzyme T4 DNA Ligase is then used to covalently join the DNA backbone by forming phosphodiester bonds. This creates a stable recombinant plasmid containing the inserted DNA fragment. The ligated plasmid is introduced into competent cells of Escherichia coli through transformation. Inside the bacteria, the plasmid is replicated, allowing amplification of the inserted gene. The cells are grown on selective media to identify those that have successfully taken up the plasmid. Finally, colonies are screened and verified to confirm that the correct DNA insert is present.

I will be using Benchling for this simulation.

Part B

don’t have access to kernel yet…