Week 06 HW -Genetic Circuits Part I: Assembly Technologies

‘Week 6 — Genetic Circuits Part I: Assembly Technologies’

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Homework: Genetic Circuits Part I: Assembly Technologies

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?
(Phusion High-Fidelity PCR Master Mix にはどのような成分が含まれていて、それぞれの目的は何ですか。)

https://www.youtube.com/watch?v=c07_5BfIDTw&t=115s

https://www.neb.com/ja-jp/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531

  • Phusion High-Fidelity DNA Polymerase

    The enzyme that drives DNA synthesis with high fidelity. It has 3’→5’ exonuclease (proofreading) activity, which reduces the error rate during PCR.

  • dNTP（A, T, G, C）

    These are the nucleotide building blocks used to synthesize new DNA strands during PCR.

  • HF Buffer（High-Fidelity Buffer）

    This is an optimized reaction buffer that maintains the proper pH and ionic conditions for high-fidelity DNA amplification.

  • MgCl₂

    Magnesium ions are an essential cofactor for DNA polymerase activity and are provided at an optimal concentration (1.5 mM in the 1X final reaction).

Together, these components provide the enzyme, substrates, and chemical environment needed for accurate DNA amplification.

2. What are some factors that determine primer annealing temperature during PCR?
(PCR におけるプライマーのアニーリング温度は、どのような要因によって決まりますか。)

https://www.nippongene.com/siyaku/product/pcr/cat_pcr.pdf

The primer annealing temperature should be set about 5°C lower than the Tm, typically around 55–60 (50- 65?)°C. Higher annealing temperatures increase specificity. At a primer concentration of 0.2 µmol/L, annealing occurs within a few seconds

The extension reaction is commonly carried out at 72°C, and depending on other reaction conditions, the synthesis rate is approximately 35–100 nucleotides per second

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.
(この授業で線状DNA断片を作る方法は2つあります。PCR と制限酵素消化です。
これら2つの方法について、プロトコルの面と、どのような場合にどちらがより適しているかという面の両方から、比較して説明しなさい。)

PCR obtains a desired DNA fragment by using primers to flank and amplify a specific region, requiring primers, dNTPs, and polymerase

Restriction enzyme digestion involves enzymes (e.g., EcoRI) that recognize specific restriction sites and cut the DNA

If no such sites exist in the vector or DNA sequence, digestion is not possible

Thus, PCR is used when the target restriction site is absent, while restriction digestion is preferred if the vector already contains suitable restriction sites and high reproducibility is desired

4. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
(消化したDNA配列およびPCRで増幅したDNA配列が、Gibsonクローニングに適したものになるようにするには、どうすればよいですか。)

https://docs.google.com/document/d/1_aSV7w8iRYc3EDmbueJ_hSEGy_jHLDfxT2wAezEtC4c/edit?tab=t.0#heading=h.a157u2dx9dhb

https://www.neb.com/ja-jp/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly

https://docs.google.com/document/d/1_aSV7w8iRYc3EDmbueJ_hSEGy_jHLDfxT2wAezEtC4c/edit?tab=t.0#heading=h.ysntjrikaygh

  • Overlap Design

    Confirm that each fragment has the correct 20–40 bp overlaps required for Gibson Assembly

  • Fragment Size and Sequence Accuracy

    Check via gel electrophoresis (for size) and, if necessary, sequencing (for the correctness of overlap regions or introduced mutations)

  • Orientation and Molar Ratios

    Ensure each fragment is in the proper 5′→3′ orientation and use an appropriate insert-to-vector molar ratio (commonly 2:1) to maximize assembly efficiency

5. How does the plasmid DNA enter the E. coli cells during transformation?
(形質転換の際、プラスミドDNAはどのようにして E. coli 細胞の中に入りますか。)

https://www.thermofisher.com/jp/ja/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-workflow.html

In order to introduce DNA into cells, it is necessary to temporarily increase the permeability of the cell membrane

Generally, there are two main methods for transforming E. coli: chemical transformation (e.g., the CaCl₂ method) and electroporation

(A) Chemical Transformation

  1. Mix chemically competent E. coli cells (prepared with CaCl₂, etc.) with ligated DNA and incubate for a set period
  2. Briefly subject the mixture to a high temperature (e.g., 42°C) for heat shock, creating temporary pores in the cell membrane through which the DNA can enter
  3. Transfer the cells into a recovery medium afterward to allow them to recover

(B) Electroporation

  1. Add purified DNA to electrocompetent E. coli cells
  2. Apply a high-voltage pulse (e.g., ~15 kV/cm), which forms transient micro-pores in the cell membrane, enabling the DNA to enter
  3. Finally, move the cells to a recovery medium to allow them to recuperate
6. Describe another assembly method in detail (such as Golden Gate Assembly)
別のアセンブリ法を1つ詳しく説明しなさい（例：Golden Gate Assembly）。

(1) Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).
(その方法を 5〜7文で説明し、図も付けなさい（手描きでも、オンラインの図でも可）。)

(2) Model this assembly method with Benchling or Asimov Kernel!
(Benchling または Asimov Kernel を使って、このアセンブリ法をモデル化しなさい。)

https://www.youtube.com/watch?v=NzQdLQ44I7w

https://www.youtube.com/watch?v=EpHeu44hitI

  1. Golden Gate Assembly is a method that employs Type IIS restriction enzymes to cleave DNA and efficiently ligate multiple fragments using custom-designed overhangs

    ※ An overhang refers to the single-stranded extension of DNA that protrudes from one strand when the DNA is cleaved

  2. Prepare each fragment so that it contains a Type IIS site—using primer design and PCR—ensuring the desired overhangs appear upon enzyme digestion

  3. Next, combine all fragments in a single tube with the Type IIS restriction enzyme (e.g., BsaI) and T4 DNA ligase, enabling digestion and ligation to occur simultaneously

  1. Under these reaction conditions, the enzyme repeatedly cuts the DNA to create transient overhangs, which then anneal, and the ligase seals the nicks

  2. Incorrect assemblies are recut, so only the properly matching fragments remain ligated, resulting in an efficient multi-fragment assembly in the correct order

  1. Because Type IIS enzymes cleave outside their recognition sequences, the final product is “scarless,” with no leftover restriction sites

  2. After the reaction, transform the assembled plasmid into E. coli, and confirm the intended construct by sequencing if necessary