Week 6 HW: Genetic Circuits Part I

Assignment: DNA Assembly

  1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? The NEB 2X master mix includes engineered thermostable DNApol to synthesize complimentary strands during the extension phase of PCR, with low error rate and high expressivity to enable faster, more accurate DNA replication.

It includes a buffer to maintain optimum pH for enzymatic catalysis and prevent the denaturation of enzyme or nucleic acids, as well as ${Mg}{Cl}_{2}$ to provide $Mg^{2+}$ as a cofactor for DNApol.

  1. What are some factors that determine primer annealing temperature during PCR? Primer sequence (specifically, the number of complimentary base pairs and how many are C-G as opposed to A-T): If a primer sequence contains more complimentary base pairs (eg because the forward primer is longer, say, or has higher sequence homology with the sense strand) the annealing temperature will be higher. If the primer contains more CG as opposed to AT content, the annealing temperature will be higher.

Buffer: A higher concentration of salt can lead to elevated ion levels, favoring the salting out of proteins and DNA which might, to some extent, facilitate annealing at a lower temperature.

  1. 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 and Restriction Enzyme Digests produce linear dsDNA and can be highly specific for the target region if designed properly.

However, while PCR aims to amplify a target sequence, restriction enzyme digests aim to isolate a target sequence from a given amount of DNA sample. PCR involves thermocycling while restriction enzyme digests are conducted at a constant optimum temperature. PCR requires DNA primers while restriction enzyme digests do not require additional DNA material on top of the sample itself.

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

Firstly, we can check their purity using a 260/280 absorbimetry assay, and verify the predominant fragment length using gel electrophoresis.

Secondly, we can use Sanger sequencing the verify the sequence of linear DNA produced.

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

Either through chemical means in heat shock treatment, or electrical means in electroporation, the bacterial cell membrane and cell wall are modified to grant plasmid DNA greater permeability - whether through the alteration of membrane thickness, structure and charge in chemical competence, or the electrical stress-induced poration of the bacterial cell membrane in electroporation.

  1. Describe another assembly method in detail (such as Golden Gate Assembly)

Golden Gate Assembly relies on Type IIS as opposed to Type IIP restriction enzymes to enable one-step syntheses of multipart constructs. In Golden Gate Assembly, the specific type of restriction enzymes used doesn’t depend on palindromic recognition sequences, allowing greater versatility in engineering restriction sites, and doesn’t leave parts of the restriction site in the final construct.

In Golden Gate Assembly, the respective genetic components are first checked to ensure they don’t contain any internal Type IIS recognition sites, and a unique 4-base overhang is engineered to dictate the correct order of assembly. Afterwards, the genetic components are added to a one pot reaction mixture containing the specific Type IIS restriction enzyme used, DNA ligase and buffer solution.

  1. Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).

^ See above

  1. Model this assembly method with Benchling or Asimov Kernel!

Sure thing!

Assignment: Asimov Kernel

Refer to this notebook for the constructs and repressilator

Lab Homework

I’m a remote committed listener so don’t have access to a physical lab