Week 6: Genetic Circuits I

Class Assignment

DNA Assembly

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

We used NEB’s Q5 2X Hot Start Master Mix for our lab, but like the Phusion MM, it contains a DNA polymerase (for the extension step of PCR), as well as dNTPs and Mg2+ as polymerase cofactors and a buffer.

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

The make up of nucleotides in the primer sequence (more GC content means a higher annealing temperature as it takes more energy to form the three hydrogen bonds between the base pairs) as well as the overall primer length.

  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.

PCR denatures DNA to pull the two strands apart, uses primers designed to target regions to anneal and extend certain regions, and amplifies the target product. Restriction Digests cut the template DNA at specific recognition sites based on the employed enzyme. PCR is preferred to produce high-quantity outputs of DNA for a particular region, whereas restriction digests can be used to produce cut DNA with ends compatible for assembling with another piece of DNA not from the template (such as a plasmid with complementary cut ends).

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

PCR cleanups and assessing through Nanodrop scores can be used to make sure there is not additional junk (such as from the polymerases in the PCR solution reaction) in the product and that there is sufficient concentration of DNA for an efficient cloning reaction.

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

Either electroporation or chemical transformation can work with E. coli - for our lab, we used chemical transformation with the NEB PCR Cloning kit.

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

Golden Gate Assembly uses Type II Restriction Enzymes that cut outside of their recognition sites to create 4 bp overhangs. Fragments are designed so that the resulting base pair overhangs after a restriction digest are complementary to the overhangs of the neighboring fragments so that the DNA parts can assemble like puzzle pieces (using a ligase). This assembly tool is modeled with my final project.

Asimov Kernel

Work documented in Asimov Kernel notebook.