Genetic Circuits Part I: Assembly Technologies

Assignment: DNA Assembly

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

The high Phusion High-fidelity polymerase, MgCl2, dNTPs, etc. The polymerase enable low error in the polymerization reaction.

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

how much Hidrogen bombs are present (type of bases and how much).

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.

Restriction enzyme linearization could be more sequence dependent, and also not super escalable. In comparison, PCR linearization is more independent in those aspects, but have the downsides of primer design.

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

By amplifying with exacts overlaps fragments that flank the desire product.

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

By making the cells competetent by (normally) two ways: electroporation and heatshock, inducing pores into the membrane that enables the DNA to entry into the cells.

Describe another assembly method in detail (such as Golden Gate Assembly). Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online). Model this assembly method with Benchling or Asimov Kernel!

Golden Gate Assembly is a method of cloning that uses IIs restriction enzymes. Thesee enzymes cut not in but but close the recognition site, allowing to make specifics overhangs to assembly the parts. There entry vectors and overhangs to the parts, containing the correspond recognition sites and future overhangs.

This method allow to cloning up to 50 parts in one reaction, useful for multigene and from library-parts assembly. There is a common notation to the overhangs based on the type of parts, enabling MoClo (modular cloning) (leves 0: parts of a gene assembly; level 1: multigene assembly; level 2: multi-multigene assembly).

THe reaction usually is the following: (5 minutes 40 C, 5 minutes 16 C)x50, 5 minutes 50 C. The entry vectors usually have a sequence of Lacz for blue-white colony selection, where the whites have final insert.

For the Asimov (Kernel) i didn´t have the acces to the API to do the homework