Week 6 — Genetic Circuits Part I: Assembly Technologies

Phusion Master Mix Components This mix contains a high-fidelity polymerase with proofreading activity (3 to 5 exonuclease) to minimize sequence errors. It also includes dNTPs as DNA building blocks, an optimized buffer with salts like magnesium chloride that act as enzymatic cofactors, and stabilizers to maintain the pH and ionic strength required for the reaction.

Factors Determining Annealing Temperature The annealing temperature primarily depends on the melting temperature (T_m) of the primers, which is influenced by sequence length and GC content. Other external factors include the concentration of salts (monovalent and divalent) in the PCR buffer and the concentration of the primers themselves in the mixture.

Comparison: PCR vs. Restriction Digest PCR exponentially amplifies specific sequences using a polymerase and thermal cycles, while digestion cuts pre-existing DNA at specific recognition sites using endonucleases. PCR is preferable when there is a low amount of template or when mutations and overlap sites need to be introduced; digestion is faster for verifying fragments or when pure genomic/plasmid DNA with exact cleavage sites is available.

Ensuring Fragments for Gibson Assembly It is essential for fragments to share overlap sequences at their ends (usually 20 to 40 base pairs) so that the exonuclease can generate the necessary cohesive ends. Additionally, post-PCR DNA purification must be performed, and the original template should be treated with DpnI to eliminate the background of circular parental plasmid that lacks the designed overlaps.

DNA Entry into E. coli Plasmid DNA enters through the creation of temporary pores in the cell membrane induced by thermal or electrical shock. In the heat shock method, the sudden temperature change alters the fluidity of the lipid bilayer, allowing negatively charged DNA to cross the cell wall and membrane into the cytoplasm via diffusion.

Golden Gate Assembly Golden Gate Assembly uses Type IIS restriction enzymes that cut outside their recognition site, generating unique and programmable cohesive ends. This allows for the assembly of multiple fragments in a single step and a “single-tube” reaction through cycles of digestion and ligation. Unlike Gibson, the design requires the cleavage sites to be removed during ligation, leaving a specific scar or none at all. It is the preferred method for building combinatorial libraries or complex genetic circuits modullarly.

Modeling in Asimov Kernel To model this process in a language like Asimov Kernel, fragments would be defined as sequences with BsaI sites in opposite orientations. The code would represent the combination of parts through ligation operators that recognize the Type IIS cleavage sites, simulating the formation of the final circular plasmid without the original restriction sites.