The mechanical engineering part of the project focused on the development of a low-cost, open-source thermal heating block.  An Arduino controlled Peltier system was designed and constructed to isothermally heat PCR tubes for the Gibson Assembly reaction at 50oC and Cre-mediated recombination reaction at 37oC.  The system consisted of a relay setup to regulate the thermoelectric cooling unit, with a thermistor to monitor the temperature of the copper heating block and lid.

For the biology and chemical engineering part of the project, students worked on several methods for the synthetic plasmids.  The first was Gibson assembly, optimizing the established single-step multi-enzyme reaction to produce circularized plasmids from PCR amplified fragments.  This, however, proved to be thermodynamically infeasible due to entropic limitations, as confirmed by AFM imaging.  A second method, Cre-LoxP recombination, was also assessed to produce circular product.  PCR products were designed with LoxP sites, amplifying a gene expression cassette with flanking Cre sites and then running a Cre reaction to circularize it.  While the 1’ PCR was successful, we were unable to circularize the product with Cre recombinase.  Consequently, we also investigated a third method, MIDGE (Minimalistic, Immunologically Defined Gene Expression).  This reaction creates linear DNA, but with hairpins at the ends which still prevent exonuclease digestion.  Confirmed by AFM imaging and transfections in PC3 cells, this method was partially successful, with at least one end of the fragment having proper hairpin formation.  Further optimization of the primers and reaction is necessary to ensure all product is functional.

To optimize recovery of PCR products from a gel, we developed a novel gel extraction process: 2D gel electrophoresis.  While traditional methods rely on silica spin columns to extract DNA from melted agarose, our method allows the user to electrophoretically move the desired DNA band into a liquid volume for direct extraction with a pipette.  This involved an initial electrophoresis separation (the first dimension), followed by rotating the electric field perpendicular to the initial (the second dimension), and then running a second electrophoresis into a water-filled capture well.  This method provided pure product but had issues of yield loss due to leakage.