Team:CUNY Kingsborough/Design
Design
Ethidium Bromide Spot Protocol
Accurate and quick determination of DNA concentration is critical for the assembly of synthetic constructs, as well as a multitude of other experiments. We sought to optimize an under-utilized and inexpensive approach for determining DNA concentration: a spotting technique that uses the intercalating dye Ethidium Bromide. This technique does not require specialized equipment such as a spectrophotometer, but instead relies on visualization of dye-DNA complex fluorescence when excited by UV light. We modelled and tested a range of parameters for dye concentration and spot size, finding that 15uL spots with 1.0ug/mL Ethidium Bromide produced the most reliable standard curve. More importantly, we hope that our approach can help other labs optimize this protocol for their own experimental setup. Adoption of this technique may help enable development of iGEM teams in resource limited environments and laboratories which do not or cannot employ a satisfactory method for determining DNA concentration.
Kill Switch for Engineered Bacteria
In 2016, we engineered E.coli to break down nitrogenous waste in Jamaica Bay. In 2017, we designed a light-activated kill switch to prevent our bacteria from surviving in saltwater outside of the waste treatment plant. We had two iterations of this design - one producing MazF which causes high cell death and one producing LacZ which allowed us to collect data and analyze expression levels without having to worry about high toxicity. Both versions utilized the pDawn promoter (BBa K1616019) which we characterized this year using models.
Design of Light-Activated Kill Switch
Part# K2268006 (MazF)
Part# K2268005 (LacZ)
