The design process of RNA thermometers centered around utilizing the online tool IDToligoanalyzer3.1 which is freely available from Integrated DNA Technologies. This software allows for input of a DNA sequence which will be transcribed into an RNA strand. This RNA strand is then used to predict a model that visualizes potential secondary structures based on complimentary bonding between the nucleotides of the RNA strand. The predictive model allows for the manipulation of individual nucleotides to ultimately alter the melting temperature of the RNA strand to reach the target temperature range.

With our chassis organism in mind, DH5α e. coli, a temperature range of between thirty and forty-two degrees Celsius was chosen based upon e. coli’s most favorable temperatures for proper metabolic function. After several design brainstorming sessions, the biggest obstacle faced was the presence of cytosine and guanine bonding which greatly increases the melting temperature. The Umass-Dartmouth team then began designing DNA sequences primarily of three nucleotides, excluding either cytosine or guanine except when necessary such as the biobrick prefix and suffix. The exclusion of a singular nucleotide from a DNA sequence led to more promising and potentially repeatable results.