On July 20, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace created tickets by aliquoting Ars 1.1 circuits into wells with various other reagents. This was done to test the in-paper efficacy of the circuits that the Cadets2Vets team engineered.

To these ends, the Cadets2Vets team aliquoted their circuit and reaction solution without arsenic ions into each well of the negative control. The Cadets2Vets team aliquoted their circuit and reaction solution with arsenic ions into each well of the positive control.

The reaction solution contained 7.5 µL S30 Extract, 10 µL S30 Promega, 2.5 µL amino (+), 0.5 µL amino (-), and 2 µL molecular water. For the positive test, 2.5 µL of our circuit plasmid and machinery was added, and the 2 µL molecular water contained arsenite and arsenate at a 150 µM concentration. 2 µL of water (molecular for negative control, arsenic ion and water mixture for positive test) was added to wells in column 2, and 4 µL of water (molecular for negative control, arsenic ion and water mixture for positive test) was added to wells in column 3. The wells in the second column had an arsenic ion concentration of 82.5 µM and the wells in the third column has an arsenic ion concentration of 208.3 µM for the positive test ticket.

These tickets were then incubated overnight (~18 hours) at ~40 degrees Celsius, at which point they were analyzed qualitatively using a backlight (Fig 1.1). Although measures were taken to try to prevent the tickets from drying up by supplying a source of humidity in the petri dishes, the Negative Control Paper Circuit unfortunately dried out slightly, which is why some of the wells seem lighter than the others. Despite this, no fluorescence was detected on any of the negative control wells. The positive test exhibited fluorescence in the first column of wells, but the second and third wells exhibited no fluorescence. The Cadets2Vets team currently hypothesizes that this may be a result of arsenic ions saturating the pores of the ticket, inhibiting the function of the cellular machinery by destabilizing GFP conformation, or negatively influencing ligand effects, preventing the production of GFP.

Moving forward, the Cadets2Vets team plans to amplify the signal of the positive test by spiking in more regulatory circuits to the abiotic wells. The team hypothesizes that this allow more of the arsenic ions to be functionalized by the regulatory proteins to produce GFP, increasing the ratio of GFP created. The Cadets2Vets team also plans to alter the percentage-based concentrations of arsenic in the positive test wells to determine the current upper abiotic LOD. Lastly, the Cadets2Vets team will test new methods to keep the tickets from drying out during incubation to find a (hopefully) more effective method than what is currently being used.

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Fig 3.1 – Fluorescence seen on positive test ticket (right), with no fluorescence on negative control ticket. Discoloration is due to drying of ticket and biological variability.