Difference between revisions of "Team:Cadets2Vets/Demonstrate"

On July 25, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace ran a second ticket assay to increase the power of the previous test and provide clearer results.

To these ends, the Cadets2Vets team aliquoted their circuit and reaction solution without arsenic ions to 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. The negative control ticket used only 2 µL of reaction mixture for the first two columns of wells on the left, and the other two columns of wells were filled with 2 µL of molecular water. The positive test ticket used 2 µL of the test mixture in the first column of wells, and 4 µL of test mixture in the second column of wells. The other two columns of wells were filled with 2 µL of molecular water.

Fig 4.1 and Fig 4.2 - The Positive Test Circuit (top) with first row containing 2 µL of test mixture, and the second row containing 4 µL of test mixture. Possible dose response is visible. The Negative Control Circuit (bottom), containing 2 µL of reaction mixture in first and second columns.

These tickets were then let to incubate overnight (~18 hours) at ~40 degrees Celsius, at which point they were analyzed qualitatively using a backlight (Fig 1.1 and Fig 1.2). Once again, no fluorescence was detected on any of the negative control wells. The positive test exhibited fluorescence in the first and second columns of wells, and qualitatively it appears that there may have been some form of dose response, as the second column of wells appears brighter. The negative control appears to have dried again slightly, and the Cadets2Vets team will be aliquoting more reaction mixture into each well during later tests to try and combat this issue.

Cadets2Vets has built a biological arsenic circuit that uses a combination of native DH5-alpha E. coli genes to detect arsenic ions in solution and genetic elements capable of producing GFP (Green Fluorescent Protein). After reading through academic literature, our team found a negative repressor that could be used as an arsenic regulator known as ArsR. Sensitive to arsenic ions, this protein will bind to an operon or promoter sequence and inhibit the transcription of the downstream gene in the presence of a transcription factor (in our case arsenic). Expressing a conformation change, this strategy allows us to control GFP expression using a protein sensitive to and capable of bonding with arsenic ions in solution.

Integrated DNA Technologies, an iGEM sponsor, provided us with free synthetic DNA to build our circuit. This sponsorship allowed us to select promoters and genes that fit our project. The molecular cloning strategy was to request two GBlocks that would contain a promoter-ribosome binding site-CDS-terminator sequence for constitutively expressing ArsR and regulating the expression of GFP via ArsR. We also added in a restriction enzyme site to both GBlocks that enabled us to combine the two GBlocks together.

Before running the actual circuit, we ran control experiments to determine whether or not the GFP would indeed function as a reporter, meaning it is expressed by the plasmid.

The first control experiment drove GFP using a constitutive promoter: meaning that the GFP would always be produced. This was essentially only used to ensure that the GFP works in the paper ticket.

The second control experiment was regulated, meaning that GFP expression was dependent on a trigger. This tested if the GFP could be controlled/ regulated.

These experiments were performed using a commercial in vitro transcription/translation kit by Promega.

The results from both of these experiments are shown in the pictures below.