Team:Cadets2Vets/Notebook July
July Notebook
07/20/2017
On July 19, 2017, senior technician Ryan Brisban with the help of high school interns Keshava Katti, Kian Croston, and Josh Grace analyzed a second acrylamide protein assay. This was done in order to qualitatively demonstrate the presence of GFP in situ for Cadets2Vets’ iGEM arsenic-sensing circuit, and increase the power of the evidence collected from the previous acrylamide protein assay whose gel broke.
Fig 2.1 – Acrylamide protein gel assay. First ladder (far left) is a standard. Second, third, and fourth are reaction replicates of an unregulated GFP promoter complex, with two strongly visible bands (bottom of latter bands demonstrate presence of reaction proteins at 13.3 kilo-Daltons; top of ladder bands demonstrate presence of GFP at 32.7 kilo-Daltons). The fifth ladder is a reaction control with no plasmid (bottom band presenting for reaction proteins). The sixth, seventh, and eighth ladders contain reaction replicates of the Cadets2Vets team’s regulated GFP plasmid. Variability demonstrated is due to varying amounts of regulatory protein and variability.
The second acrylamide protein gel assay presents strong qualitative evidence for the presence of GFP and known regulatory proteins in situ. The fact that the Cadets2Vets team’s regulated plasmids appear to have a high degree of variability (Fig 1.1) and protein smearing leads the team to believe that a shielding mechanism is occurring where the excess regulatory protein in situ prevents GFP from presenting as strongly as it otherwise could.
The Cadets2Vets team plans to next perform ticketing and brainstorm methods to increase GFP signaling efficiency.
07/21/2017
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.
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.
07/26/2017
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.
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