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Revision as of 20:50, 4 August 2017

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Notebook-July - Home


Notebook



JULY 14, 2017



INTER-TEAM MEETING





In an attempt to learn the methods used by other IGEM teams we participated in an inter-team Skype call. We chose to meet with the other teams sponsored by the United States Armed forces because we share many of the same challenges. Our notes were as follows:

  • Every member attending the Jamboree needs to be able to describe an answer questions about the entire project. It could possibly be a handicap to send people who aren't fully on board with the project.
  • One of the Navy programmers mentioned some sort of a universal page that affects all of our team pages. Josh, maybe you can look for that and apply some of our sidebar stuff there.
  • For our presentation, we should go as broad as we can without being boring and then get specific when judges and others ask.
  • The judges really like lots of pictures and a pretty layout for the poster and powerpoint presentation.
  • We still need to fill out our InterLab Excel files and go through the protocols.
  • We need to submit parts. I didn't get the chance to ask how we can check if a part is working, but both teams said we could feel free to email them if we had questions. The Air Force team email is igem@carrollhs.org.
  • The judges use a VGA cord to attach to computers (one of the bigger ones that needs to be screwed in). No internet is provided, but we could use hotspot if needed.
  • Conceptual models make good pictures, even if we can only model a piece of our project. There are good poster examples on iGEM.
  • For our procedures on our presentation and poster, we should include project-specific protocols (e.g. the lyophilization process or testing for GFP) before anything more basic like PCR, but it might be good to specify our specific reagents and primers used for more common things.
  • A lot of projects don't end up working, but some teams cheat and do a project for multiple years in order to make it work, or they stack a team with a ridiculous number of people. It seems judges might (?) be a little bit lenient with those who fudge the rules.
  • The Carroll College team has already gone through the InterLab procedures.
  • We need to specify our part for the GFP we're using.
  • For best human practices integration, a lot of teams go all out and serve food or draw pictures with GFP, so we need to do similar if we want to be competitive. Could we incorporate the Science Breakfasts somehow? We could also practice talking about our project to people at these events.


JULY 18, 2017





With the help of high school interns Keshava Katti, Kian Croston, and Josh Grace, senior technician Ryan Brisban performed multiple quantitative and qualitative protein assays to assess the presence of GFP in situ. To these ends, 8 circuit samples were put in a Light Cycler to incubate for 8 hours at 37 degrees Celsius. 4 samples were run with no Arsenic regulator present, meaning that GFP should have been produced continuously for the entire 8 hour period. 3 were run as controls with only broth and no circuit, and 1 control of pure molecular water was run as a baseline measurement. After 8 hours, the normalized fluorescence of the 4 samples containing GFP was significantly higher than any of the controls. To supplement these findings, the Cadets2Vets team ran an acrylamide protein gel on the samples, and although the gel broke in processing, there appeared to be a protein band corresponding to GFP’s known molecular size in kilo-Daltons. A second acrylamide protein gel assay is currently being run to increase the number of trials and provide better evidence than the broken gel. A second assay was also run using Qubit’s total protein assay. All samples were spec’d after creating a linear model of RFUs using standards, but they exceeded the range of the sensor, and thus needed to be diluted. After dilution, the protein concentration was determined to be in the ~1000s of ng/µL for each of the seven samples containing broth, whereas the sample containing molecular water exhibited control values too low for the Qubit to read. The 4 samples containing no Arsenic regulator with the circuit appeared to have lower protein concentrations by ~100-200 ng/ µL than the 3 samples containing only reagents and water. This finding was unexpected to the Cadets2Vets team, who hope that further analysis and measurement will determine the source of the extra protein. The current hypothesis is that the samples containing our circuit functionalized more of the reactants from the reaction matrix.



JULY 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. 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.



Fig 1.1 – 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.



July 20, 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 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 S30 Extract, 10 S30 Promega, 2.5 amino (+), 0.5 amino (-), and 2 molecular water. For the positive test, 2.5 of our circuit plasmid and machinery was added, and the 2 molecular water contained arsenite and arsenate at a 150 concentration. 2 of water (molecular for negative control, arsenic ion and water mixture for positive test) was added to wells in column 2, and 4 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 and the wells in the third column has an arsenic ion concentration of 208.3 for the positive test ticket.

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



July 25, 0217





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 LS30 Extract, 10 LS30 Promega, 2.5 Lamino (+), 0.5 Lamino (-), and 2 Lmolecular water. For the positive test, 2.5 Lof our circuit plasmid and machinery was added, and the 2 Lmolecular water contained arsenite and arsenate at a 150 M concentration. The negative control ticket used only 2 Lof reaction mixture for the first two columns of wells on the left, and the other two columns of wells were filled with 2 Lof molecular water. The positive test ticket used 2 Lof the test mixture in the first column of wells, and 4Lof test mixture in the second column of wells. The other two columns of wells were filled with 2Lof molecular water.

​

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.




Fig 1.1 and Fig 1.2 - The Positive Test Circuit (left) with first row containing 2 Lof test mixture, and the second row containing 4 Lof test mixture. Possible dose response is visible. The Negative Control Circuit (right), containing 2 L of reaction mixture in first and second columns.



Image Gallery



In this chart we can clearly see Positive Controls 1 and 2 expressing fluorescent signatures



Initial text of circuit in the presence of 25 um arsenite and arsenate solution we were able to generate fluorescent signature.


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