Team:Cadets2Vets/Notebook August

On August 2 and 8, 2017, Keshava Katti and Kian Croston ran two ticket assays to determine the effect of adjusting plasmid DNA proportionality. The past two tickets contained equal proportions of PcArsR and PARsGFP (1:1 ratio).

The Cadets2Vets team set up two tickets, positive and negative tests, on August 2. The positive ticket tested the dosage response at varied volumes of arsenic water; the negative ticket substituted all instances of arsenic water with molecular water.

Column 1: 2 µL Master Mix + 2 µL water (molec/arsenic)

Column 2: 2 µL Master Mix + 4 µL water (molec/arsenic)

Column 3: 2 µL Master Mix + 6 µL water (molec/arsenic)

Column 4: 2 µL Master Mix

Positive Master Mix contained:

12 µL S30 Extract
16 µL S30 Promega
4 µL amino (-Cys)
0.8 µL amino (-Leu)
3.2 µL arsenic water
4 µL of PcArsR + Ars1.1 circuit plasmid

Negative Master Mix contained:

12 µL S30 Extract
16 µL S30 Promega
4 µL amino (-Cys)
0.8 µL amino (-Leu)
3.2 µL molecular water
4 µL of PcArsR + Ars1.1 circuit plasmid

The image (above) shows the results of our tickets. As expected, our control ticket with no arsenic displayed no GFP expression. On the other hand, our positive ticket showed the same lack of fluorescence, which was attributed to the excess regulatory protein. Unfortunately, the Cadets2Vets team did not realize that Ars1.1 was actually causing increased production of regulatory protein until after our fourth ticket assay on August 8.

Team Cadets2Vets created 8uL of PcArsR + Ars1.1 plasmid DNA by combining 5.33 µL of Ars1.1 and 2.67 µL of PcArsR (2:1 ratio, respectively). There was a fundamental misunderstanding behind this combination. Our team believed that Ars1.1 was our GFP-producing plasmid DNA; in reality, Ars1.1 was our constitutive promoter, producing our ArsR regulatory protein, and GFP plasmid DNA combined.

The original idea was to increase the amount of promoter binding sites for ArsR, which is how GFP expression is regulated in the arsenic circuit. The additional PArsGFP plasmid was supposed to prevent ArsR from being free in solution and binding to free arsenic ions, thereby unintentionally sequestering our arsenic ions and ArsR proteins away from the regulatory mechanism we designed. Instead, we accidentally ended up increasing the amount of constitutive promoter DNA (Ars1.1 added unwanted constitutive promoter DNA), thereby increasing the overall production of ArsR regulatory protein.

The August 8 assay was a replicate of the setup outlined above. The only difference was increased troubleshooting in terms of ticket humidity. As shown in the image below (left), our team created a “boat” made from parafilm. The intended goal for this boat was to prevent excess liquid from the wet Kimwipe from touching the ticket. We continued using damp kimwipes in order to ensure ticket hydration. The image below (right) shows that our positive ticket continued to show no fluorescence, leading the Cadets2Vets team to conclude that the lack of fluorescence was not due to lack of humidity. In our next ticket assay, our team plans to isolate the GFP plasmid DNA to see whether there is a fundamental problem with our GFP gene.

On August 18, 2017, Keshava Katti and Brendan Studebaker ran a ticket assay to test the difference between tickets recently blotted with 5% Bovine Serum Albumin (BSA) and tickets blotted one month ago.

The Cadets2Vets team prepared four reaction conditions; two conditions evaluated the functionality of the circuit, one condition was a positive control (PArsGFP), and one condition was a negative control (water only).

Column 1: 2 µL (Master Mix + Ars1.1) + 2 µL (water)
Column 2: 2 µL (Master Mix + Ars1.1) + 2 µL (150 µM arsenic)
Column 3: 2 µL (Master Mix + PArsGFP) + 2 µL (water)
Column 4: 4 µL (water)

There were two reaction solutions involved in this ticket, one using Ars1.1 and one using PArsGFP plasmid.

The Ars1.1 master mix contained:

12 µL S30 Extract
16 µL S30 Promega
4 µL amino (-Cys)
0.8 µL amino (-Leu)
2.8 µL molecular water
4.4 µL of our Ars1.1 circuit plasmid.

The PArsGFP master mix contained:

6 µL S30 Extract
8 µL S30 Promega
2 µL amino (-Cys)
0.4 µL amino (-Leu)
2.3 µL molecular water
1.3 µL of our PArsGFP plasmid.

All DNA calculations were made to provide 10ng of DNA per 1 µL of Master Mix. The final concentration of arsenic ions in the reaction was 75 µM.

The reaction mix was pipetted onto the paper ticket in the columns and volumes written above. These tickets were then incubated overnight (~24 hours) at ~37 degrees Celsius. A change we made was to incubate the tickets in a petri dish inside a humidified tissue culture incubator, instead of humidifying the petri dish itself. After incubation, we qualitatively analyzed the tickets using a handheld UV light source and also imaged the tickets using a UV gel imaging system. The expressed GFP on both tickets lead the Cadets2Vets team to conclude achieving ambient humidity was no longer a problem and that a dry ticket works better than a wet ticket. The differences between the results of the newly blotted ticket (right) and the one-month blotted ticket (left) were almost unnoticeable; thus, we concluded that BSA was not playing a large role in the procedure and we may proceed with the old blotted tickets. Unfortunately, we received an unexpected positive test result from column 1 in each ticket. No arsenic was aliquoted into column 1, yet the column still showed fluorescence on both tickets. The Cadets2Vets team hypothesizes that there is a leaky promoter in our Ars1.1 DNA that allows GFP to express in an unregulated manner; to check this problem, we plan to use component parts (PArsGFP and PcArsR) instead of Ars1.1 in our next ticket assay.

On August 22, 2017, Keshava Katti and Brendan Studebaker ran a ticket assay to test the component parts of our circuit in hopes of identifying which part of Ars1.1 resulted in unexpected GFP expression and, consequently, the false positive test results.

The Cadets2Vets team tested each functional component of Ars1.1: PcArsR and PArsGFP. Then we combined the components together to make the equivalent of Ars1.1. We also changed the master mix solution to contain equivalent, 6% amounts of amino acid -Cys and -Leu mixes.

The PcArsR master mix contained:

6 µL S30 Extract
8 µL S30 Promega
1.2 µL amino acid mix (-Cys)
1.2 µL amino acid mix (-Leu)
2.4 µL molecular water
1.2 µL of PcArsR circuit plasmid.

The PArsGFP master mix contained:

6 µL S30 Extract
8 µL S30 Promega
1.2 µL amino mix (-Cys)
1.2 µL amino mix (-Leu)
2.3 µL molecular water
1.3 µL of PArsGFP plasmid.

Our target DNA concentration was 10 ng/µL and final arsenate/ite concentration was 75 uM.

Column 1: 2 µL of PArs GFP master mix and 2 µL of molecular water
Column 2: 2 µL of Pc ArsR master mix and 2 µL of molecular water
Column 3: 1 µL of PArs GFP master mix, 1 µL of Pc ArsR master mix, and 2 µL of molecular water
Column 4: 1 µL of PArs GFP master mix, 1 µL of Pc ArsR master mix, and 2 µL of arsenate/ite

These tickets were then incubated overnight (~24 hours) at ~37 degrees Celsius in the TC incubator, at which point they were examined qualitatively using a handheld UV light and also imaged using a UV gel imaging system. Every well in this ticket seemed to fluoresce, which led the Cadets2Vets team to conclude that perhaps one of our miniprep DNA microcentrifuge tubes had been mislabeled. Our hypothesis is that we may have accidentally used PArsGFP in all the reactions, which would have caused all wells to express GFP. As a result, the Cadets2Vets team agreed to go back to our glycerol stocks, regrow our E. coli cultures, and extract new DNA for our next ticket.

On August 24, 2017, Keshava Katti, Brendan Studebaker, Kian Croston, Zoey Shisler, and Kristina Ilyovska, overseen by Judy Nguyen and Amanda Galuszka, prepared DNA from fresh overnight cultures of E. coli containing Ars1.1 and PcArsR. The report of DNA concentration and quality obtained from using a Nanodrop are as follows:

Sample #	Plasmid Name	Concentration	A260	A260/280
1	PcArsR	130.2 ng/µL	2.605	1.9
2	PcArsR	147.3 ng/µL	2.946	1.89
3	Ars1.1	45.3 ng/µL	0.905	1.82
4	Ars1.1	14.1 ng/µL	0.282	2.01

Sample #4 had low concentration because the DNA was accidentally eluted in a larger volume than usual. We noticed that Ars1.1 had lower DNA yield compared to PcArsR even though they have the same vector backbone. We are not sure what this means but if we need to make more Ars1.1 DNA later, then we should increase the volume of bacteria culture that we use.

The Cadets2Vets team then set up a ticket assay to test the newly purified Ars1.1 and PcArsR DNA.

Column 1: 2 µL of Ars1.1 master mix and 2 µL arsenic water
Column 2: 2 µL of Ars1.1 master mix and 2 µL of molecular water
Column 3: 2 µL of PcArsR master mix and 2 µL of molecular water
Column 4: 4 µL of molecular water

The PcArsR master mix contained:

3 µL S30 Extract
4 µL S30 Promega Premix
0.6 µL amino acid mix (-Cys)
0.6 µL amino acid mix (-Leu)
1.1 µL molecular water
0.7 µL of PcArsR plasmid.

The Ars1.1 master mix contained:

6 µL S30 Extract
8 µL S30 Promega Premix
1.2 µL amino mix (-Cys)
1.2 µL amino mix (-Leu)
0 µL molecular water
4.4 µL of Ars1.1 plasmid.

These tickets were then incubated overnight (~24 hours) at ~37 degrees Celsius, at which point they were examined qualitatively using a backlight and imaged using an UV gel imager. This ticket did not end up producing any significant GFP signal. There is some fluorescence in Columns 1-3, but it was not statistically significant as compared to the Column 4 negative control. We hypothesize that the amino acids in the master mix might be contributing background fluorescence.

Even though the results were not completely what was expected, our team was happy to see that there were no longer any false positive test results. On the other hand, it meant that something was curtailing the ability of our circuit to produce GFP in the presence of the arsenic trigger. Because we do not believe we made mistakes in setting up the ticket assay, the Cadets2Vets team hypothesizes that we have exceeded the upper bound limitation for arsenic that our circuit can tolerate before denaturing. To address this, our team plans to create a new 150 µM stock solution of arsenate/ite water to be sure that we are measuring the arsenic concentrations correctly and we will dilute it down to three different working solutions of lesser concentration to run a ticket. Another possibility is that we had cross contamination of PArsGFP in our previous Ars1.1 miniprep.

Dr. Aleksandr Miklos from Edgewood Chemical and Biological Center mailed Cadets2Vets a prototype of a camera that can be used to image tickets. It is currently configured to take pictures illuminated by white LEDs and our hardware team, Jonathan Zacarias and Trevor Lowe, will work on creating a model that uses UV LEDs.

Team:Cadets2Vets/Notebook August

Our Progress

August Notebook

08/02/2017

08/08/2017

08/18/2017

08/22/2017

08/24/2017

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