Team:Cadets2Vets/Experiments

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Experiments - Home


EXPERIMENTS



The majority of the wet lab work is performed at the Readiness Acceleration and Innovation Network (RAIN) Incubator, a startup non-profit in Tacoma, Washington with ties to University of Washington Tacoma (UWT) and Edgewood Chemical Biological Center (ECBC). Some work was also performed at UWT laboratories. We have designed our arsenic circuit, cloned the inserts into the pSB1C3 vector, and transformed them into E. coli DH5a cells.


We will evaluate the circuit by measuring GFP expression in cells and the activation of the circuit using arsenic. While approaching our project in different directions, we ultimately decided to utilize the following protocols to create and test our arsenic circuit.




Protocol



Cloning



We used IDT gBlocks to synthesize the DNA we selected from the iGEM Parts Registry. Because we were creating a regulatory protein and a reporter, we made two separate pieces of synthetic DNA and later joined them with a restriction enzyme site to create a biscistronic plasmid. We also added the BioBrick Prefix and Suffix to the ends of our synthetic DNA so that we can insert it into the BioBrick assmbly plasmid pSB1C3. Information about our Parts can be found on our Parts Page.


Standard protocols for restriction enzyme digests, ligations, and transformationss were found on the iGEM Protocols page.


After transformation, two colonies were selected for screening from the transformation plates and subject to the miniprep protocol put forth by Promega Wizard Plus SV Miniprep DNA Purification system.

Sourced from:
https://www.promega.com/resources/protocols/technical-bulletins/0/wizard-plus-sv-minipreps-dna-purification-system-protocol/


The resulting plasmid DNA was screened using restriction enzyme digest, and the fragments produced were compared against in silico digestions performed using Genome Compiler and also our own predictions based on the DNA sequence.



testing e. coli plasmids using a plate reader



We took advantage of the skills that we learned performing the Interlab study and applied a similar protocol to our plasmids. We modified the Interlab protocol for our purposes by evaluating conditions such as the starting O.D., the duration of the time course, and the necessity of washing out LB before reading the cell cultures on the plate reader.

​

DH5a cells that were transformed with our plasmids were grown overnight in LB with 25ug/uL chloramphenicol. The next day, the cultures were diluted down to a specified O.D. The first time point was taken here as time 0, and we continued to culture the cells over 4 hours, taking time points as specified. The GFP and Abs600 readings were taken using a Molecular Devices FilterMax F5 multimode plate reader. When the desired reaction conditions were identified, we repeated those parameters but used cultures containing the biscistronic Arsenic Circuit spiked with a range of arsenic concentrations.



Testing plasmids in cell-free lysates using paper ticket



The optimal conditions for GFP expression in the plate reader is expected to represent the upper limits of what we can expect using the paper assay. This is because we expect it to be slower and less efficient for the reaction to occur in live cells, as compared to a cell-free system where all the necessary proteins are readily available. We start with the conditions understood from the plate reader experiments and titrate the duration and amount of arsenic down. The cell-free assay is performed using the manufacturer’s recommended protocol for the Promega E. coli S30 Extract System for Circular DNA.


Sourced from:https://www.promega.com/products/protein-expression/protein-expression-kits-and-reagents/e_-coli-s30-extract-system-for-circular-dna/?catNum=L1020

The reaction volumes were scaled such that 2-4uL of reaction volume was applied to a well on the paper ticket, with the plasmid DNA added at 10ng/uL. We incubated the ticket in a humidified incubator up to overnight. Then we took a picture of the ticket using a UV imager.



Quantification of reaction in paper ticket.



In order to quantify the results of our paper ticket assay, we utilized ImageJ software to select the reaction wells and measure the raw intensity of the fluorescence.

​

We took a photo of the ticket under UV light and made sure that the fluorescence signal was not saturating and saved as a .tiff or .png file. Small adjustments were made, such as rotating the image so that the ticket columns are level or to label the photo (Title: Date/Column numbers).


The protocol for using ImageJ was as follows:

1. Open photo in ImageJ program.

2. Open “Analyze” menu and select “Set Measurements”

3. Ensure only “Integrated Density,” is selected.

4. Hit “ok”

5. Select Oval tool. (This is more preferred method)

6. Create an Oval around the first test well. Be sure all pixels of the well are encompassed. Be sure to measure each individual well.

7. Open “Analyze” menu, and select Measure. Quick keys: Ctrl+M

8. (New window will open containing the Raw/Integrated Density measurement)

9. Use arrow keys to move the original Oval to the next well in line within that column.

10. (Repeat this process until all 16 wells have been measured)

11. Save the window containing Raw/Integrated Density Measurements.

12. Export data to Excel.

13. Organize and and label each column.

14. Calculate the mean and standard deviation of each individual column separately.

15. Highlight each column head and all four Raw Raw Density numbers at once.

16. Select “Insert” menu. In the Charts menu, be sure to use scatter plot.

17. Remove vertical grid lines.

18. Title Chart.

19. Ensure x-axis is labeled only with 1-4.

20. Right click on the chart. Select Edit Data. Click the Add button.

21. Label new data. “Mean.” Select Column 1 for x-axis. Select first Mean result for y-axis. (Hit add again and repeat process till all four column means are added for the chart.)

22. Adjust plot colors to distinguish Mean versus individual data points.

23. Make sure first mean point is selected. Open “Design,” menu. In the upper left is “Add Element,” drop down Menu. Select add error lines.

24. In the options for the error lines, find and select the “custom lines.” It will bring up a wind. Clear and select the first columns Standard Deviation. Clear the second entry and select that same Standard Deviation. (Repeat this same process for each mean of all four columns.)

25. Be sure to delete the lateral error lines that form automatically. You only want the vertical lines to remain.

26. Make any final edits to the chart for aesthetic purposes.



Development of Project



Some of our initial experiments had unclear results, which is to be expected when attempting experiments for the first time. These experiments included examining GFP expression using a Light Cycler and uninduced and induced GFP expression using a protein gel. Knowing that our goal was to demonstrate the circuit in the paper ticket, and that the reaction conditions of the paper ticket were much different than in the other experiments, we forged ahead because it appeared that plasmids were expressing GFP.


The paper ticket produced a lot of unexpected challenges, one being the to incubate the ticket in a way that prevented the ticket from drying out and also not get wet. We tried wet tissues, saturated salt solutions, and parafilm boats to try to get the right balance of humidity. In the end, the most reliable method was to incubate the ticket inside a petri dish using a tissue culture incubator that contained a water bath inside of it.


When we finally figured out this more reliable way to incubate the paper ticket, we discovered that we were getting GFP signal in the negative controls. We hypothesized that the plasmid DNAs we were working with may have gotten mixed up, and so we made new minipreps from our glycerol stocks. We also double checked the DNA sequences and to our surprise, the DNA sequence was not as expected. Although we had selected the appropriate Parts from the iGEM Registry, the sequence information for the arsenic regulator gene was incorrect. This meant we had to remake our plasmids. Luckily, we were able to synthesize new DNA from IDT and try again. The downside is that at this point, school had started for our team members and so we had less time to perform experiments. This trial and error process has allowed us to learn a lot of wet lab skills, and this will help us greatly in the future.


The idea of a paper-based arsenic sensor came from our ECBC partners and military team members. Service members put their lives at risk every time they deploy and we have a responsibility to ensure that we can provide them with the best equipment and protective measures possible. As we learned more about arsenic, we realized how big of a problem arsenic contamination is in the Tacoma area. Cadets2Vets felt that this project was a perfect fit to address both the needs of the military and the needs of our community. As we worked to share information about our project during Public Engagement and Outreach events, we met many people who would share personal stories about the history of arsenic in the area or their struggle to find access to arsenic testing. Hearing these stories made us feel that our vision of the arsenic circuit was one that would be truly appreciated by the public.


We started our research by first talking with our advisors, Drs. Hirschberg, Lux, and Emanuel, and Mr. Brisbin, and then reading literature published by peer-reviewed journals, reports by federal and state environmental agencies, and local newspaper articles. A select number of publications are listed below. We started with these articles and searched for more literature to follow up on additional questions we had about ArsR, GFP, different ways other people created biological sensors, and different types of sensor materials. The iGEM website itself was very helpful in finding information about DNA sequences and protocols.

The wet lab team started working intensely during the summer months on experiments to test their repeat experiments with purpose, clarity, and speed.



Bibliography



  1. Bereza-Malcolm LT, Mann G, Franks AE (2015) Environmental Sensing of Heavy Metals Through Whole Cell Microbial Biosensors: A Synthetic Biology Approach. ACS Synth Biol 4: 535–546.
  2. Carlin A, Shi W, Dey S, Rosen BP, Carlin A, Shi W, Dey S, Rosen BP (1995) The ars operon of Escherichia coli confers arsenical and antimonial resistance . These include : The ars Operon of Escherichia coli Confers Arsenical and Antimonial Resistance. J Bacteriol 177: 981–986.
  3. French CE, de Mora K, Joshi N, et al. (2011) Synthetic Biology and the Art of the Biosensor Design. Institute of Medicine (US) Forum on Microbial Threats. The Science and Applications of Synthetic and Systems Biology: Workshop Summary. National Academies Press (US); 2011. A5. Available from: https://www.ncbi.nlm.nih.gov/books/NBK84465/
  4. Martinez AW, Phillips ST, Whitesides GM (2008) Three-dimensional microfluidic devices fabricated in layered paper and tape. Proc Natl Acad Sci 105: 19606–19611. Available from: http://www.pnas.org/cgi/doi/10.1073/pnas.0810903105
  5. Nunnally D Three decades after the Asarco smelter shutdown, its toxic legacy surprises Tacoma newcomers | The News Tribune. News Trib Available from: http://www.thenewstribune.com/news/local/article43503663.html
  6. Pardee K, Green AA, Ferrante T, Cameron DE, Daleykeyser A, Yin P, Collins JJ (2014) Paper-based synthetic gene networks. Cell 159: 940–954. Available from: http://dx.doi.org/10.1016/j.cell.2014.10.004
  7. Rowe S (2014) State begins cleanup of Tacoma’s Vassault Park | The News Tribune. News Trib Available from: http://www.thenewstribune.com/news/local/article25871182.html
  8. Tacoma-Pierce County Health Department: Dirt Alert. Available from: http://www.tpchd.org/environment/healthy-environment/dirt-alert/​
  9. Schneider, C. A.; Rasband, W. S. & Eliceiri, K. W. (2012), "NIH Image to ImageJ: 25 years of image analysis", Nature methods 9(7): 671-675, PMID 22930834

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