iGEM 2017 — Team Cadets2Vets

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# Team:Cadets2Vets/Notebook

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Notebook

[*Notebook Overview*](https://2017.igem.org/Team%3ACadets2Vets/Notebook)
[*June Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_June)
[*July Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_July)
[*August Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_August)
[*September Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_September)
[*October Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_October)

[InterLab](https://2017.igem.org/Team%3ACadets2Vets/InterLab)
[Results](https://2017.igem.org/Team%3ACadets2Vets/Results)
[Demonstrate](https://2017.igem.org/Team%3ACadets2Vets/Demonstrate)
[Improve](https://2017.igem.org/Team%3ACadets2Vets/Improve)
[Attributions](https://2017.igem.org/Team%3ACadets2Vets/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ACadets2Vets/Parts)
[Basic Parts](https://2017.igem.org/Team%3ACadets2Vets/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ACadets2Vets/Composite_Part)

[SAFETY](https://2017.igem.org/Team%3ACadets2Vets/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ACadets2Vets/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ACadets2Vets/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ACadets2Vets/Engagement)

AWARDS

[Hardware](https://2017.igem.org/Team%3ACadets2Vets/Hardware)
[Environmental Project](https://2017.igem.org/Team%3ACadets2Vets/EnvironmentalProject)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Cadets2Vets)

Notebook - Home

* [Home](index.html)
* [Collaborations](collaborations.html)
* [Description](description.html)
* [Design](design.html)
* [Experiments](experiments.html)
* [Notebook](notebook.html)
* [Notebook August](notebook-august.html)
* [Notebook July](notebook-july.html)
* [Notebook June](notebook-june.html)
* [Public Engagement](public-engagement.html)
* [Safety](safety.html)
* [Team](team.html)
* [Basic Parts](basic-parts.html)
* [Composite Parts](composite-parts.html)
* [Parts](parts.html)
* [Interlab](interlab.html)
* [Our Partners](our-partners.html)
* [Notebook September](notebook-september.html)
* [Environmental Project](environmental-project.html)
* [Hardware](hardware.html)
* [Demonstrate](demonstrate.html)
* [Improve](improve.html)
* [Results](results.html)
* [Attribution](attribution.html)
* [Notebook October](notebook-october.html)
* [Silver HP](silver-hp.html)
* [Integrated and Gold](integrated-and-gold.html)

## Our Progress

[Notebook Overview](https://2017.igem.org/Team%3ACadets2Vets/Notebook)

[June Reports](https://2017.igem.org/Team%3ACadets2Vets/Notebook_June)

[July Reports](https://2017.igem.org/Team%3ACadets2Vets/Notebook_July)

[August Reports](https://2017.igem.org/Team%3ACadets2Vets/Notebook_August)

[September Reports](https://2017.igem.org/Team%3ACadets2Vets/Notebook_September)

[October Reports](https://2017.igem.org/Team%3ACadets2Vets/Notebook_October)

# Overview

## Follow us on Social Media!

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# Team:Cadets2Vets/Experiments

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PROJECT

[Description](https://2017.igem.org/Team%3ACadets2Vets/Description)
[Design](https://2017.igem.org/Team%3ACadets2Vets/Design)
[Experiments](https://2017.igem.org/Team%3ACadets2Vets/Experiments)

Notebook

[*Notebook Overview*](https://2017.igem.org/Team%3ACadets2Vets/Notebook)
[*June Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_June)
[*July Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_July)
[*August Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_August)
[*September Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_September)
[*October Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_October)

[InterLab](https://2017.igem.org/Team%3ACadets2Vets/InterLab)
[Results](https://2017.igem.org/Team%3ACadets2Vets/Results)
[Demonstrate](https://2017.igem.org/Team%3ACadets2Vets/Demonstrate)
[Improve](https://2017.igem.org/Team%3ACadets2Vets/Improve)
[Attributions](https://2017.igem.org/Team%3ACadets2Vets/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ACadets2Vets/Parts)
[Basic Parts](https://2017.igem.org/Team%3ACadets2Vets/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ACadets2Vets/Composite_Part)

[SAFETY](https://2017.igem.org/Team%3ACadets2Vets/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ACadets2Vets/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ACadets2Vets/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ACadets2Vets/Engagement)

AWARDS

[Hardware](https://2017.igem.org/Team%3ACadets2Vets/Hardware)
[Environmental Project](https://2017.igem.org/Team%3ACadets2Vets/EnvironmentalProject)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Cadets2Vets)

Experiments - Home

* [Home](index.html)
* [Collaborations](collaborations.html)
* [Description](description.html)
* [Design](design.html)
* [Experiments](experiments.html)
* [Notebook](notebook.html)
* [Notebook August](notebook-august.html)
* [Notebook July](notebook-july.html)
* [Notebook June](notebook-june.html)
* [Public Engagement](public-engagement.html)
* [Safety](safety.html)
* [Team](team.html)
* [Basic Parts](basic-parts.html)
* [Composite Parts](composite-parts.html)
* [Parts](parts.html)
* [Interlab](interlab.html)
* [Our Partners](our-partners.html)
* [Notebook September](notebook-september.html)
* [Environmental Project](environmental-project.html)
* [Hardware](hardware.html)
* [Demonstrate](demonstrate.html)
* [Improve](improve.html)
* [Results](results.html)
* [Attribution](attribution.html)
* [Notebook October](notebook-october.html)
* [Silver HP](silver-hp.html)
* [Integrated and Gold](integrated-and-gold.html)

# EXPERIMENTS

## 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.](https://2017.igem.org/Team%3ACadets2Vets/Parts)

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.

## 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.

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](https://2017.igem.org/Team%3ACadets2Vets/Engagement) and [Outreach](https://2017.igem.org/Team%3ACadets2Vets/HP/Silver) 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.

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# Team:Cadets2Vets/Results

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PROJECT

[Description](https://2017.igem.org/Team%3ACadets2Vets/Description)
[Design](https://2017.igem.org/Team%3ACadets2Vets/Design)
[Experiments](https://2017.igem.org/Team%3ACadets2Vets/Experiments)

Notebook

[*Notebook Overview*](https://2017.igem.org/Team%3ACadets2Vets/Notebook)
[*June Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_June)
[*July Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_July)
[*August Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_August)
[*September Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_September)
[*October Notebook*](https://2017.igem.org/Team%3ACadets2Vets/Notebook_October)

[InterLab](https://2017.igem.org/Team%3ACadets2Vets/InterLab)
[Results](https://2017.igem.org/Team%3ACadets2Vets/Results)
[Demonstrate](https://2017.igem.org/Team%3ACadets2Vets/Demonstrate)
[Improve](https://2017.igem.org/Team%3ACadets2Vets/Improve)
[Attributions](https://2017.igem.org/Team%3ACadets2Vets/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ACadets2Vets/Parts)
[Basic Parts](https://2017.igem.org/Team%3ACadets2Vets/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ACadets2Vets/Composite_Part)

[SAFETY](https://2017.igem.org/Team%3ACadets2Vets/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ACadets2Vets/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ACadets2Vets/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ACadets2Vets/Engagement)

AWARDS

[Hardware](https://2017.igem.org/Team%3ACadets2Vets/Hardware)
[Environmental Project](https://2017.igem.org/Team%3ACadets2Vets/EnvironmentalProject)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Cadets2Vets)

Results - Home

* [Home](index.html)
* [Collaborations](collaborations.html)
* [Description](description.html)
* [Design](design.html)
* [Experiments](experiments.html)
* [Notebook](notebook.html)
* [Notebook August](notebook-august.html)
* [Notebook July](notebook-july.html)
* [Notebook June](notebook-june.html)
* [Public Engagement](public-engagement.html)
* [Safety](safety.html)
* [Team](team.html)
* [Basic Parts](basic-parts.html)
* [Composite Parts](composite-parts.html)
* [Parts](parts.html)
* [Interlab](interlab.html)
* [Our Partners](our-partners.html)
* [Notebook September](notebook-september.html)
* [Environmental Project](environmental-project.html)
* [Hardware](hardware.html)
* [Demonstrate](demonstrate.html)
* [Improve](improve.html)
* [Results](results.html)
* [Attribution](attribution.html)
* [Notebook October](notebook-october.html)
* [Silver HP](silver-hp.html)
* [Integrated and Gold](integrated-and-gold.html)

# Results

what our research led to

## Cloning

The Arsenic 1.1 plasmids that Cadets2Vets worked with in June and July were synthesized by GeneWiz in a PUC57 vector. Preliminary experiments were performed using these plasmids until unexpected results in July forced us to examine the DNA more closely.

We analyzed the sequences that were sent to GeneWiz for synthesis and compared them against the NCBI BLAST database and the iGEM Registry, and found that the sequences sent to GeneWiz were not the sequences that we had intended to synthesize. The regulator we used appeared to be a generic transcription factor and the GFP had amino acid substitutions that were unexpected. We do not think the mutations affected GFP expression but because it did not match the Part in the Registry, we needed to correct that gene.

Cadets2Vets redesigned the plasmids using the appropriate Parts and successfully cloned synthetic DNA into the pSB1C3 vector backbone using the BioBrick Assembly method. The ligation of the inserts into the backbone was verified using restriction enzyme digest (left).

We screened 2 clones for every transformation performed during the cloning of the Arsenic Circuit. The DNA gel on the left shows the products from inserting PcArsR or PArsRGFP into the pSB1C3 vector. The plasmids were digested with the specified restriction enzymes and we expected PcArsR to create a 569bp fragment and PArsRGFP to create a 969bp fragment. The backbone is 2070bp.

​

The DNA gel on the right is from 2 clones where we attempted to ligate PcArsR and PArsRGFP into the pSB1C3 backbone. We used restriction enzymes to cut PArsR out of the vector, and found that the vector fragment is different sizes for the two clones we screened. We expected to have ~3000bp vector (2070 + 969bp), which is consistent with clone #1. Clone #2 is only ~2000bp, indicating that the PArsRGFP insert did not integrate. Clone #1 is the correct plasmid to keep.

## future work

For our next steps, we need to test whether the presence of ArsR will turn off GFP expression. We will do this by combining the two plasmids, BBa\_K2248001 and BBa\_K2248002. We will test this by measuring GFP fluorescence using the Light Cycler and the Promega S30 Extract kit. When we understand how much DNA is needed, then we will start adding arsenic to de-repress expression of GFP.
Additional experiments that we may need to perform to verify that all the parts are working as expected are a protein gel or western blot, to see that ArsR is being expressed and that it’s the right size. We could also imitate the experiment performed by López-Maury, et al (2003) where they looked at the specificity of arsenic and antimony binding to the ArsR binding site using an electrophoretic mobility shift assay. This assay looks at how the mobility of proteins is slower when a protein is bound to DNA.
When these experiments are worked out, we can compare this data against the combined arsenic circuit plasmid. That way, even if the complete circuit is not as efficient at sensing arsenic, we can still use the component parts and have the same effect.
We will continue to refine the VK3 camera system so that it is very user-friendly and will be able to take pictures and perform an analysis. We will also improve the hardware so that the UV and white light will be combined into one piece of housing and that the user can decide which light to turn on.

## Interlab Study

The [InterLab study](https://2017.igem.org/Team%3ACadets2Vets/InterLab) tested 6 different Devices, with a positive and negative control. This is the order of devices from the highest fluorescence to lowest at the 6 hour time point:

* Positive control
* Device 2
* Device 1
* Device 4
* LB-chloramphenicol
* Device 5
* Device 6
* Device 3
* Negative control

These results tell us that the positive control had the highest amount of fluorescence and the negative control had the least amount of fluorescence; these appear to be very good controls for the experiment.

Devices 2, 1, and 4 had fluorescence higher than the autofluorescence from the LB-chloramphenicol. These promoter/RBS combinations are good at expressing GFP.

Devices 5, 6, and 3 did not have fluorescence higher than the background levels in the culture media. We would not use these promoter/RBS combinations to look at GFP fluorescence if the GFP was being measured in cells growing in LB because GFP production appears inefficient.

The Abs600 readings gave us confidence that any lack of GFP expression was not due to dead cells. The increased Abs600 reading told us which bacteria cultures continued to grow over the duration of the experiment. The cultures with the least GFP expression actually replicated much more often than the cells expressing high levels of GFP. We think this means that the cells were able to use their energy for replicating, instead of making GFP like the other cultures. The positive control had the most GFP fluorescence but the least amount of Abs600; this supports our hypothesis that cellular energy was used to make GFP instead of replicating.

Fig. 1 and Fig. 2 show graphs of our results from the Interlab.

## Fig. 2

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