iGEM 2020 — Team Cornell

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# Team:Cornell/Notebook

Team:Cornell - 2020.igem.org

[![logo](https://static.igem.org/mediawiki/2020/a/a5/T--Cornell--igem-logo-white.png)](https://2020.igem.org/Team%3ACornell)

TEAM

[BIOS](https://2020.igem.org/Team%3ACornell/Bios)
[SPONSORS](https://2020.igem.org/Team%3ACornell/Sponsors)
[ATTRIBUTIONS](https://2020.igem.org/Team%3ACornell/Attributions)

OUTREACH

[EDUCATION & ENGAGEMENT](https://2020.igem.org/Team%3ACornell/Education)
[COLLABORATIONS](https://2020.igem.org/Team%3ACornell/Collaborations)

HUMAN PRACTICES

[INTEGRATED PRACTICES](https://2020.igem.org/Team%3ACornell/Human_Practices)
[POLICIES](https://2020.igem.org/Team%3ACornell/Policies)
[ENTREPRENEURSHIP](https://2020.igem.org/Team%3ACornell/Entrepreneurship)

PRODUCT DEVELOPMENT

[MODELING](https://2020.igem.org/Team%3ACornell/Model)
[ENGINEERING SUCCESS](https://2020.igem.org/Team%3ACornell/Engineering)
[HARDWARE](https://2020.igem.org/Team%3ACornell/Hardware)
[IMPLEMENTATION](https://2020.igem.org/Team%3ACornell/Implementation)

TOOLKIT

* **WET LAB**
* [FOUNDATIONS](https://2020.igem.org/Team%3ACornell/Foundations)
* [DEMONSTRATE](https://2020.igem.org/Team%3ACornell/Demonstrate)
* [PARTS](https://2020.igem.org/Team%3ACornell/Parts)
* [BASIC PARTS](https://2020.igem.org/Team%3ACornell/Basic_Parts)
* [COMPOSITE PARTS](https://2020.igem.org/Team%3ACornell/Composite_Parts)

* **DOCUMENTATION**
* [NOTEBOOK](https://2020.igem.org/Team%3ACornell/Notebook)
* [SAFETY](https://2020.igem.org/Team%3ACornell/Safety)
* [CONTRIBUTION](https://2020.igem.org/Team%3ACornell/Contribution)

DESCRIPTION

HOME

![notebook banner](https://static.igem.org/mediawiki/2020/1/1f/T--Cornell--notebookbanner.png)

* [February 2020](#pageheading1)
* [March-April 2020](#pageheading2)
* [May-June 2020](#pageheading3)
* [July 2020](#pageheading4)
* [August-October 2020](#pageheading5)

February 2020

---

February 2nd to February 8th
Everyone participated in a lab safety tour with Dr. Archer. We discussed transformation, including the lab techniques involved in each step. We also laid out expectations for the project timeline and member assignments.

February 9th to February 15th
We began lab training with running 5 uL of practice PCR reactions and worked on a sequence planning activity in preparation for sequence planning required for the project. We also cleaned up the lab space and organized materials.

February 16th to February 22nd
We practiced gel electrophoresis preparation and loading. We also participated in gel analysis exercise and troubleshooting for possible errors, and reviewed gel electrophoresis applications in connection with the project.

February 22rd to February 29th
We worked on training for bacterial transformation. We also went over all the aspects of sterile technique and plating. We reviewed the steps of Golden Gate Assembly and practiced using the nanodrop.

March-April 2020

---

March 1st to March 7th
We ran a Golden Gate Assembly reaction to practice the protocol. We did training for experimental design and discussed the roles that controls play in various studies, as well as how we would incorporate them into future experiments for our future project.

March 8th to April 11th
Sent home for covid

April 12th to April 18th
We did virtual transformation training by going through the steps and information we need for understanding the protocol. To practice making sequences, we searched for suitable promoters for a part of our genetic circuit. We ended training by walking through the steps of primer creation for Golden Gate Assembly using Benchling.

April 19th to April 25th
We continued our work on making sequences for parts of the genetic circuit including the toxin, antitoxin, therapeutic, and mCardinal.

April 26th to May 2nd
We finalized the lactate-inducible promoter and antitoxin sequences since we were unable to finish these sequences in the previous week.

May-June 2020

---

May 3rd to May 9th
We did MATLAB training by going through demonstrations of manipulating arrays and entering equations for our models. We learned how to important data on MATLAB and use this data to generate a script. We began briefly taking a look at the differential equations we would use to model our toxin-antitoxin system.

May 10th to June 13th
Break for final exams.

June 14th to June 20th
We reviewed background information about Michaelis-Menten kinetics. We also discussed using MATLAB to model gene expression with an activator.

June 21st to June 27th
We researched the overall mechanism and lethal dose to kill bacteria for GhoS/GhoT Toxin-Antitoxin system. We also determined differential equations, using Michaelis Menten Kinetics for modeling production and degradation of toxin and antitoxin, as well as mRNA production and degradation. Finally, we found some parameters and modeled the differential equations for each component of the system using MATLAB.

June 28th to July 4th
We chose trichosanthin as our final therapeutic and modeled its basal expression, degradation and export to determine the lethal concentration required to inactivate ribosomes in tumor cells. The steady state in the bacterial cells was 35.9 µM and 0.00955 µM once it diffuses into the cancer cell. Additionally, we modelled the production of mCardinal production in E. Coli cells. We determined the steady state concentration of the mCardinal to be 0.0158 μM, using parameters such as DNA transcription rate, mRNA translation rate, and degradation rate.

July 2020

---

July 5th to July 11th
We worked on improving our modeling for the GhoT/GhoS toxin-antitoxin system and searched for more parameters. We also researched typical metastasis locations and determined how that would impact the modeling.

July 12th to July 18th
We modeled bacterial growth in an average sized tumor and a smaller tumor (metastasis). We determined that bacteria will grow within tumor bounds with high local therapeutic concentrations for the average tumor, and will persist long term in the smaller tumors. This allowed us to show that the treatment may function in the long term until the tumor shrinks and eventually disappears. We also collaborated with Product Development to do some general information about cancer treatments.

July 19th to July 25th
We did general background research on drug delivery methods and use of other cancer treatments along with ours. We found injection to be the most efficient method, and determined that our therapeutic would work best as a supplement to other cancer treatment methods. We looked into how we could incorporate the Asd obligate gene into our plasmid and found that we could use Lambda Red recombineering, Golden Gate assembly, and subsequent plate testing. We found plasmids for this process that we would be able to purchase online.

July 26th to August 1st
We found a better toxin-antitoxin system to use for our genetic circuit: the holin-antiholin system. Our modeling showed that it would be more successful for our goals compared to the GhoS/GhoT system. We also created a diagram of our plasmid in SnapGene.

August-October 2020

---

August 2nd to August 8th
We were able to find a protocol for incorporating the Asd obligate gene into our plasmid, which we may use if we are able to get into the lab. We also explored the possibility of using the LuxR to increase the expression of mCardinal to improve our fluorescence imaging abilities.

August 9th to August 15th
We worked with our Product Development team to research more information about light absorption for different skin tones. We came up with a plan of 5 plasmids that we would theoretically use in experiments, and we wrote up experimental procedures using each plasmid. The experiments we wrote up tested our improved part, trichosanthin production, and bacterial survival at different lactate concentrations.

August 16th to August 22nd
We began compiling our results from our summer modeling to put onto the Wiki. We coordinated times to enter the lab under restricted COVID-19 regulations.

August 23rd to September 12th
Break

September 13th to September 19th
Some members began going into the lab to finish the training that got cut short due to being sent home for COVID.

September 20th to September 26th
We consolidated our figures and cleaned up our MATLAB scripts.

September 27th to October 3rd
We wrote up registry pages for all of the parts that required them, including our improved kill switch, mCardinal, trichosanthin, and Asd.

October 4th to October 10th
We worked on editing all of the sections that will be included on the Wiki.

October 11th to October 17th
We finalized our drafts for the Wiki pages.

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# Team:Cornell/Engineering

Team:Cornell - 2020.igem.org

[![logo](https://static.igem.org/mediawiki/2020/a/a5/T--Cornell--igem-logo-white.png)](https://2020.igem.org/Team%3ACornell)

TEAM

[BIOS](https://2020.igem.org/Team%3ACornell/Bios)
[SPONSORS](https://2020.igem.org/Team%3ACornell/Sponsors)
[ATTRIBUTIONS](https://2020.igem.org/Team%3ACornell/Attributions)

OUTREACH

[EDUCATION & ENGAGEMENT](https://2020.igem.org/Team%3ACornell/Education)
[COLLABORATIONS](https://2020.igem.org/Team%3ACornell/Collaborations)

HUMAN PRACTICES

[INTEGRATED PRACTICES](https://2020.igem.org/Team%3ACornell/Human_Practices)
[POLICIES](https://2020.igem.org/Team%3ACornell/Policies)
[ENTREPRENEURSHIP](https://2020.igem.org/Team%3ACornell/Entrepreneurship)

PRODUCT DEVELOPMENT

[MODELING](https://2020.igem.org/Team%3ACornell/Model)
[ENGINEERING SUCCESS](https://2020.igem.org/Team%3ACornell/Engineering)
[HARDWARE](https://2020.igem.org/Team%3ACornell/Hardware)
[IMPLEMENTATION](https://2020.igem.org/Team%3ACornell/Implementation)

TOOLKIT

* **WET LAB**
* [FOUNDATIONS](https://2020.igem.org/Team%3ACornell/Foundations)
* [DEMONSTRATE](https://2020.igem.org/Team%3ACornell/Demonstrate)
* [PARTS](https://2020.igem.org/Team%3ACornell/Parts)
* [BASIC PARTS](https://2020.igem.org/Team%3ACornell/Basic_Parts)
* [COMPOSITE PARTS](https://2020.igem.org/Team%3ACornell/Composite_Parts)

* **DOCUMENTATION**
* [NOTEBOOK](https://2020.igem.org/Team%3ACornell/Notebook)
* [SAFETY](https://2020.igem.org/Team%3ACornell/Safety)
* [CONTRIBUTION](https://2020.igem.org/Team%3ACornell/Contribution)

DESCRIPTION

HOME

![engineering success banner](https://static.igem.org/mediawiki/2020/5/56/T--Cornell--engineeringbanner.png)

* [Ideation](#pageheading1)
* [Design Process](#pageheading2)
* [Implementation](#pageheading3)
* [Sources](#pageheading4)

Ideation

---

Not only did our team create a cancer therapeutic, but we also wanted to design a monitoring system. Our team began by proposing a self-scanner, as this would be a convenient and easy-to-use approach. We were unsure how to actually create this, so started by researching existing technologies that can be used for imaging. We found many bioluminescence readers already in use to track gene expression and disease progression [1]. We found that bioluminescence is typically seen as more accurate than fluorescence when it can be measured properly because it eliminates the possibility of intervening autofluorescent tissue signals [2]. From here, we started to consider the actual reader construction and noticed bioluminescence would also be superior to fluorescence from a design perspective because we would not need a light source to excite the particles, nor would we need specific wavelength filters that add significantly to cost.

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Design Process

---

Although this was a good starting point, as we began to research how to actually implement this design, we ran into one main problem. We found that most of the systems in use are pretty large structures that fit the entire animal body (usually a mouse) inside the reader in order to make sure zero outside light can interfere with the reading [1]. Others are similarly “light-tight” even if the entire body does not fit inside. Trying to have no outside light and reading with a low grade camera would be a difficult feat, so we began to research alternatives to bioluminescence.

From this point, we saw that some of the bioluminescent readers also read fluorescence, so we researched whether that would be a better way to accomplish our goal. We found many more low-cost fluorescent readers created by various researchers [3,4]. Multiple of these also included open-source software that we could use as a reference [3,4]. With fluorescence, we would not need to be as worried about excess light getting in. We could excite the fluorophores using specific frequencies as well as read specific frequencies. We discussed with our wetlab subteam to determine what would be possible and looked at various fluorescent markers. We found comparable values in papers and decided there would be significantly high enough mCardinal presence to be feasibly detected, so we pursued a fluorescence-based reader design.

![]()

Implementation

---

Weighing pros and cons of various types of structures and features for our reader, we decided that using a Raspberry Pi camera with mCardinal specific wavelength emission and excitation filters, as well as a dichroic mirror would be ideal. We chose this idea because we essentially needed to copy over the design of a fluorescent microscope without the magnification. From here, we then created a CAD of what the reader would look like and found parts to order (raspberry pi + camera, mirror/filter block, light source, ABS plastic to 3D print). Because of COVID-19, we did not build the reader, but instead used mathematical modeling to predict what depth we could read at and answer various other questions that arose.

The modeling for our reader can be found under [hardware](https://2020.igem.org/Team%3ACornell/Hardware#pageheading3).

After creating the model, we used it to try to optimize our design. Specifically, the [power graphs](https://2020.igem.org/Team%3ACornell/Hardware#pageheading5) helped us pick a light intensity for deep reading depth that is still safe for human skin.

When we actually build the reader, the basic testing we will do can be found under [hardware](https://2020.igem.org/Team%3ACornell/Hardware#pageheading7).

After basic testing, we can use this data (such as max reading depth and intensity at each depth) to help improve the accuracy of our model. From here, we could then use our model to change other variables and predict what will improve reading depth. This testing process above will then be repeated to test various other factors in order to optimize our design. We hope to test the efficacy of emission and excitation filters in order to see whether the readable depth increases or decreases in their presence. In addition, we discussed polarization of light, diameter of reading tube, and use of magnifying lenses as other variables to try. We will conduct thorough research on every aspect we intend to experiment with and incorporate it into our model to predict outcomes prior to actually rebuilding the reader, incorporating them, and retesting.

Following this design process cycle, we preserve as many resources as possible by only building and testing after researching, designing, and modeling. All of our steps also build on each other such as the testing improving the modeling. Through this cycle, we believe we can create the best product possible. If there are any unexpected results in testing along the way, we have all of the previous steps to work backwards from. We would first look at the model to see what variables could be causing these results and if it cannot be found there, we will look back to the physical design limitations. If we cannot find the mistake there, we will move back to the research stage to try to learn more about the system before continuing.

An example of an unexpected result could be that the readable depth is about a quarter of a millimeter lower than predicted. We could look at our model and see what constants went into this prediction that would cause that effect, maybe the 10% loss through the dichroic mirror block (without this in at all, the readable depth is ~0.5mm deeper). Although the part specification gives this value [5], this would cause a slightly-off readable depth (rather than other variables that may change it to more or less extreme effects). We could easily shine light through the block and measure the loss to determine whether this is the cause. If this is the cause, we could fix our model and research more about what kinds of filters/brands let the most light through, given this is clearly an area where there is the potential for free variables. If this is not the cause, we would go through other values in our model, testing those similarly. From here, if we had not found the cause, we would consider other limitations besides variables in our model (is the reader being held at an angle, etc.). If the cause for the unexpected result has still not been identified, we would research other fluorescent readers and atypical tissue interaction to look if other researchers had run into similar findings. One fix could be, as mentioned, the polarization of the light. We might observe that too much light is reflected off of the skin, or even at the boundary between different layers in the skin that have different absorptive indices and scattering coefficients. Excessive reflection would lower our readable depth. Because we can polarize our laser light, we can reduce this reflection based on research into skin brewster angles [6]. After researching this and determining what has been optimal in other experiments, we could then move back to our model and try incorporating this to see whether our model now matches our tests. If this is the case, we would use our research to determine the optimal polarization, edit our design, and run through testing again. If it is still not the cause, we would continue to research. When we eventually find what is causing this result, we will move back to our model, learn from our research and unexpected outcome, and continue to optimize our product.

![]()

Sources

---

[1] IVIS Instrument, Spectrum, 120V, Andor C-124262|PerkinElmer. (2020). Retrieved 5 October 2020, from https://www.perkinelmer.com/product/ivis-spectrum-imaging-system-120v-124262

[2] John, S., Rolnick, K., Wilson, L. et al. Bioluminescence for in vivo detection of cell-type-specific inflammation in a mouse model of uveitis. Sci Rep 10, 11377 (2020). https://doi.org/10.1038/s41598-020-68227-4

[3] Maia Chagas, A., Prieto-Godino, L. L., Arrenberg, A. B., & Baden, T. (2017). The €100 lab: A 3D-printable open-source platform for fluorescence microscopy, optogenetics, and accurate temperature control during behaviour of zebrafish, Drosophila, and Caenorhabditis elegans. PLoS biology, 15(7), e2002702. https://doi.org/10.1371/journal.pbio.2002702

[4] Nuñez I, Matute T, Herrera R, Keymer J, Marzullo T, Rudge T, et al. (2017) Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering. PLoS ONE 12(11): e0187163. https://doi.org/10.1371/journal.pone.0187163

[5] 45° Red Dichroic Filter Mounted in C-Mount Cube | Edmund Optics. (2020). Retrieved 18 October 2020, from https://www.edmundoptics.com/p/45deg-red-dichroic-filter-mounted-in-c-mount-cube/10195/

[6] Y. Su, W. Wang, K. Xu, and C. Jiang. The optical properties of skin. InOptics in Health Careand Biomedical Optics: Diagnostics and Treatment, pages 299–304. SPIE, vol. 4916, 2002

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# Team:Cornell/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Cornell/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)