iGEM 2016 — Team Cornell_NY

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# Team:Cornell NY/Notebook

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* [HOME](https://2016.igem.org/Team%3ACornell_NY/defaulthome)
* [ABOUT](https://2016.igem.org/Team%3ACornell_NY/About)
* TECHNOLOGY
  + - WET LAB
    - [Bacteriocins](https://2016.igem.org/Team%3ACornell_NY/Proof)
    - [Parts](https://2016.igem.org/Team%3ACornell_NY/Parts%20)
    - [Protocols](https://2016.igem.org/Team%3ACornell_NY/Protocols%20)
  + - PRODUCT DEVELOPMENT
    - [Customizable Shell](https://2016.igem.org/Team%3ACornell_NY/Demonstrate)
    - [App](https://2016.igem.org/Team%3ACornell_NY/Software)
  + - MATHEMATICAL MODELING
    - [Modeling](https://2016.igem.org/Team%3ACornell_NY/Model)
    - [Animation](https://2016.igem.org/Team%3ACornell_NY/Animation)
  + - DOCUMENTATION
    - [Notebook](https://2016.igem.org/Team%3ACornell_NY/Notebook)
    - [Safety](https://2016.igem.org/Team%3ACornell_NY/Safety)
* HUMAN CENTERED DESIGN
  + - [PRACTICES](https://2016.igem.org/Team%3ACornell_NY/Integrated_Practices)
    - [POLICIES](https://2016.igem.org/Team%3ACornell_NY/HP/Silver)
    - [ENTREPRENEURSHIP](https://2016.igem.org/Team%3ACornell_NY/Entrepreneurship%20)
    - [COLLABORATIONS](https://2016.igem.org/Team%3ACornell_NY/Collaborations%20)
* [PUBLIC ENGAGEMENT](https://2016.igem.org/Team%3ACornell_NY/Engagement)
* [OUR TEAM](https://2016.igem.org/Team%3ACornell_NY/Team)

Header 2

# Notebook

* [March](#march)
* [April](#april)
* [May](#may)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#sept)
* [October](#oct)
Open navigation

### March 21st to 27th

**P&P:** We presented our fishPHARM project from last year to faculty members at the BioExpo at Cornell.

### April 11th to 17th

**P&P:** We talked about what a project team is, as well as explained last year’s fishPHARM project to students from class of 2020 at the Science and Engineering Fair.

### April 18th to 25th

**P&P:** We sent out emails to experts, professors, and outreach event coordinators with whom we wanted to collaborate and to whom we wanted to ask questions about mastitis. We also taught 7th to 12th graders from Splash! At Cornell about synthetic biology, fishPHARM, how to load gels for gel electrophoresis, as well as led a discussion about the ethics behind synthetic biology.

### April 26th to May 3rd

**P&P:** We met with Caroline Potter and Dr. Lynch from PRO-Dairy, a joint venture of the New York State Department of Agriculture and Markets and Cornell University’s College of Agriculture and Life Sciences that works on educational programming for farmers, and they answered our questions about mastitis, as well as gave us the name of other experts whom we should contact. We also presented about fishPHARM at the RAW Expo at Cornell, an event for teams to share different projects across all disciplines.

### May 8th to 14th

**Wet Lab:** We transformed the terminator sequence BBa\_B1006 in pSB1C3 into E.coli BL21. We also made cultures of the colonies that grew, and miniprepped the cultures.

**Product Development:** We completed a morphological chart of what we hope to accomplish with our design and sketched out a few designs and voted on the best ones.

**P&P:** We met with Daryl Nydam, who told us a lot about the bacteria that cause mastitis, about the traditional approach of using antibiotics to treat it, and about how we would know if our approach of using bacteriocins would show promise in treating mastitis.
We visited Snofarm, and the owner of the farm walked us through and showed us the milking process, and we learned that the most intensive part of mastitis was disease prevention, so the cows and their environment must be kept very clean.
We reached out to Dr. Neubauer, who is the president of the National Mastitis Council as well as part of Zoetis, one of the world’s largest pharmaceutical companies that develops medicine and vaccination for animals, and he was able to point our product development team in the right direction for our milking device design.
We visited the Vet Dairy Teaching Barn, which is close to campus, and after talking to Charles, one of the employees, we were inspired to develop a prototype that would assist during the milking period and help with detecting mastitis.

### June 12th to 18th

**Wet Lab:** We resuspended 15 gBlocks in dH2O with final concentration of 50 ng/uL. We also transformed terminator sequences BBa\_B0015 in pSB1C3 and BBa\_B1006 in pSB1C3 into E.coli BL21.

**Product Development:** We outlined ideas from last date and decided to focus on prevention (Product Development - modular milking nozzle) and detection (CS/ECE team). Functional morph charts were created for each design.

**CS/ECE:** We narrowed down ideas with product development into two solid ideas: an improved milking nozzle to minimize risk of infection, and a box connected to an app to allow farmers to get fast results from milk samples without the need for a lab.

**P&P:** We spoke with a 4-H coordinator who agreed to let us be in the Youth Building for the Great NY State Fair in August!

### June 19th to 25th

**Wet Lab:** We made cultures of BBa\_B1006 and miniprepped, and made cultures of H and miniprepped. We then digested H miniprep with EcoRI and PstI and gel purified. We also digested BBa\_B1006 with XbaI and PstI. We also made chemicompetent cells.

**Product Development:** We researched more into a possible iodine module and typical milking machine prices and planned on having at least 4 possible modules (iodine, uv lights, temperature sensor, and cold shock).

**CS/ECE:** We began Autodesk Fusion 360 project. We decided to include a data collection/calculation/presentation app with three subfeatures. One of these subfeatures is the ability to connect to a box that will allow the farmer to do cow-side calculations of somatic cell count and milk amyloid A content. Our ideas were to use congo red dye and a photoresistor as well as a mass measurement to extract the data needed. The app will take the data and calculate/display the results.

**P&P:** We started preparing for the 4-H Career Explorations Conference as well by creating handouts and a presentation.

### June 26th to July 2nd

**Wet Lab:** We performed PCR of gBlocks and did a restriction digest of each gBlock. We attempted ligation of some gBlocks but was unsuccessful. We continued miniprepping and digesting H, and digesting BBa\_B1006.

**Product Development:** We decided on silicone material to create our own milking liner, and chose to start building an injection molding machine. In addition, we sketched design of modules and came to consensus on modules. We decided to pursue UV light, iodine, temperature sensor, cold shock, flow rate sensor, milking completion indicator, and blank functionless modules.

**CS/ECE:** We looked at preliminary sketches and discussed the Preliminary Design Review. In our discussion, we realized need to talk to farmers about the App and Box (What they want, etc. ) and decided we needed to brainstorm questions for feedback before we jump in. We also filmed for the iGem video
.

**P&P:** YOURS students were introduced to the iGEM program and learned how to measure with pipettes and graduated cylinders.
We planned and led a 2 day workshop with the 4-H Career Explorations Conference, which bridged the gap of entrepreneurship and science for youth.

### July 3rd to July 9th

**Wet Lab:** We continued PCR and restriction digest of gBlocks. We continued miniprepping and digesting H, and digesting BBa\_B1006. We attempted ligation, but was unsuccessful.

**Product Development:** We researched materials for injection molding machine and sketched design for machine. We also created feedback questions about our project for P&P to ask farmers. And we created Preliminary Design Review of project.

**CS/ECE:** We edited first draft of iGem video, created sketches of the app, and worked on Fusion 360 projects. After further research into the methods used to count somatic cells in milk, we decided to create a cow-side microscope that farmers could use to take SCC. This would replace the need to send samples into labs where technicians look at the milk under microscopes using a haemocytometer.

**P&P:** We taught the YOURS students about DNA and helped from extract DNA from strawberries and proteins from milk.
We began reaching out to farmers for farm visits later this month.

### July 10th to July 16th

**Wet Lab:** We successfully cloned 4 genes - Nisin F, Nisin U, Nisin F-MBP, and Subtilin MBP.

**Product Development:** We finalized module placements and ordered materials for injection molding machine

**CS/ECE:** We began getting supplies to build a microscope that utilizes the smartphone camera, which would eventually allow users to save the photos and count the cells. We made a powerpoint for the design review, edited the iGem video, cut the plexiglass for microscope, and worked on the Fusion 360 projects.

**P&P:** We reached out to Tom Votny, a representative of Milkrite, and we learned about the current state of the milking machine market, as well as the technologies that had already been developed for shells and liners. This knowledge helped point us in the right direction for further developing our milking shell.
The YOURS students learned how and why molecules separate in both gel electrophoresis and chromatography. They separated the colors of dyes in M&Ms and Sharpie markers.

### July 17th to July 23rd

**Wet Lab:** We successfully cloned 2 more genes - Nisin U-MBP and MBP. We ran into problems gel purifying H, but was able to fix the problems.

**Product Development:** We began construction of the injection molding machine, we determined the dimensions of the liner, and we prototyped the method through which the modules would fit together.

**CS/ECE:** We finalized app designs and worked on the Fusion 360 projects. We continued building the Cowscope parts.

**P&P:** Using concepts of engineering, the YOURS students created contraptions to save a falling egg from cracking.
We contacted Cambridge iGEM, who said that they’d be really interested in helping and allowing us to modify their Openscope towards more of a mastitis-oriented project.

### July 24th to July 30th

**Wet Lab:** We made chemicompetent cells. We attempted ligation and transformation of some gBlocks.

**Product Development:** We created a design matrix to help decide on the details of each module. We also created CADs of the modules without connections and 3D printed them. Finally, we attempted to CAD the modules with threads, but were unsuccessful due to the inaccuracy of the 3D printer.

**CS/ECE:** We consulted farmers and experts on app designs and ideas. We got into contact with Cambridge iGEM 2015, who 3D printed their own microscope design last year. We talked to them about modifying their design and specifying it for a cow-side somatic cell counter.

**P&P:** YOURS students visited the Cornell Vet Dairy Farm to learn about milking cows, the dairy industry, and why Cornell iGEM’s project focusing on mastitis is important.
We reached out to Blane Murray and visited the Muranda Cheese Company. He thought that our idea as a whole was valuable because it is a preventative measure for mastitis, and he emphasized that the most important aspect of marketing our product to farmers would be its cost effectiveness. In addition, he pointed us to a few bigger and more technologically advanced farms that we would later visit.
We visited Daryl Nydam of Quality Milk Production Services again, and he gave us feedback on the various aspects of our project - bacteriocins, milking shell, and the app - and gave us infected milk samples so that we could test our bacteriocins. With more feedback in hand, we worked on modifying our modules and app and did more testing on our bacteriocins.

### July 31st to August 6th

**Wet Lab:** We successfully cloned 2 more genes - Bactofencin A-MBP and Enterocin E760-MBP.

**Product Development:** We started a mathematical modeling of each module and molded the liner out of liquid silicone using a 2-part mold.

**CS/ECE:** We consulted farmers and Dairy One on app designs and started the app project file. We also presented iGem to tour guides.

**P&P:** We celebrated the end of our YOURS program with a tour of the DairyBar and eating some ice cream. We also conducted one last experiment using the reaction between baking soda.
We pitched iGEM to the campus tour guides at Cornell University, informing them about our project so that they could mention it to prospective students.
We visited Windstott Farm, a robot farm that Blane Murray had referred us to, and Bill Kilcer showed us how the robotic milking machines worked. Through our talk with Bill, we found that our project would be most helpful for bigger traditional dairy farms.
We reached out to Bill Morgan, who looks over the operations of Scipio Springs Dairy, and he was very interested in implementing bacteriocins on his farm and in the UV light and temperature sensor modules of the milking shell.
We spoke with John Tauzel, John Gloss, and George Cudoc of Dairy One, a dairy data management company that helps farmers in making decisions on their farms. They provided us with feedback on our Somatic Cell Counter idea as well as the cost calculator.

### August 7th to August 13th

**Wet Lab:** We prepped BBa\_B1006 terminator sequence in bulk, digested with XbaI and PstI, as well as pSB1C3, digested with EcoRI and PstI. We also dephosphorylated digested vectors.

**P&P:** We presented at the Office of Undergraduate Biology’s Summer Institute for Life Sciences Undergraduate Symposium to share our work in progress with other undergraduate researchers on campus.

### August 14th to August 20th

**Wet Lab:** WWe evaluated ligation accuracy via colony PCR.

**CS/ECE:** We worked on the app and designed 3D printable parts for the microscope.

**P&P:** We brainstormed activity ideas for the Great NY State Fair and purchased supplies.

### August 21st to August 27th

**Wet Lab:** We successfully cloned Colicin M. All other ligations failed.

**Product Development:** Discussed the integration of wet lab by making an ergonomic Caulk gun syringe for the wetlab treatment

**CS/ECE:** We presented iGem at the project fest and began 3D printing our microscope pieces.

**P&P:** We ran a booth at the Great NY State Fair with an activity and a photo campaign, and talked to kids from all over NY and PA about synthetic biology.

### August 28th to September 3rd

**Wet Lab:** We began cloning of Nisin A, Nisin Z, and Lysostaphin into pSB1C3. We obtained infected milk samples from Dr. Anja Sipka. We began purification of Colicin M, but determined that it was lethal to the engineered chassis.

**Product Development:** We decided on how to wire electronics and ordered materials to start building the module.

### September 4th to September 10th

**Wet Lab:** We successfully cloned Nisin A, Nisin Z, and Lysostaphin into pSB1C3. We attempted pETDuet cloning with Emr lab enzymes, but were unsuccessful.

**Product Development:** We tested the iodine module and decided on a 2-part sponge.

**CS/ECE:** We worked on app and microscope assembly.

**P&P:** We co-hosted an event at the Ithaca Sciencenter in collaboration with Building with Biology, an initiative founded behind sparking conversations about synthetic biology.

### September 11th to September 17th

**Wet Lab:** We prepared standard BSA curve via Bradford Assay.

**Product Development:** We made a CAD of the temperature sensor and iodine module, and designed a syringe applicator as another module.

**CS/ECE:** We sketched the Add Cow page

**P&P:** We reached out to the Muranda Cheese Company and Scipio Spring Farms for our second round of farm visits.

### September 18th to September 24th

**Wet Lab:** We plated 38 zone of inhibition plates. We attempted to purify truncated Lysostaphin, but did not observe it. We determined that expression was induced by IPTG instead of arabinose.

**Product Development:** We began machining shell and modules, and discussed how to test UV light efficiency.

**CS/ECE:** We worked on the Add Cow page and figured out how to pull up Add Cow page programmatically.

**P&P:** We presented our iGEM project at an alumni showcase for project teams. We wrote up next questions to ask the farmers for our next farm visits.

### September 25th to October 1st

**Wet Lab:** We prepared and inoculated cultures of Aureocin and Lysostaphin in Terrific Broth. We extracted proteins and performed zone of inhibition testing.

**Product Development:** We tested the iodine module prototype and began machining the milking shell. We discussed how we want to present at competition. We also received feedback from farmers about prototype

**CS/ECE:** We continued working on the Add Cow page.

**P&P:** We went back to talk to Blane Murray of the Muranda Cheese Company with our prototype of the milking shell, and he gave us feedback on the feasibility and appearance of the shell.
We also once again visited Bill Morgan of Scipio Springs Dairy would gave us praise for certain components of our milking shell as well as insight on how to improve the technical details. He further explained how much the cost of mastitis is, and how much he would be willing to pay for the various parts of our project.

### October 2nd to October 8th

**Wet Lab:** We continued to perform zone of inhibition testing for E.coli, S. aureus and S. epidermidis with Lysostaphin and Colicin M. We extracted proteins for Aureocin A53, Enterocin E760, Bactofencin A. We then performed zone of inhibition assays using the newly extracted proteins on S. aureus, S. epidermidis, E.coli, C. freundii, K. pneumoniae, P. aeruginosa.

**Product Development:** We decided not to implement the syringe module design because it was unfounded in design research. We machined the temperature sensor mechanism and the top part of the shell. We also CADed the top shell with holes for UV lights so that we could practice wiring them, and CADed a new version of the iodine module

**CS/ECE:** We worked on the app

### October 9th to October 15th

**Wet Lab:** We tested the efficacy of Bactofencin A, Enterocin E760, and Colicin M with growth curve assays. In addition, we continued with cloning Epidermicin NI01, Microcin E492, and Colicin 10 into pSB1C3 with the goal of characterizing them with zone of inhibition assays.

**Product Development:** We tested the UV-C lights. We also discussed and designed ways to display the shell at competition. The temperature sensor was ready to attach onto shell. We started machining the bottom piece of the shell and iodine module.

**CS/ECE:** We worked on the app.

### October 16th to October 19th

**Product Development:** We finished the wiki page this week. We also finished the machining for the top piece of the shell and most of the bottom piece.

**CS/ECE:** We worked on the app.

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