iGEM 2017 — Team Cornell

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Cornell/Notebook

<!DOCTYPE html>

Bios

[![Oxyponics](https://static.igem.org/mediawiki/2017/1/1d/CornellOxyponicsLogo.png)](https://2017.igem.org/Team%3ACornell)

* [HOME](https://2017.igem.org/Team%3ACornell)
* [ABOUT](https://2017.igem.org/Team%3ACornell/Description)
* TOOLKIT
  + WET LAB
    - [FOUNDATIONS](https://2017.igem.org/Team%3ACornell/Results)
    - [DEMONSTRATE](https://2017.igem.org/Team%3ACornell/Demonstrate)
    - [CONTRIBUTION](https://2017.igem.org/Team%3ACornell/Contribution)
    - [PARTS](https://2017.igem.org/Team%3ACornell/Parts)
    - [BASIC PARTS](https://2017.igem.org/Team%3ACornell/Basic_Part)
    - [PROTOCOLS](https://2017.igem.org/Team%3ACornell/Protocol)
  + PRODUCT DEVELOPMENT
    - [DESIGN PROCESS](https://2017.igem.org/Team%3ACornell/Design)
    - [APPLIED DESIGN](https://2017.igem.org/Team%3ACornell/Applied_Design)
    - [SOFTWARE](https://2017.igem.org/Team%3ACornell/Software)
    - [MODELING](https://2017.igem.org/Team%3ACornell/Model)
  + DOCUMENTATION
    - [NOTEBOOK](https://2017.igem.org/Team%3ACornell/Notebook)
    - [SAFETY](https://2017.igem.org/Team%3ACornell/Safety)
* HUMAN CENTERED DESIGN
  + [PRACTICES](https://2017.igem.org/Team%3ACornell/HP/Gold_Integrated)
  + [POLICIES](https://2017.igem.org/Team%3ACornell/Policies)
  + [ENTREPRENEURSHIP](https://2017.igem.org/Team%3ACornell/Entrepreneurship)
* OUTREACH
  + [COLLABORATIONS](https://2017.igem.org/Team%3ACornell/Collaborations)
  + [PUBLIC ENGAGEMENT](https://2017.igem.org/Team%3ACornell/Engagement)
* TEAM
  + [BIOS](https://2017.igem.org/Team%3ACornell/Team)
  + [SPONSORS](https://2017.igem.org/Team%3ACornell/Sponsors)
  + [ATTRIBUTIONS](https://2017.igem.org/Team%3ACornell/Attributions)

Notebook

FEBRUARY 27TH TO MARCH 5TH

**Wet Lab:** Wet Lab members began training, starting with sterile technique, growing cultures, miniprep, and PCR.

**Product Development:** Product Development members began familiarizing themselves with Fusion 360.

**Wiki:** Wiki subteam learned the basics of HTML/CSS during an hour-long HTML class, and worked on an HTML/CSS Project due Thursday, 3/16.

MARCH 6TH TO MARCH 12TH

**Wet Lab:** Wet Lab members split up into training groups and performed PCR reactions of gblocks used for last year’s bacteriocin project. Team members also performed miniprep to extract the H plasmid on cells.

**Product Development:**Team members began working on an exercise to create a CAD design of their phones.

**P & P:**P&P discussed the function of P&P as a subteam, goals for the upcoming year and started brainstorming ideas for outreach.

**Wiki:** Wiki subteam learned about Typography, White Space, and Color during an hour-long Design class. They worked on an exercise in Adobe Illustrator.

MARCH 13TH TO MARCH 19TH

**Wet Lab:** Wet lab members learned about restriction digests, ligation, agarose gel electrophoresis and cleanup, as well as OE PCR, Gibson, and 3A assembly.

**Product Development:** Team members learned how to create 3D objects in Fusion 360.

**P&P:** P&P researched successful P&P applications of past projects from other teams and discussed the pros and cons of those projects.

**Wiki:** Wiki subteam learned about JavaScript and jQuery during an hour-long HTML class. They successfully submitted their HTML/CSS Project due Thursday, 3/16. They started working on their JavaScript and jQuery project.

**Business:** Team members reviewed their requirements and spoke about expectations for the upcoming year.

MARCH 20TH TO MARCH 26TH

**Wet Lab:** Wet lab members practiced restriction digest, agarose gel electrophoresis, and ligation of bacteriocin genes into a vector.

**Product Development:** Product Development decided to work in a Hardware Accelerator program at Rev: Ithaca Startup Works.

**P&P:** P&P started planning for Splash! at Cornell, our first outreach event.

**Wiki:** Wiki subteam learned about Composition during an hour-long Design class. They successfully submitted their JavaScript and jQuery project Milestone 1.

**Business:** Team members wrote their sponsorship letters and started preparing the sponsorship packet.

MARCH 27TH TO APRIL 2ND

**Wet Lab:** Wet lab members learned about restriction digests, ligation, agarose gel electrophoresis and cleanup, as well as OE PCR, Gibson, and 3A assembly.

**Product Development:** Team members learned how to create 3D objects in Fusion 360.

**P&P:** P&P continued to work on the presentation for Splash!.

**Wiki:** Wiki subteam successfully submitted their JavaScript and jQuery project Milestone 2.

**Business:** Team members explored different ways to raise funds for the upcoming year.

APRIL 10TH TO APRIL 16TH

**Wet Lab:** Wet lab members learned about DNA sequencing and plasmid transformation into competent cells.

**Product Development:** Product Development began to learn how to use Arduino by making an LED flash at certain time intervals.

**P&P:** P&P finalized the presentation and planned our hands-on experiments for Splash!. We began preparing for our next outreach events: Raw Expo, MakerFaire, and a Lab Demo/Tabling for potential Cornell students in the Women in Engineering program (WiE). We also began brainstorming redox sensor applications, including air pollution, water quality, soil chemistry, and medicine.

**Wiki:** Wiki subteam learned about Interaction & Motion during an hour-long Design class. They began working on their Bootstrap and GitHub project due 4/28.

**Business:** Team members edited each other’s sponsorship letters and edited the content for the sponsorship packet.

APRIL 17TH TO APRIL 23RD

**Wet Lab:** Wet lab members trained in transforming their ligated plasmids into competent cells.

**Product Development:** Team members continued to learn how to use Arduino with some example exercises.

**P&P:** P&P did the lab demo and tabling for the WiE program. We finished all the preparation for Splash!.

**Business:** Team members wrote their sponsorship letters and started preparing the sponsorship packet.

APRIL 24TH TO APRIL 30TH

**Wet Lab:** The team members researched oxidative stress and possible applications to pursue.

**P&P:** On April 27th, the team showcased last year’s project, Legendairy, amongst a diverse range of other undergraduate projects at Cornell, including architecture, fashion design, engineering, and agriculture. On April 29th, the team showcased Legendairy at Makerfaire amongst other engineering projects and project teams. On April 29th at Splash!, the team taught local high schoolers about synthetic biology, teaching them how to load a gel and encouraging conversation about bioethics. P&P continued brainstorming applications for the redox sensor and started contacting researchers about our project.

**Wiki:** Wiki subteam successfully submitted their Bootstrap and GitHub project.

**Business:** Team members made final edits to their sponsorship letters, explored mailing tools such as mailchimp, and discussed possibility of obtaining food sponsors.

MAY 1ST TO MAY 7TH

**Product Development:** Product Development discussed possible designs for a redox sensor.

**P&P:** P&P continued following up with contacts regarding an application for the redox sensor. We narrowed down the choices to water quality, beer/wine fermentation, and air pollution.

**Wiki:** Wiki subteam learned the competition rules and coding rules for the iGEM wiki.

**Business:** Team members reached out to old sponsors to update them about our progress and started designing team t-shirts.

MAY 8TH TO MAY 14TH

**Product Development:** Product Development collaborated with the P&P subteam to discuss applications, brainstorm interview questions, and interviewee list.

**P&P:** P&P met with PD to discuss feasibility of the possible applications. We settled on air pollution, specifically tobacco smoke. We started brainstorming potential contacts for this application, from researchers to local Ithacans. P&P also discussed who was in charge of social media pages.

**Business:** Team members discussed summer plans and started preparing for the crowdfunding campaign.

MAY 28TH TO JUNE 3RD

**Product Development:** Product Development participated in the hardware accelerator training program and learned the procedure to conduct customer outreach correctly.

**P&P:** P&P spoke with Dr. Gregory Wood at Frostburg State University to hear about the social impact of smoking. Dr. Wood studied smoking in the workplace throughout the 20th century and social factors that led to its decline to current levels.

JUNE 4TH TO JUNE 7TH

**Product Development:** Product Development conducted interviews with various hydroponic farmers and gathered data on how to build a mechanical component that would be most useful to them.

**P&P:** Together, P&P and Product Development spoke to Dr. Bill Miller at Cornell about the feasibility of our project as well as major aspects and issues with hydroponic agriculture. Furthermore, the team reached out to Rebecca Ireland-Perry at the Cornell Cooperative Extension to learn about hydroponic education in the community, and her interactions with growers, both traditional and hydroponic. Eli at Planted Earth Hydroponics gave team members a better idea of the operations and obstacles of commercial hydroponics. Through Product Development’s work at Rev, the team was able to make contact with PureSpinach, a hydroponic startup aimed at growing hydroponic spinach more efficiently. They spoke to the team about setting up a hydroponic system and their model.

Interviews: Alex Liu (Plant Sciences Undergraduate at Cornell), Gabriel Green (PureSpinach), Kyle Valachovic (PureSpinach), Dr. Bill Miller (Cornell School of Integrative Plant Science), Eli (Planted Earth Hydroponics), Rebecca Ireland-Perry (Cornell Cooperative Extension), Michael (Petal (Rev))

JUNE 8TH TO JUNE 10TH

**P&P:** Product development and P&P continued to interview hydroponic growers. The team received important feedback from them, including their needs. One key theme we found in our interviews was a need for a simple, continuous, real-time monitoring system. Furthermore, they learned that there were several sources of abiotic stress, including excess heat, insects, humidity, and pH fluctuations. They also found that many hydroponic growers were open to testing new methods and technology. The group also reached out to the Rasheed Hislop Community Garden in New York City to consider the ramifications of placing genetically-engineered bacteria in such close proximity to people’s food. While it isn’t common, team members did find that some hydroponic growers, such as those at
Terrapin Farms in Maryland, do use bacteria in their setup to provide benefits to crops.

Interviews: Craig Ellins (10 Mile Hydroponics), Jenny Harris (AmHydro), John McMahon (Schuyler Greens), Cameron Willingham (Vertical Harvest Hydroponics), Chester Bullock (HydroStackers), Jeremy Moon (Fresh Coasts), Kohlie Frantzen (Helical Outpost), Omega Gardens, Terrapin Farms, Rasheed Hislop Community Gardens

**Business:** Subteam lead reached out to crowdfunding campaign organizers and formed a more concrete plan for the campaign.

JUNE 11TH TO JUNE 14TH

**P&P:** Interviews continued, now with a greater diversity of consumers. The team reached out to some traditional and non-traditional hydroponic growers, as well as hydroponic researcher Dr. Neil Mattson at Cornell. Dr. Mattson discussed some of the specific disease problems that arise in hydroponic agriculture as well as the role of oxidative stress in those diseases. Members of product development and P&P also spoke with more hydroponic growers, and they learned that disease can kill hydroponic crops extremely quickly. Many of the non-traditional growers, such as J.J. Reidy at Urban Pastoral, who uses an old shipping container for his setup, had similar complaints about energy usage and monitoring as the traditional hydroponic growers did. Another hydroponic farm, Urban Crop Solutions, told the team about its own use of oxidants such as ozone to sterilize the hydroponic solution.

Interviews: Andrew Blume (Agritecture), Nathaniel Shaw (Karma Farms), Dr. Neil Mattson (School of Integrative Plant Science), ShuShan Valley HydroFarm, Filipous Charalambous (Urban Crop Solutions), Henry (Agritecture), JJ Reidy (Urban Pastoral)

JUNE 15TH TO JUNE 18TH

**Product Development:** Product Development created a list of possible ways to incorporate redox-sensitive bacteria into a hydroponic system. After researching and interviewing nearly 30 farmers and businesses, we decided that a deep-water culture system would work best due to its slow circulation of water.

**P&P:** Interviews continued, now with a greater diversity of consumers. The team reached out to some traditional and non-traditional hydroponic growers, as well as hydroponic researcher Dr. Neil Mattson at Cornell. Dr. Mattson discussed some of the specific disease problems that arise in hydroponic agriculture as well as the role of oxidative stress in those diseases. Members of product development and P&P also spoke with more hydroponic growers, and they learned that disease can kill hydroponic crops extremely quickly. Many of the non-traditional growers, such as J.J. Reidy at Urban Pastoral, who uses an old shipping container for his setup, had similar complaints about energy usage and monitoring as the traditional hydroponic growers did. Another hydroponic farm, Urban Crop Solutions, told the team about its own use of oxidants such as ozone to sterilize the hydroponic solution.

Interviews: Andrew Blume (Agritecture), Nathaniel Shaw (Karma Farms), Dr. Neil Mattson (School of Integrative Plant Science), ShuShan Valley HydroFarm, Filipous Charalambous (Urban Crop Solutions), Henry (Agritecture), JJ Reidy (Urban Pastoral)

**Business:** Subteam lead worked on creating crowdfunding campaign content.

JUNE 19TH TO JUNE 25TH

**Wet Lab:** Team ran PCR reactions to amplify pR and FixK2 promoters as well as Hydrazinase and NADPH gblocks. The team verified these PCR reactions with agarose gel electrophoresis followed by PCR cleanup. Primarily 1.5% agarose gels were prepared. The PCRs for GSH Independent Formaldehyde, 2-Nitropropane Dioxygenase, and L-Asparaginase were not successful.

**Product Development:** Product Development began prototyping the planter cups and assembling the hydroponic tank. Team members also setup the Raspberry Pi and created the first web design mock-ups.

**P&P:** P&P designed a lesson plan for 4H Career Explorations focusing on the the principles of synthetic biology, biology entrepreneurship, and designing a biobrick device. The lesson involved wheat germ DNA extraction using the Building with Biology kit for a hands-on experiment. There was also an interview with Maria Harrison specializing the symbiosis between plants, fungi, and bacteria.

JUNE 26TH TO JULY 2ND

**Wet Lab:** Successfully performed PCR with Tsa2, RxRFP and RoGFP2 gBlocks. Ran failed orp1 PCR. Reran PCR on the GSH-independent formaldehyde dehydrogenase and L-asparaginase gBlocks. Troubleshooting of PCR reactions throughout the week.

**Product Development:** Product Development began their second round of customer discovery. They also began brainstorming ideas for waterproofing the electronics and how to move the sensor around the tank.

**P&P:**  On June 28th and June 29th, the team taught a class for the 4H Career Explorations Program. We focused on the intersection between biology and business, teaching children how to design a plasmid and helping them create a business model for said plasmid. For our hands-on experiment, we extracted DNA from wheat germ.

**Business:** Subteam lead worked on crowdfunding campaign content.

JULY 3RD TO JULY 9TH

**Wet Lab:** Attempted overlap extension PCR to create fusion proteins of promoter and gblock (pR-2ND, for example). Troubleshooting overlap extension PCR and successfully performed PCR on orp1.

**Product Development:** Product Development decided to have a waterproof case to hold the electronics. We made a CAD of a mount to hold the camera and Raspberry Pi.

**P&P:** P&P decided to split up the contacts into categories, such as researchers, legislators, and farmers. We made initial contact with Stony Brook and began discussing a possible collaboration. We started trying to figure out how to build a small hydroponics setup.

**Business:** Team Lead met with Dr. Jocelyn Rose to talk about Biotech Sponsorship.

JULY 10TH TO JULY 16TH

**Wet Lab:** Ligated rxRFP into pSB1C3 vector. Transformed into competent cells and grew on chloramphenicol plates. The bacteria did not grow. Continued troubleshooting overlap extension PCR of promoter and gblocks. (pR - NADPH, Fixk2 - hydrazinase, etc.). Attempted PCR of YF1 and FixJ, was unsuccessful.

**Product Development:** Product Development set-up the server and project configuration for the dashboard.

**P&P:** On July 11th, P&P had a skype call with Stony Brook to discuss collaborations. We decided to create a survey to to assess attitudes towards synthetic biology in Long Island and Ithaca. We began filming our short, educational videos for our YouTube channel.

JULY 17TH TO JULY 23RD

**Wet Lab:** Changed from phusion polymerase to Q5 polymerase. Attempted once again to ligate rxRFP into vector and transformed into competent cells. Succeeded in performing first step of OE PCR for roGFP2-Orp1 and rxRFP-Tsa2. Successfully performed PCR for FixJ and YF1.

**Product Development:** Product Development setup the router, individual pages, profile, and login system for the dashboard.

**P&P:** P&P brainstormed questions for the survey with Stony Brook. We began sorting out logistics for the New York State Fair during the Fall semester and discussed the possibility of attending the World MakerFaire in NYC. P&P discussed goals for the rest of the summer.

**Wiki:** Wiki subteam learned about project and discussed possible designs.

**Business:**  Members proofread content for crowdfunding campaign.

JULY 24TH TO JULY 30TH

**Wet Lab:** Attempted second step of OE PCR for roGFP2-Orp1 and rxRFP-Tsa2. Attempted OE PCR of YF1 and FixJ. When run on a gel, both overlap reactions seemed to produce products and dimerize primers, but no bands were present at the desired fragment lengths. Gels also smeared, producing no distinct bands.

**Product Development:** Product Development learned React to be used for building the UI of the dashboard. PD decided to use servos to rotate the sensors to maximize coverage of the plants.

**P&P:** P&P continued filming videos.

**Wiki:** Wiki subteam presented possible wiki designs for home page and regular page. They decided on a final design for the wiki homepage & standard page with a few tweaks.

**Business:**  Subteam lead added final edits to the crowdfunding campaign. Team members were invited as Ambassadors for the campaign.

JULY 31ST TO AUGUST 6TH

**Wet Lab:** Continued OE PCR troubleshooting with modified PCR protocols and asymmetric PCR before OE PCR of rxRFP-Tsa2. Performed touchdown PCR of Fixk2, hydrazinase, and Tsa2. Separate annealing and extension PCR reactions were performed, yet the samples still formed smears when run on a gel

**Product Development:** Product Development implemented the general layout of the dashboard.

**P&P:** P&P had multiple calls with Stony Brook to finalize the questions on the survey. We made contact with Dr. Sparks regarding the hydroponics application and visited Dr. Turkon at Ithaca College to see her hydroponics set up again. We talked to Dr. Hong from Dr. Roeder’s lab for insight on hydroponics growing solutions.

**Wiki:** Wiki subteam presented final designs for team page & home page and made collaborative edits to those designs.

**Business:**  Team members went through their Ambassador training for the crowdfunding campaign.

AUGUST 7TH TO AUGUST 13TH

**Wet Lab:** Found some success with touchdown PCR for PCR of Hydrazinase gBlock with Q5 Polymerase.

**Product Development:** Product Development worked on a pitch deck to present to Rev, and finalized the content.

**Business:**  Subteam lead added bios for project owners and created levels for crowdfunding campaign.

AUGUST 14TH TO AUGUST 20TH

**Wiki:**  Wiki subteam decided on final designs for the team page, homepage, and standard page.

**Business:**  Subteam lead added finishing touches to the crowdfunding campaign.

AUGUST 21ST TO AUGUST 27TH

**Wet Lab:** Began brainstorming for new approaches for cloning the gBlocks into the plasmids.

**Product Development:** Product Development brainstormed the next steps for the development of the final product.

**P&P:** Policy and Practices worked this week to do one last check on the survey with Stony Brook, and began administration of the survey. We traveled to the Ithaca Farmers’ Market and collected responses from 30 people. We also began planning a public debate with Debates in Science and Health, another student organization on campus.

**Wiki:** Wiki team hosted the first wiki workday! We uploaded pics to the wiki server, added font files to the homepage, transferred homepage to wiki, and began coding the standard page. We finalized delegating assignments for all the wiki writing and then set this week’s writing action items for each subteam. We also met with subteams to assess which writings would be possible to write in the coming weeks based on the progress of the project.

**Business:** The Business Team brainstormed the business plan’s organization, and contacted other subteams about the progress over the summer. Subteam lead got ready for the launch of crowdfunding and had a call with Jon Crenshaw to make any last minute edits to the campaign.

AUGUST 28TH TO SEPTEMBER 3RD

**Wet Lab:** Cloned Orp1 and Superoxide Dismutase into pSB1C3. Began the characterization of a BioBrick, BBa\_K1758335, a lead inducible promoter under the control of a T7 promoter linked to mRFP for detection. Wrote a protocol to obtain fluorescence and colony counts for in vivo testing.

**Product Development:** Product Development made a plan to print the hardware component case and waterproof it. We also started the CAD of the railing system for the hydroponic pond.

**P&P:** P&P tabled at the NY State Fair on Sept 2nd. We gathered more responses for our survey and engaged the public about synthetic biology. We took photos for our campaign, Humans of SynBio, and we brought a small activity for kids to complete.

**Wiki:** Wiki team hosted the second wiki workday! We created all the links to the nav bar and linked them in the nav bar itself. We transferred the homepage to the wiki website and made edits to the homepage. We started coding the notebook page, team page, and standard page. We started designing Policy & Practice website. We set weekly writing action items for each team.

**Business:** The business subteam worked to finalize the first draft of the business plan.

SEPTEMBER 4TH TO SEPTEMBER 10TH

**Wet Lab:** Began the characterization of the lead-inducible promoter BBa\_K1758335 by starting to grow the colonies. Cloned Tsa2

**Product Development:** PD worked on the development for the code for the camera.

**P&P:** The policy and practice team collaborated with organization “Dignity and Justice” on campus and rehearsed the workshop for minority middle schoolers. The workshop will take place on Oct 19th.

**Wiki:** Wiki team finished coding bios page. We started coding sponsors section in bios page and attributions page. We are working on finalizing Policy & Practice page design.

**Business:** The business team used the business canvas model as an interactive visualization of the finalized business plan draft. We also listed potential professors to contact for feedback on our business plan.

SEPTEMBER 11TH TO SEPTEMBER 17TH

**Wet Lab:** Cloned YF1, FixJ, pR, and NADPH gBlocks into pSB1C3. Performed the digestion of Tsa2 gblock with HindiIII and EcoRV and PCRed.

**Product Development:** PD participated in JavaScript training for coding the camera to recognize the intensity level of the light it detects.

**P&P:** The Policy & Practice team started our new outreach event with Ithaca High School. We contacted the AP bio teacher at IHS and started planning our meeting and mentorship workshop with them.

**Wiki:** We added the sponsors to the bios page, finished the attributions page, and finalized the Policy & Practices page design. We checked on Policy & Practices, Product Dev, and Business team’s progress for wiki action items. We sent out weekly action items for all four subteams. We are working on finalizing the standard page, making edits to the fonts, designing the business page, and coding the Notebook page.

**Business:** The business team contacted other team members to ensure the business plan aligns with the progress of each subteam. After getting feedback and edits from the rest of the team, we finalized our business plan and sent it out to professors to receive feedback.

SEPTEMBER 18TH TO SEPTEMBER 24TH

**Wet Lab:** Began germination of basil seeds. Attempted to grow colonies of bacteria transformed with pR and NADPH oxidase.

**Product development:** Product Development team CADed and 3-D printed waterproofing component to house electronics. The software team also worked on code for detecting red light and ignoring any external noise.

**P&P:** We contacted the workshop along with on campus organization “Dignity and Justice” on Oct.19th. The workshop aimed to introduce local high schoolers to synthetic biology and its application and try to inspire them to become bio-engineers.

**Wiki:** We updated the bios page, homepage, and attributions with new styling. We checked in with P&P - they finished most of Public Engagement, but need to work on writing the interviews for Practices. We set a deadline of Oct 15th for Wet Lab Wiki Content. We worked with the business team to finish the business design. We sent weekly action items to each subteam. We will be coding the Notebook page, business page, Safety page, Protocols page, and P&P page and making edits to the bio page, homepage, and attributions page this week. We will also update the nav bar with the right links and updated wiki structure and transfer the standard page to the wiki.

**Business:** The business team received feedback from Professor Callister on the team’s business plan. The business team is starting to edit the business team based on the feedback.

SEPTEMBER 25TH TO OCTOBER 1ST

**Wet Lab:** Began cultures of rxRFP and roGFP2-His. Plated colonies transformed with all of the plasmids that we ordered. Performed a touchdown PCR of L-Asparaginase GBlock. Double digested hydrazinase and ligated it into pSB1C3. Started a liquid culture of roGFP-Orp1 bacteria.

**Product Development:** Product Development CADded the railing system for a proof of concept. The software team continues refining the code for the red light detection.

**P&P:** The policy and practice team had a phone interview with NY Assemblywoman Barbara Lifton to ask her questions on hydroponic regulations.

**Wiki:** The bio page, attributions page, safety page, and protocols page are done. Banner pic was updated on homepage. Navigation bar was updated on all created pages (attributions, bio, safety page). We’re working on the business page, P&P page, About page, Public Engagement page, and gathering banner pics.

**Business:** Team members continued to work on Business plan. The team also explored the option of getting hats as a new team item.

OCTOBER 2ND TO OCTOBER 8TH

**Wet Lab:** Miniprepped rxRFP and roGFP in plasmids from cultures.

**Product Development:** Software team finished up the code for the detection of red light by the camera.

**P&P:** On October 2nd, Policy and Practices team went to Apple Fest at Ithaca Commons and collected more data on the survey collaborating with StonyBrook iGEM team.
Our team also made our first trip to Ithaca High School where we got to know the High School students interested in BioBuilder competition and helped them brainstorm their initial idea.

**Wiki:** We continued developing the business page, P&P page, About page, and Public Engagement page and continued gathering banner pics.

**Business:** Continued refining the business plan and entrepreneurship wiki content. Also came up with more designs for the wiki business page.

OCTOBER 9TH TO OCTOBER 15TH

**Wet Lab:** Biobricking hydrazinase gblock into pSB1C3. Performed a quickchange rxFP to remove the NcoI cut-site and performed PCR to remove the insert. Cut roGFP-orp1 with NcoI and SpeI to get rid of the roGFP2 insert, then ligated and transformed into DH5A.

**Product Development:** Product Development made some edits to the code for the camera and made a video rendering of the hardware components and assembly.

**P&P:** Policy and Practices team began working on data analysis and interpretation of the Stony Brook-Cornell public perception survey.

**Wiki:** Wiki team finished About page. Wiki team worked on wiki fixes to ensure designs is consistent throughout all pages. Wiki team continued to work on P&P page, business page, Public Engagement page (due to in-progress content), and standard page.

OCTOBER 16TH TO OCTOBER 23RD

**Wet Lab:** Characterized the lead-sensitive red fluorescent protein qualitatively by observing the appearance of the cells when grown in media with and without lead. Tested the time and dose dependence of pDawn. Grew a 1L culture of rxRFP-Orp1 in BL21 cells. Grew mRFP for testing the camera, since rxRFP was not expressing strongly enough.

**Product Development:** Developed computer assisted designs for the waterproof box and printed more prototypes.

**P&P:** Policy and Practices team finished up data analysis and interpretation of the survey.

**Wiki:** Wiki team worked on standardizing page styling into 1 CSS style sheet and uploading that to the wiki. Wiki team communicated with other sub teams about writing deadlines, judging form, and gathering other content such as videos and images.

OCTOBER 24TH TO OCTOBER 31ST

**Product Development:** The product development team maintained the hydroponic setup. We worked with the wetlab team to further progress the fluorescing bacteria for testing.

**Wiki:** Wiki team communicated with other subteams to finalize content and transferred content to pages on the wiki.

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# Team:Cornell/Experiments

<!DOCTYPE html>

Wet Lab

[![Oxyponics](https://static.igem.org/mediawiki/2017/1/1d/CornellOxyponicsLogo.png)](https://2017.igem.org/Team%3ACornell)

* [HOME](https://2017.igem.org/Team%3ACornell)
* [ABOUT](https://2017.igem.org/Team%3ACornell/Description)
* TOOLKIT
  + WET LAB
    - [FOUNDATIONS](https://2017.igem.org/Team%3ACornell/Experiments)
    - [DEMONSTRATE](https://2017.igem.org/Team%3ACornell/Demonstrate)
    - [CONTRIBUTION](https://2017.igem.org/Team%3ACornell/Contribution)
    - [PARTS](https://2017.igem.org/Team%3ACornell/Parts)
    - [BASIC PARTS](https://2017.igem.org/Team%3ACornell/Basic_Part)
    - [PROTOCOLS](https://2017.igem.org/Team%3ACornell/Protocol)
  + PRODUCT DEVELOPMENT
    - [DESIGN PROCESS](https://2017.igem.org/Team%3ACornell/Design)
    - [APPLIED DESIGN](https://2017.igem.org/Team%3ACornell/Applied_Design)
    - [SOFTWARE](https://2017.igem.org/Team%3ACornell/Software)
    - [MODELING](https://2017.igem.org/Team%3ACornell/Model)
  + DOCUMENTATION
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* HUMAN CENTERED DESIGN
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* TEAM
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Wet Lab

* [OVERVIEW](#overview)
* [REDOX SENSITIVE FLUORESCENT PROTEINS](#rfp)
* [LIGHT SENSITIVE GENETIC CIRCUITS](#geneticcircuits)
* [FUTURE WORK](#futurework)
* [REFERENCES](#references)

OVERVIEW

To tackle the problem of inconsistent yields in hydroponic farming, we decided to develop a system that would optimize oxidative stress conditions in the growth tanks. Since conventional methods of monitoring and responding to high levels of reactive oxygen species (ROS) are expensive and inefficient, we wanted to design a ROS-level controlling biocircuit centered around a genetically engineered cell that would facilitate detection and response to varying oxidative stress levels in solution. Our solution was an E. coli cell engineered with two novel components: a redox-sensitive fluorescent protein and light-controlled expression of antioxidant proteins. The fluorescent protein’s output signal varies with the ROS concentration in the bacteria’s environment, so the fluorescence can be read and interpreted to determine the oxidative conditions of the growth tank. The light-sensitive promoter can be turned on or off as needed in a non-invasive way to closely control the expression of antioxidant proteins and thus the tank ROS levels. Our bacteria are at the heart of an integrated system that will help farmers monitor and control for a parameter that has not been manipulated to date and which may prove to increase yields across the hydroponics industry.

REDOX SENSITIVE FLUORESCENT PROTEINS

Introduction

The redox environment of a cell is the balance of reductive and oxidative species in the cell. Fluorescent redox probes like roGFP have been developed in order to obtain an accurate real-time measurement. The oxidation of a disulfide bond in roGFP causes a shift in the peak excitation wavelength, producing a ratiometric reading by comparing emission from excitation at the fully-oxidized and fully-reduced peak wavelengths [1]. Ratiometric results allow the signal to be independent of the amount of probe present and are essential in obtaining an accurate, quantitative reading.

In order to have an accompanying variation in signal to a change in redox status, the probe must be partially oxidized and partially reduced in the basal environmental state, somewhat analogous to a pH buffer. For cytosolic and mitochondrial readings, the roGFP-Orp1 fusion protein satisfies this requirement. The addition of Orp1, a reactive oxygen species (ROS) scavenger, also greatly improves the sensitivity of the probe [2,3]. Other fusions with ROS scavengers exist, such as roGFP(iL)-Grx1 which has a higher reducing potential, and roGFP-Tsa2 which has even higher sensitivity than roGFP-Orp1. Our project introduces roGFP2 a variation of roGFP, and rxRFP a redox-sensitive RFP that behaves similarly to roGFP.

![roGFP oxidized](https://static.igem.org/mediawiki/2017/c/cc/T--Cornell--WetLab_roGFP_oxidized.png)

![roGFP reduced](https://static.igem.org/mediawiki/2017/a/af/T--Cornell--WetLab_roGFP_reduced.png)

Redox-sensitive green fluorescent protein (roGFP) is a mutant GFP with two cysteine residues introduced by mutagenesis into the beta-barrel structure. The two cysteines (red) are redox sensors that form a disulfide bridge under oxidizing conditions (left, PDB 1JC1). This leads to a subtle change in the barrel structure and its absorption spectrum compared to the reduced form (right, PDB 1JC0).

Associated BioBricks

Our initial approach to accessing cellular redox processes was to use roGFP2 as a reporter for the external optics system. roGFP2 is a redox-sensitive green fluorescent protein that displays characterizable peaks at 400 nm and 488 nm in its excitation spectra, whose intensities change between the oxidized and reduced states. The external LED system was then used to signal optogenetic plasmids, pDawn and pDusk, which control for gene expression. The complication with this approach is that the pDawn and pDusk are regulated by light of wavelength 470 nm. Due to overlap in the excitation spectra of roGFP2 and those of pDawn and pDusk, we opted for an alternative fluorescent protein. rxRFP is a red, redox-sensitive fluorescent protein which has excitation spectra with peaks at higher wavelengths for both oxidized and reduced states [4,5].

Our fluorescent protein constructs were fused to yeast peroxidases to enhance sensitivity. The yeast peroxidase Orp1, in response to oxidative stress, activates the YAP1 transcription factor to promote the expression of stress response pathways. A peroxidatic cysteine acts as the sensor, while the resolving cysteine acts to form a disulfide bridge [6,7]. The Tsa2 yeast peroxidase functions similarly to Orp1, but the resolving cysteine has been deleted to promote the transfer of sulfenic acid to the fluorescent protein. Similarly, we mutated the resolving cysteine of Orp1 to serine for the same purpose.

![Tsa2](https://static.igem.org/mediawiki/2017/2/27/T--Cornell--WetLab_Tsa2.png)

The yeast peroxidase Tsa2 has two cysteine residues (red) that respond to reactive oxygen species in the environment (PDB 5DVB).

![Peroxidase Mutation](https://static.igem.org/mediawiki/2017/9/95/T--Cornell--WetLab_Peroxidase_Mutation.png)

LIGHT SENSITIVE GENETIC CIRCUITS

Introduction

Optogenetics is a biological technique utilizing light to control cells, typically by genetic regulation. In many organisms, particularly plants, the response to light is crucial for an energetic reaction such as photosynthesis. Phytochromes are proteins central to the plant sensing and response to light and are involved in plant growth and development. Besides phytochromes, plants have a myriad of light-sensitive proteins, many of which can be harnessed with synthetic biology to create optogenetic (light-regulated) circuits [8]. Gene expression that is responsive to light is a versatile effector in the genetic toolkit. In particular, the pDawn/pDusk light activated promoter system features low background noise, ease of use, and reversibility in control of gene expression [9].

Gene expression can be made to be responsive to light with the pDawn and pDusk one-system plasmids. These systems are able to respond with incredible spatial and temporal precision to blue light. In addition, these plasmids are noninvasive and employ bacterial components already present in E.coli. When exposed to blue light, pDusk represses the gene it controls, whereas pDawn reverses the signal polarity of pDusk by employing an additional component to its circuitry. Oxyponics necessarily features optogenetic expression of effectors that can regulate environmental ROS. It is crucial for the function of our project because it allows the oxyponics hardware to directly activate gene expression by light, relaying the oxidative stress levels reported by redox-sensitive fluorescent proteins.

Associated BioBricks

In order to target the optimal level of oxidative stress for plant growth, our platform uses redox-sensitive fluorescent proteins as a reporter to couple an external optics system to an intracellular optogenetic transcriptional circuit. Our team assembled two plasmids that allow for light-sensitive gene expression. The pDusk construct allows for transcription in the absence of light.

This system takes advantage of the YF1 and FixJ. YF1 is a histidine kinase that, in the absence of blue light, phosphorylates the regulator FixJ, which proceeds to enhance the expression of genes downstream of the FixK2 promoter.

![PDawn](https://static.igem.org/mediawiki/2017/e/e5/PDawn.png)

The pDawn system adds onto the pDusk system by incorporating the lambda repressor downstream of the FixK2 promoter. This repressor exerts an inhibitory effect on the transcription of genes downstream of the pR promoter.

![PDUSK](https://static.igem.org/mediawiki/2017/f/f6/PDUSK.png)

We have a panel of FLAG-tagged enzymes whose expressions are regulated by pDawn and pDusk. These enzymes include superoxide dismutase, 2-nitropropane dioxygenase, glutathione-independent formaldehyde dehydrogenase, hydrazinase, L-asparaginase 2, and NADPH-dehydrogenase. These enzymes break down many of the harmful chemicals that trigger the formation of reactive oxygen species [10,11].

Results

We can characterize pDawn and pDusk by Western blot. By growing E. coli under different intensities of visible light, we can compare the relative expression levels of a FLAG-tagged superoxide dismutase under optogenetic control.

We first inserted superoxide dismutase into the pDusk circuit. We then proceeded to incorporate a cI repressor (BBa\_K2296045) and a pR promoter into the pDusk system followed by insertion of superoxide dismutase to create a light-inducible pDawn circuit.

We assessed the transcriptional activity of pDawn and pDusk in response to three different levels of light intensity from a standard laboratory fluorescent light:

![DoseResponse](https://static.igem.org/mediawiki/2017/3/31/T--Cornell--WetLab_DoseResponse.png)

Our initial characterization revealed a dose-dependence of pDusk. However, pDawn did not display a dose-dependent response in response to stimulation from fluorescent light. We suspect this to be the result of low light intensity.

Our subsequent characterization uses a significantly greater intensity of LED light:

![pDawnpDuskExpression](https://static.igem.org/mediawiki/2017/5/59/T--Cornell--WetLab_pDawnpDuskExpression.png)

Our results suggest a light-intensity dependence of pDusk and pDawn. Our results also show that our cI repressor biobrick successfully converted the light-repressible pDusk into a light-inducible pDawn.

In addition to dose-dependent characterization, we also conducted time-dependence characterization of pDawn following stimulation by 470 nm LED light (LTL3H3TBPADS1) with the following emission spectrum [12]:

![470LEDAbsSpec](https://static.igem.org/mediawiki/2017/6/6e/470LEDAbsSpec.png)

Our initial characterization was conducted with 1 second pulses and revealed a significant lag between initial exposure and translational response:

![pDawnTimeDependence](https://static.igem.org/mediawiki/2017/a/a3/T--Cornell--WetLab_pDawnTimeDependence.png)

For our subsequent characterization, we increased the intensity and pulse duration of the LED to parse out more subtle effects at earlier time points:

![pDawnTimeDependenceGraph](https://static.igem.org/mediawiki/2017/0/03/T--Cornell--WetLab_pDawnTimeDependenceGraph.png)

Our results suggest a light intensity and time-dependence of the pDawn and pDusk optogenetic circuit.

FUTURE WORK

Our system was designed with the future in mind. The pDawn/pDusk system gives researchers the power to arbitrarily modify any environmental parameter. Our initial brainstorming session also indicated that our system could be applied to water improvement, soil quality, and remediation, as heavy metals can induce oxidative stress in bacteria[13].

Although our hydroponic system is safe and there are mechanisms to contain the E. coli, some of the farmers we spoke with mentioned concerns over the use of bacteria. Therefore, another key area for future development, specifically within the scope of hydroponics and agriculture, would be to adapt our fluorescent protein genetic constructs into different expression systems, such as those for yeast (S. cerevisiae) or other naturally-occurring symbiotic bacteria that lack the stigma associated with E. coli in food production. Furthermore, we found that there exist potential applications of an oxidative stress sensor in brewing, to which yeast that can report on the environmental redox state can prove to be extremely useful and acceptable.

Fundamentally, our construct can be used to make hardier bacteria for both aerobic and anaerobic cultures. As a result, engineered bacterial circuits can allow bacteria to survive in unprecedented high-stress laboratory conditions. Our construct will also be foundational to modifying plant and other eukaryotic cells to be more resistant to oxidative stress.

REFERENCES

1. Cannon, M. B., & Remington, S. J. (2006). Re-engineering redox-sensitive green fluorescent protein for improved response rate. Protein Science, 15(1), 45-57. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242357/.
2. Delaunay, A., Pflieger, D., Barrault, M., Vinh, J., Toledano, M.B. (2002). A Thiol Peroxidase is an H2O2 Receptor and Redox-Transducer in Gene Activation. Cell, 111: 471- 481.
3. Ma, L., Takanishi, C. L., & Wood, M. J. (2007). Molecular Mechanism of Oxidative Stress Perception by the Orp1 Protein. Journal of Biological Chemistry, 282.(43), 31429-31436. Retrieved from http://www.jbc.org/content/282/43/31429.long
4. Fan, Y., Chen, Z., & Ai, H. (2015, February 10). Monitoring Redox Dynamics in Living Cells with a Redox-Sensitive Red Fluorescent Protein. Anal. Chem., 2015, 87 (5), pp 2802–2810. Retrieved from http://pubs.acs.org/doi/abs/10.1021/ac5041988
5. Ren, W., Ai, H. (2013). Genetically Encoded Fluorescent Redox Probes. Sensors, 13: 15422-15433.
6. Gutscher, M., Sobotta, M. C., Wabnitz, G. H., Ballikaya, S., Meyer, A. J., Samstag, Y., & Dick, T. P. (2009). Proximity-based Protein Thiol Oxidation by H2O2-scavenging Peroxidases. Journal of Biological Chemistry,284(46), 31532-31540. Retrieved from http://www.jbc.org/content/284/46/31532.long
7. Morgan, B., Van Laer, K., Owusu, T.N.E., Ezerina, D., Pastor-Flores, D., Amponsah, P.S., Tursch, A., Dick, T.P. (2016). Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes. Nature Chemical Biology 12: 437-445
8. Tabor, J.J., Levskaya, A., Voigt, C.A. (2011). Multichromatic control of gene expression in Escherichia coli. J Mol Biol 405(2): 315-324.
9. Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K., & Moglich, A. (2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. Journal of Molecular Biology, 416,  534-542.
10. Birben, E., Sahiner, U.M., Sackesen, C., Erzurum, S., Kalayci, O. (2012). Oxidative Stress and Antioxidant Defense. WAO Journal,  9-19.
11. Mukhopadhyay, S., Schellhorn, H.E. (1997). Identification and Characterization of Hydrogen Peroxide-Sensitive Mutants of Escherichia coli: Genes That Require OxyR for Expression. Journal of Bacteriology,  179(2): 330-338.
12. Lite-On Technology Corporation. Information Data Sheet for LED Lamp LTL3H3TBPADS1-132A. Retrieved from http://www.mouser.com/ds/2/239/Lite-On\_LTL3H3TBPADS1-132A-Ver.A-341105.pdf

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# Team:Cornell/Results

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Wet Lab

[![Oxyponics](https://static.igem.org/mediawiki/2017/1/1d/CornellOxyponicsLogo.png)](https://2017.igem.org/Team%3ACornell)

* [HOME](https://2017.igem.org/Team%3ACornell)
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Wet Lab

* [OVERVIEW](#overview)
* [REDOX-SENSITIVE FLUORESCENT PROTEINS](#rfp)
* [LIGHT-SENSITIVE GENETIC CIRCUITS](#geneticcircuits)
* [FUTURE WORK](#futurework)
* [REFERENCES](#references)

OVERVIEW

To tackle the problem of inconsistent yields in hydroponic farming, we decided to develop a system that would optimize oxidative stress conditions in the growth tanks. Since conventional methods of monitoring and responding to high levels of reactive oxygen species (ROS) are expensive and inefficient, we wanted to design a ROS-level controlling biocircuit centered around a genetically engineered cell that would facilitate detection and response to varying oxidative stress levels in solution. Our solution was an E. coli cell engineered with two novel components: a redox-sensitive fluorescent protein and light-controlled expression of antioxidant proteins. The fluorescent protein’s output signal varies with the ROS concentration in the bacteria’s environment, so the fluorescence can be read and interpreted to determine the oxidative conditions of the growth tank. The light-sensitive promoter can be turned on or off as needed in a non-invasive way to closely control the expression of antioxidant proteins and thus the tank ROS levels. Our bacteria are at the heart of an integrated system that will help farmers monitor and control for a parameter that has not been manipulated to date and which may prove to increase yields across the hydroponics industry.

REDOX-SENSITIVE FLUORESCENT PROTEINS

Introduction

The redox environment of a cell is the balance of reductive and oxidative species in the cell. Fluorescent redox probes like the green fluorescent protein, roGFP have been developed in order to obtain an accurate real-time measurement. The oxidation of a disulfide bond in roGFP causes a shift in the peak excitation wavelength, allowing a ratiometric reading by comparing emission to excitation at the fully-oxidized and fully-reduced peak wavelengths [1]. Ratiometric results allow the signal to be independent of the amount of probe present and are essential in obtaining an accurate, quantitative reading.

To respond to changes in oxidative stress, a probe must have both an oxidized and reduced form that exist in an equilibrium that shifts depending on the oxidative environment (in that sense, redox-sensitive fluorescent probes are analogous to pH-dependent dyes). The roGFP-Orp1 fusion protein satisfies this requirement, making it appropriate for cytosolic and mitochondrial redox level readings. The addition of Orp1--a reactive oxygen species (ROS) scavenger--to roGFP greatly improves the sensitivity of the probe [2,3]. Other fusions with ROS scavengers exist, such as roGFP(iL)-Grx1 which has a higher reducing potential, and roGFP-Tsa2 which has even higher sensitivity than roGFP-Orp1. Our project introduces roGFP2, a variation of roGFP, and rxRFP, a redox-sensitive red fluorescent protein that behaves similarly to roGFP.

![roGFP oxidized](https://static.igem.org/mediawiki/2017/c/cc/T--Cornell--WetLab_roGFP_oxidized.png)

![roGFP reduced](https://static.igem.org/mediawiki/2017/a/af/T--Cornell--WetLab_roGFP_reduced.png)

Redox-sensitive green fluorescent protein (roGFP) is a mutant GFP with two cysteine residues introduced by mutagenesis into the beta-barrel structure. The two cysteines (red) are redox sensors that form a disulfide bridge under oxidizing conditions (left, PDB 1JC1). This leads to a subtle change in the barrel structure and its absorption spectrum compared to the reduced form (right, PDB 1JC0).

Associated BioBricks

Our initial approach to studying cellular redox processes was to use roGFP2 to signal to the external optics system. roGFP2 is a redox-sensitive green fluorescent protein that displays characterizable peaks at 400 nm and 488 nm in its excitation spectra, whose intensities change between the oxidized and reduced states. The external LED system was then used to signal optogenetic plasmids, pDawn and pDusk, which control for gene expression. The complication with this approach is that the pDawn and pDusk are regulated by light of wavelength 470 nm. Due to overlap in the excitation spectra of roGFP2 and those of pDawn and pDusk, we opted for an alternative fluorescent protein. rxRFP is a red, redox-sensitive fluorescent protein which has excitation spectra with peaks at higher wavelengths for both oxidized and reduced states, so they do not interfere with the pDawn and pDusk pathways [4,5].

Our fluorescent protein constructs were fused to yeast peroxidases to enhance sensitivity. The yeast peroxidase Orp1, in response to oxidative stress, activates the YAP1 transcription factor to promote the expression of stress response pathways. A peroxidatic cysteine (CP) acts as the sensor, while the resolving cysteine (CR) acts to form a disulfide bridge [6,7]. The Tsa2 yeast peroxidase functions similarly to Orp1, but the resolving cysteine has been deleted to promote the transfer of sulfenic acid to the fluorescent protein. Similarly, we mutated the resolving cysteine of Orp1 to serine for the same purpose.

![Tsa2](https://static.igem.org/mediawiki/2017/2/27/T--Cornell--WetLab_Tsa2.png)

The yeast peroxidase Tsa2 has two cysteine residues (red) that respond to reactive oxygen species in the environment (PDB 5DVB).

![Peroxidase Mutation](https://static.igem.org/mediawiki/2017/9/95/T--Cornell--WetLab_Peroxidase_Mutation.png)

LIGHT-SENSITIVE GENETIC CIRCUITS

Introduction

Optogenetics is a biological technique utilizing light to control cells, typically by genetic regulation. In many organisms, particularly plants, the response to light is crucial for an energetic reaction such as photosynthesis. Phytochromes are proteins central to the plant sensing and response to light and are involved in plant growth and development. Besides phytochromes, plants have a myriad of light-sensitive proteins, many of which can be harnessed with synthetic biology to create optogenetic (light-regulated) circuits [8]. Gene expression that is responsive to light is a versatile effector in the genetic toolkit. In particular, the pDawn/pDusk light activated promoter system features low background noise, ease of use, and reversibility in control of gene expression [9].

Gene expression can be made to be responsive to light with the pDawn and pDusk one-system plasmids. These systems are able to respond with incredible spatial and temporal precision to blue light. In addition, these plasmids are noninvasive and employ bacterial components already present in E.coli. When exposed to blue light, pDusk represses the gene it controls, whereas pDawn reverses the signal polarity of pDusk by employing an additional component to its circuitry. Oxyponics necessarily features optogenetic expression of effectors that can regulate environmental ROS. It is crucial for the function of our project because it allows the oxyponics hardware to directly activate gene expression by light, relaying the oxidative stress levels reported by redox-sensitive fluorescent proteins.

Associated BioBricks

In order to target the optimal level of oxidative stress for plant growth, our platform uses redox-sensitive fluorescent proteins as a reporter to couple an external optics system to an intracellular optogenetic transcriptional circuit. Our team assembled two plasmids that allow for light-sensitive gene expression. The pDusk construct allows for transcription in the absence of light.

This system takes advantage of the YF1 and FixJ. YF1 is a histidine kinase that, in the absence of blue light, phosphorylates the regulator FixJ, which proceeds to enhance the expression of genes downstream of the FixK2 promoter.

![PDawn](https://static.igem.org/mediawiki/2017/e/e5/PDawn.png)

The pDawn system adds onto the pDusk system by incorporating the lambda repressor downstream of the FixK2 promoter. This repressor exerts an inhibitory effect on the transcription of genes downstream of the pR promoter.

![PDUSK](https://static.igem.org/mediawiki/2017/f/f6/PDUSK.png)

We have a panel of FLAG-tagged enzymes whose expressions are regulated by pDawn and pDusk. These enzymes include superoxide dismutase, 2-nitropropane dioxygenase, glutathione-independent formaldehyde dehydrogenase, hydrazinase, L-asparaginase 2, and NADPH-dehydrogenase. These enzymes break down many of the harmful chemicals that trigger the formation of reactive oxygen species [10,11].

Results

We can characterize pDawn and pDusk by Western blot. By growing E. coli under different intensities of visible light, we can compare the relative expression levels of a FLAG-tagged superoxide dismutase under optogenetic control.

We first inserted superoxide dismutase into the pDusk circuit. We then proceeded to incorporate a cI repressor (BBa\_K2296045) and a pR promoter into the pDusk system followed by insertion of superoxide dismutase to create a light-inducible pDawn circuit.

We assessed the transcriptional activity of pDawn and pDusk in response to three different levels of light intensity from a standard laboratory fluorescent light:

![DoseResponse](https://static.igem.org/mediawiki/2017/3/31/T--Cornell--WetLab_DoseResponse.png)

Our initial characterization revealed a dose-dependence of pDusk. However, pDawn did not display a dose-dependent response in response to stimulation from fluorescent light. We suspect this to be the result of low light intensity.

Our subsequent characterization uses a significantly greater intensity of LED light:

![pDawnpDuskExpression](https://static.igem.org/mediawiki/2017/5/59/T--Cornell--WetLab_pDawnpDuskExpression.png)

Our results suggest a light-intensity dependence of pDusk and pDawn. Our results also show that our cI repressor biobrick successfully converted the light-repressible pDusk into a light-inducible pDawn.

In addition to dose-dependent characterization, we also conducted time-dependence characterization of pDawn following stimulation by 470 nm LED light (LTL3H3TBPADS1) with the following emission spectrum [12]:

![470LEDAbsSpec](https://static.igem.org/mediawiki/2017/6/6e/470LEDAbsSpec.png)

Our initial characterization was conducted with 1 second pulses and revealed a significant lag between initial exposure and translational response:

![pDawnTimeDependence](https://static.igem.org/mediawiki/2017/a/a3/T--Cornell--WetLab_pDawnTimeDependence.png)

For our subsequent characterization, we increased the intensity and pulse duration of the LED to parse out more subtle effects at earlier time points:

![pDawnTimeDependenceGraph](https://static.igem.org/mediawiki/2017/0/03/T--Cornell--WetLab_pDawnTimeDependenceGraph.png)

Our results suggest a light intensity and time-dependence of the pDawn and pDusk optogenetic circuit.

FUTURE WORK

Our system was designed with the future in mind. The pDawn/pDusk system gives researchers the power to arbitrarily modify any environmental parameter. Our initial brainstorming session also indicated that our system could be applied to water improvement, soil quality, and remediation, as heavy metals can induce oxidative stress in bacteria[13].

Although our hydroponic system is safe and there are mechanisms to contain the E. coli, some of the farmers we spoke with mentioned concerns over the use of bacteria. Therefore, another key area for future development, specifically within the scope of hydroponics and agriculture, would be to adapt our fluorescent protein genetic constructs into different expression systems, such as those for yeast (S. cerevisiae) or other naturally-occurring symbiotic bacteria that lack the stigma associated with E. coli in food production. Furthermore, we found that there exist potential applications of an oxidative stress sensor in brewing, to which yeast that can report on the environmental redox state can prove to be extremely useful and acceptable.

Fundamentally, our construct can be used to make hardier bacteria for both aerobic and anaerobic cultures. As a result, engineered bacterial circuits can allow bacteria to survive in unprecedented high-stress laboratory conditions. Our construct will also be foundational to modifying plant and other eukaryotic cells to be more resistant to oxidative stress.

REFERENCES

1. Cannon, M. B., & Remington, S. J. (2006). Re-engineering redox-sensitive green fluorescent protein for improved response rate. Protein Science, 15(1), 45-57. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242357/.
2. Delaunay, A., Pflieger, D., Barrault, M., Vinh, J., Toledano, M.B. (2002). A Thiol Peroxidase is an H2O2 Receptor and Redox-Transducer in Gene Activation. Cell, 111: 471- 481.
3. Ma, L., Takanishi, C. L., & Wood, M. J. (2007). Molecular Mechanism of Oxidative Stress Perception by the Orp1 Protein. Journal of Biological Chemistry, 282.(43), 31429-31436. Retrieved from http://www.jbc.org/content/282/43/31429.long
4. Fan, Y., Chen, Z., & Ai, H. (2015, February 10). Monitoring Redox Dynamics in Living Cells with a Redox-Sensitive Red Fluorescent Protein. Anal. Chem., 2015, 87 (5), pp 2802–2810. Retrieved from http://pubs.acs.org/doi/abs/10.1021/ac5041988
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