iGEM 2024 — Team cornell

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[February](#feb)
[March](#mar)
[April](#apr)
[May](#may)
[June](#june)
[July](#july)
[August](#aug)
[September](#sep)
[October](#oct)

# February 2024

### February 4th to February 10th

**Wet Lab**: New members had a meeting to learn and discuss PCR. New members set up and ran PCR reactions in the lab with guidance. New members had a meeting to learn and discuss lab techniques including DNA cleanup, nanodrop, and transformation.

**Product Development**: Preparing to start work for the season.

**Policy and Practices**: Preparing to start work for the season.

**Business**: New members analyzed last year's winner of Best Supporting Entrepreneurship, Tec-Chihuahua AureoBus in terms of their strengths and weaknesses. The goals of this assignment were to learn from a high-quality example of the kind of work that the Cornell iGEM business subteam should be aiming for, and what things we should or should not do with our market research, corporate sponsorship, etc. New members organized all the key takeaways from the AureoBus project into a Google Slides presentation and presented it to the subteam during our weekly meeting.

**Wiki**: New members had a meeting to learn the basics of HTML, CSS, and GitHub. New members discussed wiki requirements and expectations for the brainstorming process.

### February 11th to February 17th

**Wet Lab**: New members set up and performed DNA cleanup, nanodrop, and gel electrophoresis in the lab with guidance.

**Product Development**: Subteam members researched brainstorming topics, completed ECE training, and considered the ethics, audience, and applications of their brainstorming topics. Subteam members signed up for coffee chats with team leadership.

**Policy and Practices**: The subteam began brainstorming, and discussing the different facets of the projects that should be addressed, including ethics, policy analysis and outreach events to better share and understand public perceptions. New Members were given their third assignment related to brainstorming local outreach events and different ways to engage with both the local and broader audience in an inclusive and directed manner. Old members analyzed overarching ethical dilemmas related to different fields of science that synthetic biology touches to get a better understanding of the varied discussions within each field.

**Business**: This week in the business subteam we went over market analysis and industry evaluation along with segmentation. This week the new members prepared a slideshow for their project proposals for team brainstorming.

**Wiki**: New members presented their first assignment: creating a wiki page about themselves using HTML and CSS. They learned the basics of Figma and were introduced to the second training project: creating mockups of existing websites using Figma.

### February 18th to February 24th

**Wet Lab**: Wetlab leads hosted office hours to talk about WL portions of brainstorming. New members completed the Weill Hall Access Form along with all the training required for it.

**Product Development**: Subteam members considered the business portions of their brainstorming projects and familiarized themselves with iGEM judging criteria. New subteam members completed another ECE training assignment. Subteam members attended a team social event. The whole team took team photos following a weekly meeting. Subteam members attended both a small group session for brainstorming and the aforementioned coffee chats with team leadership.

**Policy and Practices**: The subteam refined key ideas from impactful projects and expanded upon policy-based guidance for particular projects.

**Business**: Began brainstorming for round 2 of brainstorming presentations. Second Business assignment: Google doc detailing what the steps would be for each of the 4 specific forms of IP as the new members try to get them for their projects. Also went into which one, licensing or franchising, works better and went into detail about specifics.

**Wiki**: New members continued work on brainstorming assignments. Members began determining design features for each idea, such as color palettes and logos. New members continued work on the second training assignment.

# March 2024

### February 25th to March 2nd

**Wet Lab**: New members had a meeting to learn and discuss sequence design.

**Product Development**: Subteam members had mid-winter break, continued to work on brainstorming projects in the interim.

**Policy and Practices**: No specific subteam meeting was held due to mid-winter break. PnP members were encouraged to continue working on their brainstorming analysis, as well as continue thinking about different policies and other human practice related work that would affect the overarching projects.

**Business**: Learned about market analysis in the business meeting (TAM, SAM, SOM) and introduction to PEST(LE) analysis. Learned about CapIQ software and IBISWorld software and how it can be used to analyze specific industries or companies that are competitors

**Wiki**: New members presented the second training assignment and gave each other feedback. The first part of the third and final training assignment was introduced. Members were asked to being researching UI/UX features and JavaScript animations.

### March 3rd to March 9th

**Wet Lab**: Wetlab leads hosted office hours to talk about WL portions of brainstorming. New members completed a procedural design assignment and discussed it during the wet lab meeting.

**Product Development**: Subteam members focused on the PD aspects of remaining brainstorming projects. Subteam members began attending weekly subteam-specific meetings.

**Policy and Practices**: PnP new member education was completed and future subteam meeting organization were outlined for future reference. An initial interview was completed with Dr. Mikail Abbasov, an organic chemistry professor, who encouraged Oncurex as a novel use of Ursolic Acid and a potential application to cancer. An interview was completed with Dr. Wong, a PhD student studying chemical synthesis. He stated he believes that producing ursolic acid using synthetic biology was an extremely valuable potential process to address cost disparities. Members began developing ideas and topics for outreach at Splash!, including different applications of synthetic biology, creating a mock iGEM project team and hosting an ethical dilemma debate. Members continued thinking about different ways to reach a wider audience, and completed an assignment related to social media and virtual based outreach.

**Business**: All new members worked on market analysis business assignment for the week (TAM, SAM, SOM, and PEST(LE)) analysis of a specific biotech industry

**Wiki**: Members presented their third projects and received feedback from one another.

### March 10th to March 16th

**Wet Lab**: Members completed a sequence design assignment and discussed it during the wet lab meeting. Members took feedback and made revisions to the procedural design assignment. Members set up bacterial plates and perform transformations in the lab with guidance.

**Product Development**: Brainstorming continued - subteam members focused on PD aspects of various brainstorming projects.

**Policy and Practices**: Brainstorming was advanced, and interviews were discussed. PnP members began reaching out to interviewees for their assigned brainstorming ideas. An interview was completed with Dr. Kevin Siegenthaler, who elaborated upon the use of yeast as a potential model organism for ursolic acid, and that lipid extraction would be a likely pathway for extraction. Members also continued working on the conceptual ideas behind the brainstorming project as well, and analyzed next steps and stakeholder interests. Splash! Outreach event was set on April 27th. This consists of explaining different layers of synthetic biology research through the layout of Cornell iGEM’s subteam set up. members focused on making different presentations representing different facets of iGEM.

**Business**: This week the subteam worked on SWOT analysis and Porter’s 5 forces. We did this as practice with chosen biotech industries in the subteam and used this knowledge to develop our respective brainstorming projects. The business plans of the projects we are brainstorming are becoming more fully developed, which will help us when it’s time to create the entire business plan for the chosen project.

**Wiki**: New members were introduced to the second part of the third training assignment. Each member was asked to choose two of the animations they found during their previous research to implement on a wiki page from last year's competition wiki.

### March 17th to March 23rd

**Wet Lab**: Members had a meeting to learn and discuss lab techniques including colony PCR and Gibson assembly. Members conducted research on ursolic acid optimization and LNP production.

**Product Development**: Brainstorming continued - subteam members remained focused on PD aspects of the brainstorming project. Subteam members registered for the iGEM jamboree.

**Policy and Practices**: Interviewees were reached out to based on the respective project ideas in brainstorming. Each project in the brainstorming was discussed among the team for strengths and weaknesses per project, as well as the role PnP plays in each project. Research for presentations were started for Splash! and compiled for future use. Members also attended training sessions in preparation for Splash!, which included how to work with minors training, boundary training and emergency training.

**Business**: Members registered for the iGEM jamboree. Continued work on market analysis from last week

**Wiki**: New members continued work on the third training assignment. New members were introduced to the logo design project, in which they would work with a partner to create two designs for the winning project.

### March 24th to March 30th

**Wet Lab**: Members took feedback and made revisions to the sequence design assignment. Members also began modeling kinetics involved in the MVA pathway and production of UA to find the best variant of alpha amyrin synthase (AAS) and which gene to knock out for improving the yield of ursolic acid.

**Product Development**: Brainstorming continued - subteam members remained focused on PD aspects of the brainstorming project.

**Policy and Practices**: As brainstorming was completed and Oncurex was determined to be the final project, PnP continued to analyze the role stakeholders and human practices as a whole played within the project. Research was broken up into three distinct parts: the technical details behind the project, the ethics and legislation behind regulations, and overall ursolic acid details. The primary concern for PnP is understanding the role Ursolic Acid plays in different sects of medicine. Preliminary research indicated important potential in anti-cancer treatments. Interviewees were divided among PnP members with the goal of understanding the role of ursolic acid in medicine from a stakeholder perspective. An interview was completed with Dr. Agnes Slawska, who emphasized the need for ethics and pointed us to look into the specific potential Ursolic Acid has in cancer treatments as the possible main application. Presentations were planned for CUrself as an Engineer, a program encouraging highschoolers in engineering fields and the MIT Future of Biology Conference, a conference dedicated to speaking to a larger public about the role biology plays in daily life.

**Business**: Meeting was asynchronous this week as members continued to work on their market analysis projects

**Wiki**: Members worked on the third training assignment and held a virtual work session for the logo design project.

# April 2024

### March 31st to April 6th

**Wet Lab**: Subteam members were on Spring Break, so no wet lab activities took place.

**Product Development**: Subteam members were on Spring Break, so no product development activities took place.

**Policy and Practices**: Members planned outreach for the Cornell BME Banquet, focusing on the role of human practices in project creation.

**Business**: Subteam members were on Spring Break, so no business activities took place.

**Wiki**: Subteam members were on Spring Break, so no wiki activities took place.

### April 7th to April 13th

**Wet Lab**: Members took a couple days to update inventory for the Weill Hall lab space. Members completed the stochastic model, and both sensitivity analysis and graph theory models were further developed.

**Product Development**: PnP was on Spring break, members continued to reach out to stakeholders based on Dr. Slawska's guidance on anti-cancer potential. Presented an overview of bioreactor designs to the team at the weekly meeting. Created and presented a poster for the BME Banquet that walked through the organization, practices, and goals of iGEM PD.

**Policy and Practices**: Members continued reaching out to stakeholders to understand the potential of the project. PnP considered the various facets of human practices that needed to be considered from stakeholders as well, and specifically updated our stakeholder engagement to cover fields in cancer treatments, technical details, and overall ursolic acid use based upon our research.Members discussed medal criteria and the research done into this year's project. The poster was finalized for the BME Banquet, focused on the work PnP and human practices as a whole completes within the iGEM framework. An engagement event was set up at Makerfaire, and was discussed for possible outreach and understanding of the project with the greater New York community.

**Business**: The subteam got ready for giving day. Practiced mail merge by sending a test message to the other members and prepared a list of alumni to reach out to for fundraising

**Wiki**: Members presented the third training project and gave each other feedback. Members presented their logo designs and a voting form was sent out to all team members to choose the final logo. Members were introduced to the banner design and merch design assignments. Members would again be working in pairs, but with a different partner for each.

### April 14th to April 20th

**Wet Lab**: Members hosted a CUrself as an Engineer event for high schoolers curious in syn bio. Members completed the stochastic model, and both sensitivity analysis and graph theory models were further developed.

**Product Development**: Subteam members looked further into the parts needed to build a CSTR reactor. PD moved into a new location on campus – subteam members spent most of their time this week packing / moving equipment and materials between the two. Parts list needed for CSTR reactor assembled to make future purchasing easier.

**Policy and Practices**: Stakeholder outreach was continued based on Dr. Slawska’s feedback and the technical integrations of the project. Due to a lack of response from stakeholders, stakeholder outreach was reassessed. Emails and surveys that were initially sent out were reviewed for possible discrepancies, and updated to include new technical and stakeholder information. Splash! presentations were finalized and prepared for presentation. Members completed specific presentations each related to a subteam on Cornell iGEM Members presented at the BME Banquet, speaking to the Cornell community emphasizing the importance of continuous engagement and feedback of synthetic biology with society. Some members of PnP presented at the CUself as an Engineer program ran by Cornell encouraging high schoolers in engineering. The students were extremely interested in the the vast interdisciplinary potential of synthetic biology, and many showed interest in synthetic biology and joining iGEM as a program. Other members presented at the MIT Future of Biology Conference about the purpose of synthetic biology in iGEM. Many questions were focused on the last potential of iGEM, and students were walked through how to conceptualize their own project from the technical details to societal perspective

**Business**: Finished applying for the Cultivarium grant due 4/18. Attended BME banquet and helped run iGEM business stand for 30 mins. Continued organizing and assembling the grants list document. The IP group made a list of what we think of each other's sub-teams IP that needs to be protected and we will file for them accordingly soon.

**Wiki**: Members presented their banner designs and gave each other feedback. A voting form was sent out to all team members to choose the final banner. Members continued work on the merch design assignment.

### April 21st to April 27th

**Wet Lab**: New members performed calculations for a sample Gibson Assembly and set up the reactions in the lab with guidance. Members worked on a Gibson Assembly worksheet to complement the training. Members completed the stochastic model, and both sensitivity analysis and graph theory models were further developed. Members also found that the MdOSC class of AAS enzymes would have the highest activity compared to alternatives.

**Product Development**: Subteam began working on schematic for our reactor design – each member created a sketch and a final draft was created from the best parts of each.

**Policy and Practices**: An interview was completed with Dr. Matt DeLisa, who works with molecular mechanisms of protein synthesis and bioreactor structure. Dr. DeLisa gave key advice into both wet lab and product development of the project in terms of bioreactor modification, and plant byproduct formation from synthetic biology. An interview was completed with Dr. Shaoyi Jiang, who works with yeast and lipid nanoparticles. Contrasting Dr. Siegenthaler’s initial recommendation, Dr. Jiang stated the difficulties of lipid nanoparticles in our specific system, and suggested we look into the use of yeast extracellular vesicles as another pathway. Members of iGEM presented at Splash! on each subteam in Cornell iGEM. Various questions were posed to students on different fields of synthetic biology, and students created their own projects based on their interests. PnP developed infographics and flyers on three main topics for social media and Maker Faire, focused on explaining Oncurex and synthetic biology in an easy to digest manner.

**Business**: Krispy Kreme fundraiser on 4/22. Made progress towards filing the patent for the integrated bioreactor/biosensor. Applied for the Promega Grant

**Wiki**: Members presented their merch designs and gave each other feedback. A voting form was sent out to all team members to choose the final merch design.

# May 2024

### April 28th to May 4th

**Wet Lab**: Both stochastic and sensitivity models were completed, and it was concluded that knocking POR2 will lead to the highest yield of UA.

**Product Development**: Subteam looked into designing and implementing a cartridge-based sensor system, but decided against it as the idea was deemed impractical.

**Policy and Practices**: Dr. David Specht, a researcher focused on Vibrio Natriegens manipulation, was interviewed regarding our model organism and difficulties with PCR and Gibson Assembly in our project. Members of iGEM presented at Maker Faire in Syracuse, a large scale conference showcasing projects around the NY area. It was an incredibly valuable experience speaking with members of the general public from professors to students about our project. PnP held a large scale stakeholder focus meeting based on discussing next steps for stakeholder engagement and outreach.

**Business**: Applied for a provisional patent. Prepared a new sponsorship slide deck and prepared a new sponsorship packet in collaboration with Wiki

**Wiki**: Members discussed the summer timeline and expectations.

### May 5th to May 11th

**Wet Lab**: Leads discussed methods for improving yield of ursolic acid as well as methods for quantifying the yield. We decided on knocking out POR2, but low cost methods for ursolic acid quantification were not found.

**Product Development**: Subteam members spent this week and the following week preparing for final exams.

**Policy and Practices**: Members continued reaching out to stakeholders to understand the potential of the project specifically focused on cancer treatments and ursolic acid. Due to difficulties with stakeholder interview engagement, additional research via a review of literature on ursolic acid was started in the form of a Ursolic Acid Historical Handbook analyzing the origins of Ursolic Acid and it’s historical context. PnP discussed summer plans related to possible outreach events, including science educational videos, infographics, continuing the handbook, and stakeholder engagement. PnP began discussing a collaboration with Wiki for an Oncurex specific game based around “fruit ninja”. Deliverables regarding how to make the game education were drawn up.

**Business**: Presented our work for the semester at gbody. Subteam had a wrap-up meeting discussing goals for next semester

**Wiki**: Members decided which teams they wanted to work on over the summer. The subteam was divided into game development and design/web development.

### May 12th to May 18th

**Wet Lab**: Leads discussed methods for knocking out POR2, and we decided on using a PCR mediated knockout method which replaces the target gene with a sequence of choice to knock out the gene. Subteam members prepared and took final examinations.

**Product Development** : Subteam members prepared for final examinations.

**Policy and Practices**: An interview was completed with Dr. Chris Fromme, a researcher for Molecular Biology who specializes in yeast usage. Dr. Fromme helped explain to us key components of yeast genetic modifications with plasmids and provided numerous resources. An engagement event was lead at Sciencenter both based on ursolic acid and general science topics. The main audience was kid engagement, and it was noticed that many younger kids did not necessarily understand the premise of the project or synthetic biology. This encouraged us to create a Children’s Book focused on educating kids about synthetic biology.

**Business**: Subteam members prepared and took final examinations.

**Wiki**: Subteam members prepared and took final examinations.

### May 19th to June 8th

#### Summer Break

# June 2024

### June 9th to June 15th

**Wet Lab**: Subteam members read the paper discussing the engineering behind the PCR mediated knockout strategy. Leads interviewed professors for further insight into the project and they created relevant protocols from sources received.

**Product Development**: Subteam members arrived back on campus following break. Subteam members began to re-do an inventory due to materials moving in transit after the move. Established a cadence for summer meetings and expectations.

**Policy and Practices**: First priority of the summer was to focus on stakeholder engagement and feedback. PnP needed to understand the perspective of stakeholders not only in ursolic acid and cancer, but also pharmacy and other related fields, given that interviews over spring mainly yielded technical avenues and pointed us toward the continuation of cancer. PnP followed up with the interview with Dr. Chris Fromme regarding specific questions on yeast modification. He provided multiple possible directions for us. Members continued reaching out to stakeholders to understand the potential of the project specifically focused on cancer treatments and ursolic acid. Members began planning their scientific educational videos, building upon the previous series from last year.

**Business**: Subteam members had the summer off.

**Wiki**: Members had a virtual meeting to review the summer timeline. Deadlines and checkpoints were established for the entire summer. Work began on creating the 2024 Sponsorship Packet, a collaboration between the Business and Wiki subteams.

### June 16th to June 22nd

**Wet Lab**: Primers were received, and the wet lab was split into groups for parallel projects. The freezers were defrosted.

**Product Development**: Subteam members finished the inventory. Subteam members began testing sensors in our inventory to see which ones still worked. Began comparing purchase list to inventory list. Subteam members began testing our pump inventory. Subteam members looked for potential reservoirs in our inventory.

**Policy and Practices**: PnP continued reaching out for stakeholders for cancer treatments, ursolic acid research and other applications of ursolic acid to understand different applications for Oncurex. Members worked on planning scientific educational videos, choosing the topics each member will present on. Topics include growing yeast, capillary action, oxidation. Members also worked on the Ursolic Acid Historical Handbook, meeting individually as a group to plan out different topics to include. PnP started writing the Children’s book in another collaboration with Wiki after the results of Sciencenter. We decided on the journey of an e. Coli due to it’s prevalence in the synthetic biology field as a staple model organism, and hoped to emphasize the vast amount of potential synthetic biology has.

**Business**: Subteam members had the summer off.

**Wiki**: The game development team went over expectations from Policy and Practices for the game. The game would have educational popup questions that players could answer to unlock powerups. The Sponsorship Packet was fully completed and updated with all of Team Cornell’s current and past sponsors, as well as descriptions of our recent work.

### June 23th to June 29nd

**Wet Lab**: Primers were suspended and working stocks were made. Plasmids (KanMX and pRS306) and WT yeast (SEY6210) were gifted by the Fromme lab.

**Product Development**: Subteam members continued to test necessary components. It was found that we needed new pH probes. Sous-vide, oxygen bubbler tested and found to be working. Temperature probe tested and found to be working.

**Policy and Practices**:An interview was completed with Dr. LuAnn Van Campen, a bioethics specialist regarding EUDI. PnP interviewed Dr. Jini Hyun, an oncologist from Weill Cornell. She was excited to hear our project, and explained to us the process of cancer treatments and where Ursolic acid would fit in. Given the nature of the project, we found it apt to reach out to and learn from cancer patients as well, and started drafting an IRB in response. Members completed the presentations for the science educational videos based on the topics decided. The children’s book was completed and sent to Wiki for review and illustrations. Considering that at Sciencenter, we met many families whose primary language was not english, we decided that translating the children’s book to different languages would increase accessibility for many.

**Business**: Subteam members had the summer off.

**Wiki**: The game development team began familiarizing themselves with the base code that would be built upon for the game. Game development team leaders were assigned for each week of the summer. The design team worked on improving the designs made during the spring semester.

# July 2024

### June 30th to July 6th

**Wet Lab**: Liquid cultures and glycerol stocks were made. OD was measured for the liquid cultures before making glycerol stock. YPD media was made and autoclaved for the yeast glycerol stock and liquid cultures.

**Product Development**: Subteam members tested the necessary boards (Arduino, L298N motor controllers) to ensure working order and sufficient quantity. Subteam members updated documentation and researched past projects for examples.

**Policy and Practices**: An interview was completed with Dr. Ran Yin, an ursolic acid specialist regarding antioxidant and anti cancer activity of ursolic acid. Discussed Oncurex based game, and focused on introducing the educational component via science based questions and additions. PnP began reviewing past documentation of work in an effort to consolidate and connect all the information learned from past interviews and public and stakeholder engagement. Members continued preparing for science videos and working on the historical handbook. An outreach event was conducted at Thoreau Day Camp to discuss the possibilities of biology and science to children potentially not well versed. Ultimately, we found that our project was difficult to understand for those that do not have a strong science background, encouraging us to develop more concise and explorative infographics to explain our project via pictures rather than words.

**Business**: Subteam members had the summer off.

**Wiki**: Work continued on assignments for the first deadline.

### July 7th to July 13th

**Wet Lab**: Members miniprepped KanMX and measured nanodrop.

**Product Development**: Short break for the fourth of july. Continued to work on documentation updates. Investigated potential modeling softwares for the reactor.

**Policy and Practices**: PnP completed an interview with Dr. Susan Sadoughi and Dr. Joshua Zaritsky, a pediatric nephrologist to explore alternative applications of ursolic acid. Members discussed the collaborative game, finalizing the educational component as science based questions as incentives for accessory based power ups. A list of questions, accessories and backgrounds were drawn up and sent to Wiki. PnP continued finalizing the documentation of work for an internal documentation deadline focused on outlining human practices, education and inclusivity within our project

**Business**: Subteam members had the summer off.

**Wiki**: Work began on illustrations for the children’s book written by PnP. Feasibility of game features were discussed with PnP. The game development team began working on the implementation of those features. Assignments for the first deadline were completed and reviewed. The team met to discuss and modify assignments for the second deadline.

### July 14th to July 20th

**Wet Lab**: Members miniprepped the pRS306 plasmid and measured it with nanodrop. Performed PCR on the KanMX plasmid and measured it with nanodrop. Realized that some of the eppendorfs are contaminated, so they were re-autoclaved. DNA cleanup was performed on the PCR product in an attempt to raise the 260/230 ratio.

**Product Development**: Ordered and received new parts. Performed first assembly of bioreactor during a “crunch day” work session. Assembled pump circuits. Assembled sensor circuits. Calibrated pH probes. Assembled reservoir systems.

**Policy and Practices**: An interview was completed with Dr. Blessing Aderibigbe, a Ursolic Acid researcher and with Dr. Ashish Saxena, an oncologist at Weill Cornell. PnP began translating the children’s book into 5 different languages, Korean, ASL, French, Spanish and Chinese (Mandarin and Cantonese). Documentation was returned to for any revisions and updates, including elaboration and adding photos. Materials were gathered for the scientific educational videos with the intent of recording within the next few weeks. The IRB protocol was officially submitted to the Cornell Review Board for approval with interviews and surveys for cancer patients.

**Business**: Subteam members had the summer off.

**Wiki**: Work continued on checkpoints for deadline 2. Game development added popup questions to the game while design/web development continued work on graphic elements, the children’s book, and merch designs.

### July 21st to July 27th

**Wet Lab**: Members miniprepped and performed PCR on pRS306 and it was measured with nanodrop. Gel electrophoresis was run on the PCR products. It looked like there was no product for any of the PCR reactions. Reached out to mentors and decided on changing annealing temperature along with adjusting DMSO concentration. AAS thing 1 and 2 were received and members ran PCR on it. Members reran gel to see if it was the gel or the PCR products that were bad. Nothing showed up, but it was discovered that the gel was run backwards resulting in the DNA falling off the gel. PCR with KanMX was rerun with different annealing temperatures. Members attempted gradient PCR but was halted due to a missing activation key.

**Product Development**: Worked on programming for the bioreactor using the arduino IDE. Re-calibrated sensors using pH buffer. Combined sensor and pump portions of the reactor.

**Policy and Practices**: An interview was completed with Dr. Sean Nicholson from policy analysis and Dr. Ying from health consulting. They both pointed out the key markets in CDMO’s. Conversation over email with Dr. Bruce Ganem, an organic chemist, regarding the potential of modifications was held. Held an extended PnP meeting regarding any pertinent questions left in the project regarding FDA approval and clinical trials. Given the results of the Thoreau Day Camp, we decided to focus on more accessible means of explaining our work. This came in the form of infographics and images, encouraging us to develop infographics regarding our project to more easily conceptualize the process behind our work. Given the nature of the game collaboration with Wiki, PnP opted to return to the game and develop explanations to each of the questions in an effort to increase understanding of the more technical aspects of our project.

**Business**: Subteam members had the summer off.

**Wiki**: Assignments for the second deadline were completed and reviewed. Assignments for the third deadline were updated. Work continued on the children’s book. Game mechanics of the popup questions were finalized.

# August 2024

### July 28th to August 3rd

**Wet Lab**: Members packed lab equipment to prepare moving into Thurston Hall. Gel was rerun with all the PCR products. Some products were good, but others had multiple bands or bands of the wrong length. Members ran touchdown PCR on all the parts.

**Product Development**: Finished final assembly of bioreactor. Prepared reactor for test runs. Had a “frankenmeeting” with Wet Lab to discuss considerations for the sensor and reactor.

**Policy and Practices**: An interview was conducted with Dr. Colleen Carey, a health insurance and drug policy specialist. Stakeholder outreach continued in terms of understanding cancer Specialist perspectives. PnP worked on analyzing applications of the maturity model for human practices, outlining the different ways Oncurex applies. Documentation was updated for interview write ups and educational assignments

**Business**: Subteam members had the summer off.

**Wiki**: Web development met to finalize the design of the competition wiki. Research on implementation began. Work continued on final deadline assignments.

### August 4th to August 10th

**Wet Lab**: Members ran a gel on the PCR products, and KanMX had two bands, one of which was the target length. Members cut the AAS gene fragments and ligated them together.

**Product Development**: Worked on and updated documentation. Worked on modeling for the reactor, specifically measuring flow rates and researching growth kinetics for the yeast.

**Policy and Practices**: Interview completed with Dr. Oyedeji, an ursolic acid specialist. IRB protocol was sent back with comments, and PnP edited and resubmitted the protocol. Ursolic Acid handbooks were worked on, along with video recordings and other infographics. Outreach Events at Kendal and Longview were organized for September via email and zoom meetings. These meetings discussed the content of the presentations as well as intent. Documentation was updated for human practices and education.

**Business**: Subteam members had the summer off.

**Wiki**: Work continued on final deadline assignments. Priorities for the fall semester leading up to Wiki Freeze were determined.

### August 11th to August 17th

**Wet Lab**: Subteam has the summer off.

**Product Development**: Subteam had time off for the start of semester.

**Policy and Practices**: Subteam has the summer off.

**Business**: Subteam members had the summer off.

**Wiki**: End of summer break. Work continued on the children's book.

### August 18th to August 24th

**Wet Lab**: Members had summer off.

**Product Development**: Subteam had time off for the start of semester.

**Policy and Practices**: Interview completed with Dr. Bruce Lewenstein for cancer patient interviews and outreach. Farmers market tabling set up in Ithaca. Documentation continuously updated

**Business**: Subteam members had the summer off.

**Wiki**: End of summer break. Work continued on the game graphics and children’s book.

### August 25th to August 31st

**Wet Lab**: Moved things from Weill Hall to new lab space in Tang Hall. Determined ways to optimize PCR reaction using DMSO and finding ideal annealing temperature. Redesigned primers for Gibson Assembly since ligation was unsuccessful. SnapGene simulations were run for PCR and gel electrophoresis.

**Product Development**: Collapsed summer space to comply with admin-allowed space. Had to relocate and reorganize lab space and supplies. Performed initial construction of bioreactor, including assembling motors, programming Arduino, and printing a custom lid for the main reservoir.

**Policy and Practices**: Began collaboration with Queens iGEM (QGEM) regarding regulatory policy around the world.

**Business**: Subteam members had the summer off.

**Wiki**: Wiki pages were assigned to each team member. Children’s book was finalized and all translated versions were made.

# September 2024

### September 1st to September 7th

**Wet Lab**: Assigned duties to complete required trainings for Tang Hall access. Assigned experiment plan write-ups for each subgroup in WL. Arranged a time to unpack iGEM boxes in new lab space.

**Product Development**: Finished construction of bioreactor. Drafted final bioreactor design schematic. Had first meeting with Business team to discuss scale-up.

**Policy and Practices**: Finalized recordings for science videos. Continued outreach for interviews and started closing the loop interviews. Finished writing Ursolic Acid Handbook. Completed outreach event at Sciencenter. Completed interviews with Dr. Robert Weiss regarding setting up cancer patient interviews.

**Business**: Reconvened after summer break. Assigned responsibilities for the business plan to each team member.

**Wiki**: Pairs were made with current subteam members and advisors. Each pair was assigned to specific larger tasks on the wiki, such as styling and game implementation. Final promo video content was filmed. Promo video was edited and submitted.

### September 8th to September 14th

**Wet Lab**: Set up a safety lab tour with Dr. Archer for Tang Hall lab access. Assigned responsibilities to each subgroup to complete PCR reactions, resuspend newly ordered primers, and proceed with gel confirmations before transforming into yeast cells for when we get access to the lab. Assigned the subteam to work on literature review for biosensor binding agents, UA extraction methods, and encapsulation techniques. Members performing literature review had to compile their findings on a document within one week.

**Product Development**: Finalized bioreactor schematic. Began work on sterility manual with PNP team. Continued scale-up meetings with the Business team.

**Policy and Practices**: Completed outreach event at farmers market and consolidated data. Interviewed Dr. Raymond Glahn, Dr. Sophie Openshaw, Dr Li Li and Bob Riter and Monica Vakimer. Started outreach to Ithaca Breast Cancer Alliance and Cancer Resource Center. Prepared for outreach event at Long view Senior Center by making presentations. Started reviewing all documentation and making edits. Set up Elementary school outreach at Beverly J Martin Elementary school. Posted Science video #1

**Business**: Made infographics. Worked on Market Sizing. Worked on Market Analysis. Worked on IP

**Wiki**: Wiki members began meeting with other subteam leads to discuss the layouts for individual pages. Members met to clarify procedures for editing the competition wiki. This meeting also served as a work session to begin uploading content.

### September 15th to September 21st

**Wet Lab**: Safety lab tour was postponed due to conflicts, scheduled for the 22nd at noon. Shared literature review findings with entire team. Began designing plasmid for signal peptide extraction

**Product Development**: Calculated Reynold’s number for our main reservoir. Repaired reactor form move and prepared for first wet run. Began designing biosensor housing and adapting WL’s literature search for a biorecognition element.

**Policy and Practices**: Met with Ithaca Breast Cancer Alliance and Cancer Resource Center regarding collab. Met with QGEM regarding finalizing results of survey and handbook. Held outreach events at Longview and Beverly J Martin elementary school. Continued working on documentation. Finalized all write ups for interviews and closing the loop interviews. Started Cancer Patient Interviews

**Business**: Worked on Business Model Canvas. Worked on Risk Assessment. Worked on Supply Chain

**Wiki**: Work continued on content uploads for Wiki Freeze.

### September 22nd to September 28th

**Wet Lab**: Worked on documentation. Finalized plasmid design for signal peptide extraction. Calculated E-factor, PMI, and mass of total product from one liquid culture for Business subteam.

**Product Development**: Performed first wet run of the bioreactor. Calculated price per run with the Business subteam. Updated documentation for Wiki freeze. Calculated Biot number and associated dimensionless numbers for heat transfer into the main reservoir.

**Policy and Practices**: Work on updating content and finalizing documentation. Completed collab with QGEM regarding future steps. Continued working with Ithaca Breast Cancer Alliance and Cancer Resource Center regarding collaboration. Continued Cancer patient interviews. Started mock interviews with Bob Riter and Monica Vakimer

**Business**: Worked on Future Plans and Exit Strategy. Worked on Competitor Analysis. Worked on Stakeholder Analysis. Made infographics

**Wiki**: Work continued on content uploads for Wiki Freeze.

# October 2024

### September 30th to October 5th

**Wet Lab**: We gained access to lab space, resuspended primers, performed PCR, and performed gel confirmations.

**Product Development**: Finalized and printed biosensor housing. Finalized documentation for Wiki freeze. Finalized a mathematical model for biosensor response quantification.

**Policy and Practices**: Successful completion of data. Continued working with Ithaca Breast Cancer Alliance and Cancer Resource Center regarding future steps during grace period. Resubmitted IRB given advice from Breast Cancer Alliance and Cancer Resource Center specifically for survey updates

**Business**: Computed PMI. Computed E factor. Computed cost at an industrial scale. Put all info on the Entrepreneurship page and Biz Plan

**Wiki**: Work continued on content uploads for Wiki Freeze. Filming and editing began for the presentation video.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Ursolic Acid Development{% endblock %}
{% block page\_content %}

[Wet Lab Overview](#wl)
[WL Cycle 1](#wl-cycle1)
[WL Cycle 2](#wl-cycle2)
[WL Cycle 3](#wl-cycle3)
[WL Cycle 4](#wl-cycle4)
[Product Development Overview](#pd)
[PD Cycle 1](#pd-cycle1)
[PD Cycle 2](#pd-cycle2)
[PD Cycle 3](#pd-cycle3)

# Wet Lab CyclesWL Overview The goal of Oncurex is to create a synthetic biosynthesis pathway for ursolic acid (UA), an anticancer and antibacterial triterpenoid naturally found in fruit peels, such as apple and loquat, and herbs such as rosemary and sage. In plants, the precursor for ursolic acid, α-amyrin, is produced by α-amyrin synthase (AAS). By expressing AAS in Saccharomyces cerevisiae, α-amyrin can be made which ultimately allows for the production of UA by Cytochrome P450 and CRP. For biomanufacturing, it is necessary to increase yields to create a production process that can result in higher yields than plant extraction. Additionally, drug substances sourced from plant extracts are highly heterogeneous and difficult to regulate, so using synthetic biology streamlines production and allows for easier FDA approval. To this end, knocking out a gene encoding the mitochondrial transporter POR2 decreases the flow of acetyl-CoA into mitochondria, where it can be used for other anabolic purposes. Since acetyl-CoA is an early precursor in the ursolic acid synthesis pathway, this increase in cytosolic acetyl-CoA concentration intends to increase yield since ursolic acid production occurs in the cytoplasm by simultaneously decreasing the cellular usage of acetyl-CoA for the production of other compounds. Instead of solely transforming the yeast with the plasmid of interest, we have chosen to integrate the genes of interest directly into the yeast genome. This method was chosen because yeast has a relatively high rate of plasmid loss, meaning that a culture of yeast with the target plasmid would eventually lose the plasmid over generations [1]. It is crucial for our project that the gene of interest is not lost because we are using a CSTR to produce our target compound, and a high rate of plasmid loss leads to more downtime in the CSTR. The genes of interest will be incorporated into the yeast through homologous recombination. This occurs between two DNA sequences with similar nucleotide sequences and results in the exchanging of genetic information. This process can be leveraged during transformation to allow *S. cerevisiae* to incorporate the transformed DNA into its genome for insertions and knockouts [2]. Our plasmid sequences were homologous to a targeted region of the *S. cerevisiae* genome, allowing for controlled gene knockouts and integration of the genes of interest into the yeast genome. The proof of concept was established by creating a plasmid construct via Gibson Assembly with the AAS gene isolated from Malus domestica (MdOSC1) and a pRS306 backbone. This construct was then transformed into *S. cerevisiae* cells, where the production of ursolic acid was determined. Characterization of our construct occurred by testing its performance with the presence of a POR2 knockout in addition to various growth conditions such as the absence of uracil.

## WL Cycle 1: 2-Step Single Crossover Homologous Recombination

### Design

This method differs from the double crossover homologous recombination as it's composed of two separate single crossover processes with two separate plasmids: KanMX and pRS306. KanMX is a commonly used gene knockout and replacement *S. cerevisiae* plasmid that contains kanamycin resistance genes for selection. pRS306, which was donated to us by the Fromme Lab, is another commonly used plasmid used for integration of genes into the SEY6210 strain of *S. cerevisiae* and contains a URA3 sequence that is used for selection on media lacking uracil.

![](https://static.igem.wiki/teams/5059/engsuccess/figure1a.png)
![](https://static.igem.wiki/teams/5059/engsuccess/figure1b.png)

Figure 1a, 1b. Plasmid Design for KanMX (left), Plasmid Design for pRS306 (right)

### Build

Plasmids were received in Escherichia coli stab cultures, which were plated (Figure 2a) and then liquid-cultured with ampicillin. Following the liquid culture, spectrophotometry was used to verify growth before a miniprep was performed. After mini-prepping the pFA6a-kanMX6 plasmids (Figure 2b), the team noticed low 260/230 ratios, so DNA cleanup was performed to purify plasmid content.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-2.png)

Figure 2a, 2b. Plate Prep for KanMX-plasmid-containing E. Coli (left), Nanodrop for KanMX MiniPrep pre-Clean Up (right)

The pRS306 plasmid was plated (Figure 3a) and grown in a liquid culture with ampicillin. Miniprep was then conducted, and the purity of the sample was confirmed through nanodrop, with a 260/280 ratio of 1.87 (Figure 3b).

![](https://static.igem.wiki/teams/5059/engsuccess/fig-3.png)

Figure 3a, 3b. Plate Prep for pRS006-plasmid-containing E. Coli (left), Nanodrop for pRS0306 MiniPrep (right)

With purified DNA, a gel confirmation (Figure 4) was performed to verify the correct plasmid length and to check for target amplicons. As seen below, we see our PCR product at their corresponding sizes as shown by the ladder (1st well), with the pFA6a-kanMX plasmid (3rd well) at the 4kb and 1.5kb positions and the pRS306 plasmid (2nd well) at the 4kb position.

![](https://static.igem.wiki/teams/5059/engsuccess/figure4.png)

Figure 4. Gel Confirmation of pRS306 and KanMX Plasmid PCR. Ladder in lane 1. pRS306 in lane 2. KanMX in lane 3.

1.1 KanMX Crossover

To allow the transformation of the pFA6a-kanMX plasmid to have the function of knocking out POR2, it is necessary to insert a homologous POR2 sequence into the pFA6a-kanMX plasmid. To insert the homologous POR2, we performed PCR on the plasmid with primers containing homologous regions to the yeast POR2 gene to add the POR2 homology region (Figure 4).

![](https://static.igem.wiki/teams/5059/engsuccess/fig5a.jpeg)

Figure 5a. KanMX insertion into yeast genome and simultaneous POR2 knockout.

After the PCR, the plasmid was introduced into the *S. cerevisiae* through transformation. When the engineered plasmid is inserted into the *S. cerevisiae*, its internal homologous recombination mechanism will recognize the POR2 homologous sequence on the KanMX plasmid and align it with the corresponding homologous POR2 gene sequence in the *S. cerevisiae* genome. Once this crossover occurs and the KanMX cassette is inserted into the POR2 gene, it will knock out the POR2 gene.

Initially, we only got bands at around 1.5kb for the KanMX PCR product, even though the expected length is 4.5kb. After reviewing each step of our PCR protocol, we realized that this may be a more complex region of DNA, so we increased the elongation time. This improved our results immediately, resulting in the target 4.5kb band as seen in Figure 4. However, a 1.5kb band appeared alongside the 4.5kb band for each PCR. The primers themselves were very long (~60bp), and secondary structures were very prevalent. To troubleshoot this and to increase specificity, we added 3% DMSO. This did not lead to any changes in the bands formed after PCR. We further troubleshooted by performing touchdown PCR at different ranges around the recommended annealing temperature. However, this did not result in a more specific PCR amplification.

1.2 pRS306 Crossover

For the transformation of pRS306, primers were used to linearize the plasmid while adding homologous ends to the ends of α-amyrin synthase to prepare it for Gibson assembly. The α-amyrin synthase was broken up into two parts, AAS1 and AAS2, because the complexity was too high to be constructed. Then to construct one functional AAS coding region, the AAS1 and AAS2 genes were digested by an overlapping EcoRI region and ligated together using T4 ligase. PCR of the α-amyrin synthase gene with primers designed to match the ends of the gene insert with the ends of the linearized pRS306 amplified the gene insert and ensured its ends overlap with the ends of the linearized plasmid backbone. This allowed for Gibson Assembly to be used to assemble the pRS306 plasmid with the α-amyrin synthase insert, creating a new circular plasmid with both the α-amyrin synthase gene and the URA3 sequence that was already present on the pRS306 plasmid.

This plasmid was cut at the URA3 homology site with SbfI before the transformation into yeast. The internal mechanism of yeast would recognize the homologous URA3 sequence, which resulted in crossover. This crossover occurred at the nonfunctioning URA3 gene while inserting the α-amyrin synthase gene and a functioning URA3 gene. URA3 encodes Orotidine 5’-phosphate decarboxylase (ODCase), which would enable yeast to grow without the presence of uracil or uridine. Thus, a successful transformation in which the URA3 gene is disrupted would result in a lack of growth in a medium without uracil. Further success of the transformation would be confirmed by Ni-NTA chromatography as our design of AAS includes a 6x His-tag at the end of the AAS coding sequence. This is to ensure that not only is the AAS gene fragment integrated into the yeast genome but to also confirm successful production of AAS itself.

![](https://static.igem.wiki/teams/5059/engsuccess/fig5b.jpeg)

Figure 5b. pRS306 crossover with AAS and transformation into yeast using URA3 homology

The AAS1 and AAS2 as well as the pRS306 vector were successfully amplified via the PCR primers we designed. The base pair length of AAS1 and AAS2 was confirmed through our gel confirmation shown below.

![](https://static.igem.wiki/teams/5059/engsuccess/figure5c.png)

Figure 5c. Left to right: Lane 1 - Ladder, Lane 2 and 3 - AAS1 (2.3kb), Lane 4-7 - AAS2(1.1kb)

However, when it came to performing the EcoRI digest and the T4 ligase ligation, we ran into many issues. We troubleshooted with different EcoRI and T4 ligase concentrations, digest and ligation time, as well as incubation temperatures. Ultimately, we were unable to obtain the ligated product of AAS1 and AAS2. The only parameter that affected results was the concentration of starting DNA as expected, observed by the increase in nanodrop concentration and slightly brighter bands on the gel. All wells contained the ligation product achieved through different parameters. Wells 4 and 5 had increased starting concentrations of DNA, hence the slightly visible bands.

![](https://static.igem.wiki/teams/5059/engsuccess/figure5d.png)

Figure 5d. AAS1+2 digest and ligation gel confirmation

### Test and Learn

Characterization of the construct will occur through lysis and purification of ursolic acid. Given that this method relies on separate single crossover recombinations, we expect a greater success rate compared to the double crossover homologous recombinations. Based on the results, we would iterate the more efficient method allowing for further improvements on the construct and its yield.

For the KanMX, we were initially unable to obtain the target band, but we were able to overcome this by lengthening the extension step. We have learned to do this first thing when amplifying large PCR fragments such as pRS306. However, we were unable to get rid of the off-target amplicon at 1.5kb, even after many iterations of adjusting the temperature parameters. To reduce the possibility of off-target sites as well as the complexity of the target fragment, we decided to amplify a much smaller fragment from the pFA6a-kanMX plasmid containing the KanMX gene for our next cycle.

Although each genetic component was successfully amplified for the AAS insertion, we were ultimately unable to produce the target pRS306 vector with the AAS. After consulting our graduate advisors, we troubleshooted different parameters but were unable to produce the target part. We then settled on a different solution bypassing the need for the EcoRI digest and ligation step involving a three-part Gibson Assembly. This method was considered at the beginning, but our team has had poor luck with Gibson Assembly in the past, leading to the initial use of digestion and ligation instead.

Using two plasmids is not the most efficient design choice since it involves nearly double the lab work with more opportunities for mistakes. Thus, a new build design was utilized in Cycle 2 to allow each transformation step to take place at once. By utilizing gene fragments, we can leverage the ability of *S. cerevisiae* to easily perform homologous recombinations [3] without the need for plasmids resulting in faster transformations. Despite this ability, the procedure may have a higher failure rate given the nature of a triple crossover, making this procedure a trade-off between time and quality. As we are working with both cycles in parallel, the team will move forward and iterate the more efficient one once the cycles succeed.

## WL Cycle 2: Triple Crossover Homologous Recombination

### Design

We seek to accomplish two goals: to knock out the POR2 gene and to insert AAS into the yeast genome. In this iteration, we aimed to accomplish both goals in one experiment by transforming two gene fragments into yeast. One fragment was AAS, which would produce α-amyrin, and the other fragment was KanMX - a common selection marker for yeast - to confirm the insertion of the AAS gene into the POR2 coding region. Yeast cells have the mechanisms to be extremely apt at homologous recombination [3], which is the mechanism used for this cycle, but due to the necessity of three homologous recombination events occurring for the insertions to be successful, it might present a challenge to accomplish.

The KanMX gene was extracted from the pFA6a-kanMX plasmid (Figure 6a), and primers were designed to add homology regions to the POR2 gene on one end and homology to the AAS gene on the other. The AAS gene was PCR amplified with primers designed to have homology to the KanMX gene on one end and the POR2 gene on the other (Figure 6b). These fragments were transformed into yeast, and we selected for colonies that grow on G418, which the KanMX gene gives resistance to. These colonies should have had the three crossover events to result in the gene fragments inserted into the POR2 part of the genome, knocking out the gene as shown below.

![](https://static.igem.wiki/teams/5059/engsuccess/figure6a.png)

Figure 6a. pFA6a-kanMX Plasmid design

![](https://static.igem.wiki/teams/5059/engsuccess/fig6b.jpeg)

Figure 6b. Model of the triple homologous crossover and simultaneous POR2 knockout in the yeast genome.

### Build

Based on our design, the assembly of our recombination would consist of the integration of the KanMX sectioned plasmid and the AAS coding plasmid into the POR2 locus effectively knocking out the expression of POR2. Both plasmid components were first built separately to allow for individual testing, as this allows us to ensure their functionality before combining them into the final homologous construct design.

2.1 Assembly of KanMX sectioned plasmid

Similar to the 2-step single crossover homologous recombination, the extraction of the KanMX gene from the pFA6a-kanMX plasmid consists of plating the Escherichia coli stab culture containing the original plasmid and liquid cultured with ampicillin. The KanMX was obtained from a miniprep performed on the liquid culture used in Cycle 1. This was then followed by a DNA cleanup procedure.

Following the purification procedure, PCR was performed on the isolated plasmid to ensure proper amplification and ample concentration while adding the homologous ends. After ensuring ample concentration, a gel confirmation was performed to verify the length of the pFA6a-kanMX plasmid (1.5kb).

2.2 Assembly of AAS coding plasmid

To ensure the AAS gene fragment isolated from Malus domestica (MdOSC1) was able to successfully crossover with the POR2 homology region, PCR with the necessary forward and reverse primers containing the homologous regions for the POR2 gene was performed. Once more, to ensure the AAS gene fragment was at its accurate length, a gel confirmation was performed as shown in Figure 5c.

2.3 Integration of KanMX sectioned and AAS coding plasmids into POR2 homologous region

Following the gel confirmation and PCR amplification of both AAS and pFA6a-kanMX plasmid, the transformation of the *S. cerevisiae* was performed through a two-day Lithium acetate (LiAc) method. The first day consisted of prepping the necessary components needed in the transformation mix, such as Lithium Acetate, PEG MW 3350, and Single-Stranded Carrier DNA (Salmon Sperm DNA) which are all essential for the facilitation and uptake of the plasmids by the yeast cells. The cells were then inoculated in the 2XYPAD medium overnight at 30°C on a rotary shaker at 200 rpm in preparation for the main transformation procedure the following day.

![](https://static.igem.wiki/teams/5059/engsuccess/fig7a.png)

Figure 7a. Components of the transformation mix needed for the *S. cerevisiae* transformation [13]

To verify the growth of the cells, a spectrophotometer was used to determine the optical density at 600 nm. Once the desired cell density was reached, the harvesting of the yeast cells was performed by centrifugation at 3,000 ×g for 5 min and thereafter, washed and resuspended in sterile water. Finally, the cell suspension was transferred into a 1.5 mL microcentrifuge tube and further centrifuged to pellet the cells and remove any supernatant before the transformation process.

To begin the transformation process, the transformation mix prepared in advance is added to the microcentrifuge tubes containing the yeast cells and further vortexed to ensure the cells are appropriately mixed with the transformation media. Afterward, the cells are incubated in a 42 °C water bath to induce heat shock on the yeast cell walls to facilitate the uptake of the plasmids. Following the heat shock, the cells are pelleted again by centrifugation for 30 seconds and any leftover transformation mix is carefully removed to avoid losing any competent cells. Resuspending the cell pellet in 1.0 mL of sterile water, the cells are plated onto the YPD media plates containing the G418 antibiotic selective medium at three different volumes (2, 20, and 200 μL). Lastly, the plates are incubated at 30 °C for 3 - 4 days in preparation for screening and verification.

To verify the transformation of the *S. cerevisiae*, cells plated on the YPD media containing the G418 antibiotic were screened for resistance. Only cells that have successfully integrated the KanMX gene fragment are capable of surviving due to their resistance to G418. A colony PCR could also be performed by lysing the transformed yeast and using the plasmid released as a template to be PCR and eventually verified through gel confirmation.

### Test and Learn

Like the 2-step single crossover, the characterization of the construct will occur through lysis and purification of ursolic acid. Through using chromatography, the ursolic acid can be separated from any other contaminants or non-ursolic acid products to be quantified.

In this cycle, we were not able to produce the full AAS gene through digestion and ligation, similar to Cycle 1. We talked to our graduate student advisors and changed various parameters including EcoRI and T4 ligase concentrations, digest and ligation time, as well as incubation temperatures. We were however unable to obtain the ligated product of AAS1 and AAS2. Similar to Cycle 1, we will construct the AAS fragment through Gibson Assembly into pRS306 and perform PCR with the target primers from there.

## WL Cycle 3: Simulation

### Design

Our lab was required to move to a different lab space in a newly constructed building, but due to logistics, all wet lab work had to be paused until further notice. To maintain productivity during this time, we performed SnapGene simulations of the wet lab genetic engineering of our parts so that we can ensure success when we regain access to a lab space. At the time the wet lab was paused, both the Cycle 1 and Cycle 2 groups had accomplished successful minipreps of the KanMX and pRS306 plasmids with much troubleshooting of the PCR.

Since we already experimented with different temperatures for the PCR, we decided to build and order new primers for AAS, pRS306, and KanMX. The first part of the simulation involved confirming successful PCR through the virtual gel. Before designing the new primers, we decided to test the old ones for KanMX to see if it could yield a successful result. The products labeled G3 were the Cycle 2 products and the rest were the Cycle 1 products. Interestingly, all of the PCR simulations resulted in the target results with bands indicating the right length.

![](https://static.igem.wiki/teams/5059/engsuccess/fig8a.png)

Figure 8a. Simulation of PCR using Cycle 1 and Cycle 2 primers

Next, the same simulation was performed on the new set of Cycle 1 KanMX and AAS primers. Since we had difficulties ligating the AAS1 and AAS2, we decided to do a three-piece Gibson Assembly, so new primers were designed accordingly. The KanMX primers were designed to target shorter fragments flanking the KanMX gene region to decrease the possibility of off-target sites. For this, three sets of KanMX primers were designed. These new primers were designed to result in around 1.6kb amplicons each instead of the original 4kb. All of the designed primers had the PCR amplicons at the correct lengths, so these primers should work once we return to the lab.

![](https://static.igem.wiki/teams/5059/engsuccess/fig8b.png)

Figure 8b. Simulation of PCR using new Cycle 1 and Cycle 2 primers.

Since the DNA fragment is shorter, we decided to go through a double crossover cassette recombination between the yeast genome and the transformed fragment as opposed to the original single crossover recombination with a larger fragment. This may raise the failure rate due to the nature of double crossovers compared to single crossovers. However, we think that the shorter length of the target fragment would increase PCR efficiency and integration efficiency.

![](https://static.igem.wiki/teams/5059/engsuccess/fig8c.jpeg)

Figure 8c. KanMX insertion into the POR2 coding region of the yeast genome

### Build

For the second part of the simulations, we ensured that the designed primers produced PCR products that would be successful for Gibson Assembly. As in the design phase, Gibson Assembly simulations of both the old and new designs were run on SnapGene. As seen below, the PCR products yielded from both the old and new primers should result in the desired Gibson Assembly construct. However, we were unable to successfully ligate AAS1 and AAS2 together leading to the redesigning of the primers to do a three-fragment Gibson Assembly.

![](https://static.igem.wiki/teams/5059/engsuccess/fig9a.png)

Figure 9a. Simulation of new Gibson Assembly on SnapGene

![](https://static.igem.wiki/teams/5059/engsuccess/fig9b.png)

Figure 9b. Simulation of old and new Gibson Assembly with visualization through gel

### Test and Learn

Once we return to the lab, we will be performing all of the simulated experiments. Since the simulations confirmed the successful PCR and Gibson Assemblies, everything should go relatively well in the lab. This will allow us to quickly get to the transformation stage so that we can provide the PD team with the target yeast liquid culture for the CSTR. At the same time, it will allow us to further test the yield of UA.

However, the simulations have revealed that not everything that should work in practice would work in the lab. One great example of this is the KanMX PCR that we were troubleshooting in Cycle 1. Even after experimenting with annealing temperature with touchdown PCR, extending elongation time, and adding DMSO, the KanMX PCR was unsuccessful even though it was successful on the SnapGene simulations. Knowing now that this is a possibility, we adjusted best practices to prepare several sets of primers, especially for more complex and longer PCR fragments.

## WL Cycle 4: New Lab Space and Future Steps

### Design and Build

After our SnapGene simulations, we looked into designing extraction and encapsulation methods after producing ursolic acid in yeast. In regards to extraction, we looked in the literature for potential methods to extract a molecule similar to ursolic acid. We found researchers had developed a signal peptide system where a signal peptide sequence from an extracellular transport protein was integrated into a carrier protein, such as supernatant protein factor (SPF), which binds to squalene. This would essentially transport the drug extracellularly, allowing for the extraction of the bound molecule (4). Since squalene and ursolic acid are both hydrophobic molecules, we looked into the affinity of the lipid-binding domain of SPF (tSPF) and ursolic acid via MolModa, a molecular docking software. We found the Gibbs free energy of the binding event to be negative, conveying that it's spontaneous and indeed would occur. This model gave us confidence in this extraction method working with ursolic acid.

![](https://static.igem.wiki/teams/5059/engsuccess/figure10a.png)

Figure 10a. Plasmid design of the signal peptide extraction of UA utilizing pMV-LEU2 and integrating the Suc2 signal peptide with tSPF to bind to UA for extracellular export.

Next, we considered designing a method to quantify its production in the bioreactor that the PD team was producing. Through a literature review, we found that ursolic acid inhibits, through allosteric regulation, α-glucosidase, an enzyme that hydrolyzes starch and oligosaccharides into glucose (5). Since our bioreactor has a glucose sensor built in, we theorized a system where we would have ursolic acid bind to α-glucosidase and test glucose levels as a proxy to quantify the amount of ursolic acid produced in the bioreactor.

Finally, once the ursolic acid is isolated, encapsulating the ursolic acid in a method that would increase its bioavailability would be the issue at hand. The bioavailability of ursolic acid is a relevant focal point for implementing ursolic acid in clinical settings. Through a literature review and an interview with Dr. Shaoyi Jiang, we compiled three methods for encapsulating ursolic acid to maximize its bioavailability. One method takes advantage of β- or γ-cyclodextrin, oligosaccharides that form a complex wrapping around ursolic acid, increasing its hydrophilic properties and simultaneously having a great affinity to the hydrophobic ursolic acid at its inner core (6). Then, we could encapsulate this inside sEVs, fuse this with a liposome to increase drug loading capacity, and form sEV-liposome fusions with β-cyclodextrin (β-CD) bound to the UA, as observed in Figure 10b.

The second and third methods involve utilizing sEVs and liposomes to encapsulate UA. One technique is to obtain sEVs with biomarkers of interest for specific cellular targeting, then perform electroporation to eliminate its inner contents. Then, liposomes encapsulating ursolic acid can be fused with these sEVs, creating a sEV-liposome hybrid that not only contains ursolic acid but has specificity to the cellular biomarkers we are interested in (7). An alternative technique is to mix polylactic-co-glycolic acid (PLGA), a hydrophobic polymer, with ursolic acid to form nanoparticles via precipitation. Once the PLGA-UA complex is formed, we can mix them with sEVS, and self-assembled sEVs containing the hydrophobic complex will be formed (8). A fascinating feature of PLGA is that based on the amount of PLGA used, we can determine its usage since it is biodegradable, so the dosage can be controlled via the release of the drug.

![](https://static.igem.wiki/teams/5059/engsuccess/figure10b.png)

Figure 10b. A diagram summarizing the UA encapsulated in β-cyclodextrin, followed by the encapsulation of this complex in sEVs, then fused with liposomes to form sEV-liposome hybrid nanoparticles.

To build the pMV-LEU2-Suc2-tSPF construct, we would first design primers to integrate the Suc2 signal peptide sequence with the tSPF sequence via PCR-mediated homologous recombination. Then, we would integrate Suc2-tSPF to pMV-LEU2 through Gibson Assembly, similar to how we inserted the AAS sequence into the pRS306 plasmid as described earlier in Cycle 2. This would proceed with the plasmid being cut at the LEU2 homology site with EcoRI before transformation into yeast. The internal mechanism of yeast would recognize the homologous LEU2 sequence, resulting in a crossover event. This will occur at the nonfunctioning LEU2 gene while inserting the Suc2-tSPF sequence (Figure 11a). LEU2 encodes for β-isopropylmalate dehydrogenase, an enzyme crucial for the biosynthesis of leucine. Thus, a successful transformation in which the LEU2 gene is knocked out would result in bacteria growing in media without leucine.

To perform the transformation of pMV-LEU2 into yeast, primers will be used to linearize the plasmid while adding homologous ends to the ends of Suc2-tSPF to prepare it for Gibson Assembly. PCR of the Suc2-tSPF sequence with primers designed to match the ends of the gene insert with the ends of the linearized pMV-LEU2 will amplify the gene insert and ensure its ends overlap with the ends of the linearized plasmid backbone. This provides the opportunity for Gibson Assembly to be used to assemble the pMV-LEU2 plasmid with the Suc2-tSPF insert without requiring restriction enzymes. This provides the opportunity to move forward with the transformation of the new circular plasmid into yeast.

To test for successful transformation, cells plated on the YPD media containing yeast drop-out medium supplements without leucine will be screened. Only cells that have successfully integrated the Suc2-tSPF gene fragment will not be able to grow on media without leucine. A colony PCR could also be performed by lysing the transformed yeast and using the plasmid released as a template to be PCR and eventually verified through gel confirmation.

![](https://static.igem.wiki/teams/5059/engsuccess/fig11a.jpeg)

Figure 11a. Proposed insertion of signal peptide sequence Suc2 into yeast genome using pMV-LEU2 plasmid, which knocks out the LEU2 coding region upon insertion.

Once the transformation has been tested to be successful, we can transform the plasmid into the yeast cells that have been engineered to produce ursolic acid to test if the extraction is successful. One way to test if the extraction is successful is to measure the glucose levels where the ursolic acid should be excreted since ursolic acid is known to inhibit α-Glucosidase, an enzyme that converts carbohydrates to glucose [9]. When ursolic acid and α-Glucosidase are present in a solution, the ursolic acid will bind to the enzyme via hydrogen bonding, inhibiting its action and causing glucose levels to be lower when complex carbohydrates are added in the solution.

Successfully exporting ursolic acid to the extracellular environment will segue into the next step: encapsulation. Figure 11b below describes the four encapsulation methods we have determined will be the most effective delivery systems for ursolic acid considering its low bioavailability due to its strong hydrophobic properties.

| Encapsulation Method | PROS | CONS |
| --- | --- | --- |
| β-Cyclodextrin | * Amphiphilic properties increase the bioavailability of hydrophobic UA * Increases concentration of UA in the dissolved phase * Rapid enzymatic degradation of complexes allows for rapid absorption of guest molecules into circulation * Stabilizing effect on a guest molecule in the solid state * Simple and low-cost encapsulation procedure | * Solid complexes can crystallize if stored in humid conditions * Nonspecific selection |
| γ-Cyclodextrin and Metal-Organic Frameworks | * Low-cost incorporation with ball-mill * Metal-organic frameworks effectively deliver hydrophobic drugs * Proven efficacy specifically with ursolic acid | * Time-intensive, encapsulation takes up to 1 week * Must consider the toxicity of metal complexes * Little data on yield |
| sEV-Liposome Hybrid | * High specificity to biomarkers due to unique targeting abilities of sEV * Limited immunogenicity * Demonstrated target accumulation in cancer cells | * Delicate procedure as the sEV’s membrane integrity needs to be maintained during encapsulation * sEVs pose concern for transforming normal cells cancerous if the cargo isn’t removed * Isolated sEVs aren’t uniform in size |
| PLGA Nanoparticles (NPs) | * Uses reproducible single-step nanoprecipitation method * Targeted delivery by adding targeting molecules to NPs surface * Size of particles can be fine-tuned | * Low weight percentage of the drug to hybrid NPs needed to ensure sustained drug release * Must keep initial drug inputs low during encapsulation as higher inputs will decrease encapsulation yield |

Figure 11b. Pros and Cons of various ursolic acid encapsulation methods for effective delivery.

### Test and Learn

By incorporating the tSPF-Suc2 into yeast, we'd expect to see an increase in UA secretion compared to yeast without the signal peptide complex. Prior research achieved a squalene secretion of 225 mg/L by incorporating the tSPF-Suc2 into yeast [4]. Since squalene has a slightly lower binding energy to tSPF, -10.5 kcal/mol, compared to compounds similar to ursolic acid like its precursor, α-amyrin (-6.8 kcal/mol), and its isomer, oleanolic acid (-6.3 kcal/mol), we’d expect a yield around or lower than the one found for squalene.

We expect to encapsulate UA in β-CD through a stirring method involving the solubilization of β-CD in water and an excess of UA. This method shown to have achieved successful encapsulation of UA by β-CD according to research [11], which was confirmed by comparison of differential scanning calorimetry (DSC) curves of the physical mixture of UA and β-CD and its inclusion complexation, in which peaks in the UA curve were found in the UA and β-CD physical mixture curve. Additionally, a virtual screening by [12] measured UA’s binding affinity to be -4.2 kcal/mol, which indicates that β-CD encapsulation would be favorable. Further encapsulation of the UA-β-CD complex in a cancer-derived sEV-liposome hybrid fusion is expected to increase specificity to cancer cell biomarkers, as demonstrated by research by [7] showing that lung cancer cell A549-derived sEV-liposome hybrids displayed a 14-fold higher cellular uptake to A549 cells than non-hybrid sEVs.

# Product Development Cycles

## PD Overview

The goal of Product Development was to create a continuous stirred-tank bioreactor (CSTR) to produce Ursolic Acid using the genetically engineered yeast from Wet Lab. This will also increase efficiency compared to batch reactors by shortening downtime and providing a more energy efficient model that can correct parameters falling below ideal conditions in real-time to preserve yield.

Furthermore, an electrochemical biosensor was designed with the aim to be integrated with the reactor system for live UA tracking without the need for offline and expensive UA test kits on the outflow of the bioreactor. Tracking the concentration of UA is crucial for assessing reaction efficiency and controlling industrial production yield.

The methodology and modeling of the bioreactor/biosensor can be found on the Modeling page on our website, while this page will detail the DBTL cycles and progress of developing the bioreactor along with initial and future steps on the biosensor.

## PD Cycle 1: Initial Design and Gathering Individual Components

### Design

After weighing the options and confirming the goal of building a continuous stirred tank reactor, the design started. All the first-round designs showed clear consistency. The reactor generally consists of a chassis, a main reaction chamber, and inflow and outflow reservoirs.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-12a.jpg)

Figure 12: One of the first round design sketches

The main reaction chamber and the reservoirs that require temperature control are placed inside the chassis. A sous vide will be used to control the temperature of the water inside the chassis and thus the internal temperature of the containers. The containers are preferably made of glass to increase thermal conductivity. Different containers are connected through PVC tubes and peristaltic pumps. Several sensors are fixed inside the main reaction container, including a thermometer, a pH meter, a dissolved oxygen sensor, and a glucose sensor. An agitator connected to a motor will also be fixed inside the main reaction container. Finally, an Arduino connected to a computer will control the entire reactor. All circuit boards and some circuitry will be stored next to the chassis, and this part needs to be watertight.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-13a.png)

Figure 13: Final Reactor Design

We had more in-depth discussions on some of the details. First, we discussed how many reservoirs we need. It is known that outflow and inflow are necessary. After discussion, we decided to store the engineered yeast and glucose-based nutrients separately to avoid premature reactions that are difficult to control. Then, oxygen will be supplied using a bubbler placed at the bottom of the main reaction container. Secondly, we discussed the outflow position. Although there is an agitator, reaction products may still deposit at the bottom of the container. Our original design had the outflow from the top of the container, but after consideration, we decided during the design phase to have the outflow from the side at the bottom of the reaction container. Another option is to use a pump to overcome the gravity and allow outflow extracted from the bottom area of the reaction container. We finalized the design of outflow during the building.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-14.png)

Figure 14: Final design of the outflow of the bioreactor

### Build

During the building phase, we completed the testing of all our pH meters, dissolved oxygen sensors, and thermometers. Additionally, all pumps, including two high-flow pumps and several low-flow pumps, as well as the sous vide, have been tested and are functioning correctly. We have also decided to use a plastic box as the chassis and several glass jars as containers. During testing, none of the three pH meters we had provided accurate readings, so new pH meters were ordered. The remaining sensors worked properly, and we are familiar with the related Arduino setup, with the relevant code already saved.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-15a.png)

Figure 15a: Testing the thermometer

In the next steps, once the new pH meters and calibration solutions arrive, we will complete the final testing. Then, we will begin assembling the different parts together. Anticipated challenges include mounting the sous vide. Because the edges of the chassis are not flat, the sous vide clips cannot hold it steadily. Plastic plates are planned for fixing. Additionally, we need to fix the pumps onto the containers, which requires drilling holes in the glass jar lids. Secondly, sterility of the reaction containers needs to be ensured. We will ensure the integrity of the seal for each container, and after building, we also need to test the seal of the entire liquid flow area. Additionally, we will use plastic wrapping to ensure the cleanliness of the internal environment. Finally, a plastic box is prepared for watertight storage of the Arduino boards, but after fixing the circuit boards, we will need to ensure watertight sealing with plastic cover.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-15a-3.jpg)
![](https://static.igem.wiki/teams/5059/engsuccess/fig-15b-1.jpg)

Figure 15b: Testing the bubbler and motors

![](https://static.igem.wiki/teams/5059/engsuccess/fig-15b-2.png)

Figure 15c: Running all 5 motors at the same time using Arduino, controlling different speeds

![](https://static.igem.wiki/teams/5059/engsuccess/fig-15d.jpg)

Figure 15d: Rough sketch of the circuit design of the motors

### Test

The testing process of our CSTR will incorporate different stages. First, we would check if the CSTR would function overall, meaning, the fluid flows throughout the bioreactor. If this means that we need to make sure that the flow going from the input reservoir to the main tank to the output reservoir is all even so that there are no overflows. This will be mainly determined from the pumps, so we will prioritize the consistency of all the pumps that we use to build our bioreactor. We can also use food dye and add it to the input reservoir, and see if the food dye gets transferred to the output reservoir to test if the water flows through the entire bioreactor. Next, we will test the systematic control of fluid temperature with sous vide, making sure that the temperature remains constant for the reaction to occur continuously. This can be tested easily by checking the sous-vide, as we tested already if the reading on the sous-vide is the same as a thermometer’s reading. To follow that, we will test the pH, oxygen, fluid glucose levels with base/acid inflow and pH meters, a bubbler, and glucose inflow, respectively. This can be done once all the sensors arrive and are tested to show that they read accurate measurements. Once all the components have passed necessary tests, we next can go through a major test to check the continuity of the flow ( ) to make sure that the production does not stop until we manually pause it. We can first test if the bioreactor runs for a day, and then test it longer through proof of battery; this way, we do not have to waste time just observing the bioreactor run for days. We will note any important observations and use them to make adjustments in the learning process.

### Learn

From the test results, we will make proper adjustments to different parts of the bioreactor to further maximize the production of UA. This process can go in many different ways as we will change the conditions of the CSTR depending on the result and the materials we have. During this phase, we will mostly work with what we have in our inventory to keep the testing and learning process moving forward. We will also collaborate with Wet Lab to make sure the UA production is stopped while making adjustments to our bioreactor ( ). If we encounter any contamination even despite well sterilizing before, we will halt the bioreactor and remove any feed. We will troubleshoot what caused the contamination and clean the whole bioreactor thoroughly and fix the problem that caused contamination. Once we learn how to fix any errors we find on the way, the cycle will begin again with new designs for an improved bioreactor. If we find more errors, we will repeat the process again with testing and learning until we produce our desired bioreactor.

## PD Cycle 2: Combining the Components Together

### Design

After completing the first cycle of DBTL, we initiated the second cycle of design based on the experience gained from the first round. The most significant difference in the design phase is related to the pH reservoir, which was not emphasized in the initial design. Starting from our needs, we need to maintain the pH level of the solution inside the reaction container within a range optimizing the yeast growth, which is pH 4-7. As a result, we need the ability to both raise and lower the pH of the solution. Therefore, we need to set up two containers: one for storing a basic solution and another for storing an acidic solution. This introduces an additional container requirement in our originally designed bioreactor, which also implies the need for an extra set of circuits for the motor.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-16a.png)

Figure 16a: Bioreactor focusing on the pH reservoirs

Moreover, the placement of each container and its corresponding motor was also determined during this DBTL cycle. Due to considerations of container shape and stability, we chose to place the motors for the glucose reservoir and the first pH buffer solution reservoir externally, for example fixed on the chassis rather than on the container lid. Additionally, since the main container needs to accommodate many sensors and an oxygen bubbler while also ensuring seal ability, numerous holes need to be made in the lid. The original container lid could not meet these requirements, so we decided to 3D print a lid that would satisfy both the need for holes and seal ability.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-16b.png)

Figure 16b: Motors mounted on the chassis

![](https://static.igem.wiki/teams/5059/engsuccess/fig-16c.png)

Figure 16c: The 3D printed Lid

Lastly, the circuit design did not undergo many changes compared to the first DBTL cycle. The only differences are that we removed the glucose sensor and separated the oxygen sensor from the Arduino system, as we decided to promote the reaction by providing an excess of glucose. Additionally, since the oxygen bubbler itself cannot control the flow rate, we removed the oxygen sensor from the Arduino system, and purchased a sensor that can display readings independently. Regarding the circuit boards, during this cycle, we optimized the positioning of different boards. This optimization facilitates the arrangement of the wiring and connections to various motors. It is worth mentioning that the design and building processes in this cycle were intertwined, with troubleshooting being our primary method for generating new design ideas.

### Build

During the building process, encountering issues is inevitable. Troubleshooting became the main theme of our second building cycle. In this cycle, we integrated the entire motor system and sensor system onto a single Arduino board for the first time. Previously, our tests were conducted separately. After merging everything onto one Arduino, the first issue we encountered was that the pH meter could not obtain readings and consistently displayed “NaN.” To solve this problem, we conducted multiple comparative tests, including disconnecting parts of the motor system, using a different breadboard, and eventually replacing the Arduino board. We discovered that the issue was with the Arduino board. However, a new problem quickly arose—the pH meter could not provide reliable readings. It gave a higher reading in acidic solutions than in neutral solutions, which was obviously incorrect. We then updated the pH meter's code and made it display the initial voltage reading on the probe. This allowed us to determine whether the issue was with the sensor probe or the algorithm that converts the voltage readings. Finally, we succeeded in enabling the pH meter to read reasonably accurate values within the pH range of 4-7.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-17a.png)

Figure 17a: Calibrating ph meter with buffer solutions

Next, after integrating the motor circuit system and the sensor circuit system, we made improvements to the overall circuit. First, using just one Arduino board, we employed two breadboards to separate the motor and sensor circuits, preventing potential interference. Then, based on the new design, we optimized the positioning of the L298N motor driver board to ensure that the wires connected to the motors had enough space for proper connection. In terms of wiring, we first distinguished different types of wires by color and then updated the wire types to ensure stable connections, as we had encountered sensor reading abnormalities due to poor wiring connections during testing.

![](https://static.igem.wiki/teams/5059/engsuccess/figure-17.png)

Figure 17b: Final circuit

Finally, in terms of coding, we integrated the motor system code with the sensor code. After ensuring they did not interfere with each other, we developed code to maintain solution balance. The key part was controlling the flow of the two pH solutions based on the pH meter’s readings. When the pH was too low, the alkaline solution motor would activate, and vice versa. We also tested the flow rate of the motors—how much liquid they could draw per second—and integrated this value into the code. Ultimately, the code allowed us to maintain data output from the pH meter and thermometer while all motors were operating. Since the oxygen sensor was not integrated into the Arduino system, its readings were displayed separately.

### Test

During this period, the primary testing objective is to conduct a dry run on the assembled bioreactor. The first task is to ensure that all the motors and pumps are functioning correctly. Next, we need to verify through testing whether the motors’ speed and rotation direction can respond to the sensor readings while the sensors are operating properly. Specifically, by modifying the code, we adjust the target pH range to simulate how the motors respond under different pH conditions. Ideally, the motors associated with pH1 and pH2 should activate when the pH value falls below or exceeds the target range. During the testing process, we measured the flow rate of each motor, that is, the volume output per unit of time. This allows us to relate the actual flow rate to the values in the code, giving us a better understanding of control conditions.

In addition, we tested the interaction between the Arduino, the motor circuits, and the sensor circuits. On one hand, we ensured that each motor was connected properly and that no coordination issues arose after all parts were integrated. During this process, the sensors needed to continuously provide up-to-date readings. This was not only to maintain control over the reaction environment but also played a decisive role in regulating the flow rates of the different reservoirs.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-18a.png)

Figure 18a: Dry Run

After completing the dry run, we proceeded with the wet run using dyed water. The primary goals of the wet run were, first, to ensure that the water flow in the system was normal, that water could successfully flow from different reservoirs into the main chamber, and could be collected through the outflow. In this phase, we mainly checked the operation status of each motor, i.e., the pumps, and the water flow rate. Specifically, we differentiated each reservoir by using colored water and mixed them in the main chamber, with the mixture flowing into the outflow container. This also demonstrated that the bioreactor had the feature of continuous flow.

Secondly, we filled the chassis with water and conducted a sous vide test, bringing the bioreactor to the target testing temperature, which is crucial for controlling reaction conditions. Another key test involved the sensors. We needed to ensure that the sensors could operate correctly and stably in the real working environment, especially the pH meter and temperature sensors, which needed to coordinate with the entire system. In the end, the wet run went smoothly, and we achieved the expected results.

Figure 18b: Wet Run

Moving forward, we plan to conduct more wet runs, including additional tests using dyed water. We will need to pay closer attention to the efficiency of pH regulation. Since we didn’t use a pH buffer this time, we may add one in future tests to examine how well the code and motor adjustments control the pH. Additionally, we plan to perform the final tests using yeast culture, which will be a real test of how the bioreactor operates and will help with future industrial applications. Finally, we will continue to optimize the entire bioreactor system, with a greater focus on sterility level, yield, and the overall operational efficiency.

### Learn

One challenge we encountered was in pin resource allocation and cable arrangement when connecting all the wiring to a single Arduino. The placement of the three L298N boards used to control the motors had to satisfy the requirement for short connections to the Arduino while also leaving enough space for the cables to reach each motor attached to the reservoirs. Ultimately, we connected the interfaces requiring continuous signal output to the PWM pins, while connecting others to the basic digital pins. The sensors were connected to the analog inputs. This arrangement ensured that the available pin count was sufficient, and the layout was logical.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-19a.png)

Figure 19a: Arduino Pins

Additionally, during hardware integration, we realized that motor vibrations could affect structural stability, so we updated the mounting design for some of the motors on the reservoirs. We secured the motors for the pH1 and glucose reservoirs to the chassis to meet our stability requirements. Finally, we optimized some of the wiring and code naming to ensure that after the dry test, the wet test would also proceed smoothly.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-19b.png)

Figure 19b: Motors Running

During the wet run, we encountered several troubleshooting issues. First, we discovered that one of the pumps of pH reservoirs wasn’t functioning, but we found this was related to the logic of how we controlled the pH. When the sensor is active, if the pH remains within range, the motor for the pH buffer reservoir does not activate. After temporarily modifying this portion of the code, we successfully got both pumps for the pH reservoirs running. Additionally, we tested the sous vide functionality and its impact on temperature. It worked properly and corresponded with the thermometer readings. Lastly, regarding the flow rate of the entire system, the current outflow rate is quite significant. However, in order to optimize the yeast culture, we may reduce the flow rate in the future to allow the yeast in the main chamber more time to culture properly.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-19c.png)

Figure 19c: Wet Run Prep

In the future, we will focus more on interpreting the results of the wet runs. We will also optimize the testing procedures and attempt to standardize them to minimize differences between trials. Moreover, we plan to correlate this with our modeling for validation, which will aid in scaling up. Finally, we want to emphasize that this will be a long-term process of optimization and scale-up, but the current bioreactor has already demonstrated the feasibility of this strategy.

## PD Cycle 3: Biosensor Initial Design

### Design

The next step to improve our bioreactor was to add a biosensor as a new component. We began to design our biosensor, but we initially began with designing the housing for the sensor to attach to the reactor. Thus, we first considered our initial sketch of the biosensor from earlier.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-20a.png)

Figure 20a: Biosensor Sketch

We wanted the housing to be simple and cheap to keep costs and complexity down. As such, we came up with a simple box-based design in Fusion 360. Each hole was sized to commercially-available electrodes we intended to use for the sensor design with the larger one intended for an inlet.

![](https://static.igem.wiki/teams/5059/engsuccess/fig-20b.png)

Figure 20b: Biosensor Housing CAD Model

The biosensor chamber would consist of three different electrodes: counting, reference, and working. Using voltmeter and ammeter, and connecting them to Arduino, the biosensor will recognize Ursolic Acid. This is done by tracking the voltage using Ohm's law. Tracking the voltage change using the transducer will help us recognize the UA activity.

### Build

For the building portion, we will focus on making the house for the sensor. With our housing design created, we 3D-printed the biosensor housing using our Prusa Mk.4 printer and PLA filament.

![](https://static.igem.wiki/teams/5059/engsuccess/figure21.jpg)

Figure 21: Printed Biosensor Housing

We then attached the outflow hose of our bioreactor to the inlet of the housing and began to move fluid through it. The biosensor would fit right into the housing to detect the UA activity, but for now, we will focus on whether or not there are leaks in the housing.

### Test and Learn

To test, we would add water to the reactor and let it flow through it, similar to the previous tests that have been done with water and food coloring. The whole point of this test run is to check for any leaks, with the addition of the biosensor added to our device. All of our other components of the bioreactor should work properly as they were tested in the previous cycles. This cycle focuses on the proper housing of the biosensor that has been attached to the bioreactor. Once we take future steps and complete building the biosensor, we would place them into the housing we tested. We would test the functionality of the biosensor with the yeast and all the necessary components needed to get the reaction started. Using our biorecognition model, we would measure the glucose level to determine UA’s activity. If the detection of UA works, the bioreactor and the sensor will be a success. Otherwise, we will test if there is any problem with the biorecognition model and test it outside of the bioreactor.

References +

[1] Dani, G. M., & Zakian, V. A. (1983). Mitotic and meiotic stability of linear plasmids in yeast. Proceedings of the National Academy of Sciences of the United States of America, 80(11), 3406–3410.

[2] Longtine, M. S., McKenzie, A., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., & Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast (Chichester, England), 14(10), 953–961. [https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U](https://doi.org/10.1002/%28SICI%291097-0061%28199807%2914%3A10%3C953%3A%3AAID-YEA293%3E3.0.CO;2-U)

[3] Eckert-Boulet, N., Rothstein, R., & Lisby, M. (2011). Cell Biology of Homologous Recombination in Yeast. Methods in Molecular Biology (Clifton, N.J.), 745, 523–536. <https://doi.org/10.1007/978-1-61779-129-1_30>

[4] Son, S.-H., Kim, J.-E., Park, G., Ko, Y.-J., Sung, B. H., Seo, J., Oh, S. S., & Lee, J. Y. (2022). Metabolite trafficking enables membrane-impermeable-terpene secretion by yeast. Nature Communications, 13(1), 2605. <https://doi.org/10.1038/s41467-022-30312-9>

[5] Ding, H., Hu, X., Xu, X., Zhang, G., & Gong, D. (2018). Inhibitory mechanism of two allosteric inhibitors, oleanolic acid and ursolic acid on α-glucosidase. International Journal of Biological Macromolecules, 107, 1844–1855. <https://doi.org/10.1016/j.ijbiomac.2017.10.040>

[6] Cerga, O., Borcan, F., Ambrus, R., & Popovici, I. (2011). Syntheses of new cyclodextrin complexes with oleanolic and ursolic acids. J Agroaliment Proc Technol, 17.

[7] Zhang, W., Ngo, L., Tsao, S. C.-H., Liu, D., & Wang, Y. (2023). Engineered Cancer-Derived Small Extracellular Vesicle-Liposome Hybrid Delivery System for Targeted Treatment of Breast Cancer. ACS Applied Materials & Interfaces, 15(13), 16420–16433. <https://doi.org/10.1021/acsami.2c22749>

[8] Zhang, L., Chan, J. M., Gu, F. X., Rhee, J.-W., Wang, A. Z., Radovic-Moreno, A. F., Alexis, F., Langer, R., & Farokhzad, O. C. (2008). Self-Assembled Lipid−Polymer Hybrid Nanoparticles: A Robust Drug Delivery Platform. ACS Nano, 2(8), 1696–1702. <https://doi.org/10.1021/nn800275r>

[9] Wang, J., Zhao, J., Yan, Y., Liu, D., Wang, C., & Wang, H. (2020). Inhibition of glycosidase by ursolic acid: In vitro, in vivo and in silico study. Journal of the Science of Food and Agriculture, 100(3), 986–994. <https://doi.org/10.1002/jsfa.10098>

[10] Inoue, Y., Motoda, A., Tanikawa, T., Takao, K., Arce, F., Lee See, G., Ishida, Y., Nakata, D., & Terao, K. (2023). Inclusion complexes of Ursolic acid with Cyclodextrin-based metal-organic Framework-1 enhance its solubility. Journal of Drug Delivery Science and Technology, 89, 104986. <https://doi.org/10.1016/j.jddst.2023.104986>

[11] Huang, Y., Quan, P., Wang, Y., Zhang, D., Zhang, M., Li, R., & Jiang, N. (2017). Host-guest interaction of β-cyclodextrin with isomeric ursolic acid and oleanolic acid: Physicochemical characterization and molecular modeling study. Journal of Biomedical Research, 31(5), 395–407. <https://doi.org/10.7555/JBR.31.20160073>

[12] Oprean, C., Mioc, M., Csányi, E., Ambrus, R., Bojin, F., Tatu, C., Cristea, M., Ivan, A., Danciu, C., Dehelean, C., Paunescu, V., & Soica, C. (2016). Improvement of ursolic and oleanolic acids’ antitumor activity by complexation with hydrophilic cyclodextrins. Biomedicine & Pharmacotherapy, 83, 1095–1104. <https://doi.org/10.1016/j.biopha.2016.08.030>

[13] Daniel Gietz, R., & Woods, R. A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods in Enzymology, 87–96. [https://doi.org/10.1016/s0076-6879(02)50957-5](https://doi.org/10.1016/s0076-6879%2802%2950957-5)

Figure 5a, 5b, 6b, 8c, and 11a were created with BioRender.com

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## 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](https://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 and what you learned from them. 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. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}