iGEM 2023 — Team cornell

{% extends "sidenav-layout.html" %}
{% block title %}Notebook{% endblock %}
{% block header %}

# Notebook

{% endblock %}
{% block sidenavvalues %}
[February 2023](#febuary)
[March 2023](#march)
[April 2023](#april)
[May 2023](#may)
[June 2023](#june)
[July 2023](#july)
[August 2023](#august)
[September 2023](#september)
[October 2023](#october)
{% endblock %}
{% block page\_content %}

# February 2023

January 29th to February 4th:

**Wet Lab:**  The subteam began to brainstorm, and each member developed their own project idea. They familiarized themselves with the role of Wet Lab within the team and became acquainted with the lab space. Learned about iGEM competition, how it works, how the Giant Jamboree works/takes place, and stated their interests (genetic engineering, modeling, writing, etc) on a Google Form

**Product Development:** The subteam conducted their first meeting, reviewing member expectations and the grading structure for those taking the project team “class” for credit, cell biology, and new member training. Members began participating in the team-wide brainstorming process, each developing their own project idea.

**Policy and Practices:** The subteam had their first meeting where we discussed the role of PnP within the team and discussed expectations for the semester. New members were given their first assignment of training. Members were required to research previous teams who were listed as model examples for IHP and evaluate their work, including what impressed them, areas that would have approached differently, and more. Members were introduced to brainstorming teamwide and everyone began developing their own project idea.

**Wiki:** The subteam had their first meeting where they went over grading, an overview of the coming year and team member expectations, and received their first onboarding project. Learned about HTML and CSS basics and discussed key aspects of a good website layout and design.

**Business:** The subteam met to discuss the importance of commercialization and entrepreneurship in a season-based competition such as iGEM. Criteria for Best Supporting Entrepreneurship was reviewed to instill a sense of direction in the subteam. Guidance over registration was issued. The subteam read the first two chapters of the business plan manual developed by previous Cornell iGEM Business teams. The subteam discussed the application of the readings to potential projects for the coming year.

February 5th to February 11th:

**Wet Lab:** Wet Lab continued to brainstorm, narrowing down on the more impactful and feasible project ideas. New members learned how to use PCR to amplify DNA and perform gel electrophoresis to analyze the results of their PCR. They also received an equipment walkthrough of the lab.

**Product Development:** Members continued to brainstorm, refining the team’s more feasible and impactful ideas. New members learned how to work with LTspice via an online tutorial.

**Policy and Practices:** New members were given their second assignment of training where they were required to research a previous Cornell iGEM project idea and develop a set of interview questions for it. They were then asked to interview the PD and/or WL leads as a mock interview in order to get more comfortable with the interview process, start to finish, and get some feedback from the interviewees. Members continued to brainstorm as a whole team, developing their ideas.

**Wiki:** Subteam members continued to work on their first assignment, where they were promoted to redesign the homepage from the 2018 Cornell iGEM wiki.
Project had to use a different color scheme and layout, include a nav bar that has a different way of organizing the content, and change 2-3 images. Returning members had to make it mobile responsive and add CSS transitions or JavaScript animations

**Business:** The subteam finished reading the iGEM Business Plan document and turned in reflections on what they learned from reading the document, what they knew, and what they were excited to learn about entrepreneurship in a synthetic biology/iGEM Jamboree context.

February 12th to February 18th:

**Wet Lab:** Wet Lab was introduced to crucial software such as SnapGene, Benchling, NEBioCalculator, etc while learning about the sequence design process and engineering design proces.Presentation delivered on design principles (in vitro, in vivo, in silico), and the importance of wet lab’s role in overall experimental design

**Product Development:** New members received in-person training on circuitry, including high and low pass circuits, and how to implement a breadboard. Members began developing the engineering implementation behind the final designs in the brainstorm process.

**Policy and Practices:** New members were given their third assignment of training where they were told to research and write about two areas: bioethics, legislation, or safety and risk assessment in relation to a brainstorming topic of their choice.

For brainstorming, now that projects had been narrowed down, members began focusing on developing more PnP specific work, including reaching out to experts for technical assistance.

**Wiki:** Subteam had their second subteam meeting where they went over each member’s website redesign of assignment 1. Each member discussed the reasoning and motivation behind their redesign as well as gave feedback to each other. Learned about design psychology and the basics of color theory and graphic design. Subteam members were introduced to Figma and the second onboarding project focusing on design.

**Business:** In order to ensure the general and subteam’s tasks were achieved fairly, the subteam divided up into 3 subgroups, with a group working on case reviews, sponsorship outreach, and grant research. The case review team had two members make a presentation on prior winners of Best Supporting Entrepreneurship and take questions from the subteam. The sponsorship outreach team brainstormed potential sponsors to fund our project through monetary donations or in-kind donations. The grant research team began compiling a list of applicable grants

February 19th to February 25th:

**Wet Lab:** February break, no lab work was performed.

**Product Development:** Members continued to refine project ideas for the 2023 season.

**Policy and Practices:** Members continued to reach out to interviewees. Some members began conducting interviews for the remaining brainstorming projects in order to gain a better understanding of the feasibility and necessity of each project. Sciencenter event was planned for the following week, so members began preparing for the event including pre-running the activities to ensure they would run well and buying the appropriate materials.

**Wiki:** February break, no subteam meeting. Members continued to work on their project 2.

**Business:** Each member completed a case study report of previous BSE winners over fall break and worked on completing a sponsorship packet for the upcoming season.

# March 2023

February 26th to March 4th:

**Wet Lab:** Performed liquid culture and plate preparation training to prepare for mini-prep and transformation protocols respectively.

**Product Development:** New members began MATLAB training. Members continued to refine the final brainstorming ideas.

**Policy and Practices:** Members continued to reach out to interviewees for their assigned brainstorming ideas. On March 4th, we hosted our first outreach event of the year: Sciencenter. We wanted to start early this year to provide us with the opportunity to host another event where we could improve upon the feedback and results of this event. Since we had yet to decide on our final project idea for the season, we hosted three different stations, all focused on general synthetic biology concepts.

**Wiki:** Members continued to work on their design project to gain more experience with design tools such as Figma. Members were given multiple websites to choose from where they had to (1) replicate this site’s layout in Fimga then (2) redesign the website.

**Business:** Four members presented their case studies to the subteam and took questions from the remaining members. In preparation for our giving day event in March, the remaining members continued building out a sponsor reachout sheet.

March 5th to March 11th:

**Wet Lab:**  Performed DNA cleanup training.

**Product Development:**
New members finished MATLAB training while working in collaboration with other members. New members began CAD training in Fusion 360 via a virtual tutorial.. Members continued to refine brainstorming ideas.

**Policy and Practices:**
Members continued to conduct interviews and reach out for future interviewees for their assigned brainstorming project. They reached out to stakeholders this week in addition to technical interviews.

**Wiki:** Each member presented their design project and explained the motivation behind their design changes. Each team member gave feedback to each other. Started discussing merch. What type of team merchandise we wished to create this year, and what type of designs. Subteam members worked on each designing a merch design for the team.

**Business:** Our subteam created a 300 character template for linkedin messaging representatives of different companies that were in our sponsor list. Our subteam filed an application to join the Cornell Engineering Innovation Competition on 3/24. Our subteam learned mail-merging techniques to prepare for giving day

March 12th to March 18th:

**Wet Lab:**
Performed more gel electrophoresis training using old parts from last year’s MicroMurals project.

**Product Development:**
Members continued refining potential project ideas by responding to team feedback. Members wrapped up CAD training.

**Policy and Practices:**
Members began working on lesson plans and slideshows for upcoming Splash! education event. This consisted of researching various areas of synthetic biology research that have serious ethical implications and developing questions and prompts for students to have debates. Members also attended training sessions in preparation for Splash! event. This consisted of receiving training on how to work with minors training and boundaries training for educators through Cornell University’s CULearn online platform. Members also went to training specific to the event hosted by the event coordinators about logistics and safety plans for the day.

**Wiki:** Subteam learned about Javascript and JQuery. Team voted on merch designs. The one which was chosen was completed in more detail by the subteam member.

**Business:** Our subteam was focused on contacting donors via linkedin, emails, and mail-merge in anticipation of our giving day event on 3/16/23. Our successful efforts led to our team raising a record $5,579.24

March 19th to March 25th:

**Wet Lab:** Performed transformation training using the plates prepped on March 4th (the protocol was successful, all plates had transformed colonies that produced asPink chromoprotein)

**Product Development:**
ENERGEM was chosen as the final project idea for the 2023 competition. All members gathered for a meeting discussing in depth the ENERGEM development process, especially the details surrounding enzyme encapsulation, Ansys modeling, and the bioreactor. New members learned how to implement arduino circuits and continued to refine their breadboarding skills.

**Policy and Practices:**
ENERGEM was selected as the final project. The primary concern for PnP is getting a better understanding of the areas in which 7-methylxanthines can be applied. Our preliminary research indicated that it could have important implications for agriculture or respiratory diseases. Members began reaching out to experts and stakeholders in these fields to gain a better understanding of the applications of 7-methylxanthines. Conducted an interview with Dr. Li. She recommended that we focus on enzymatic efficiency when selecting our pathway.

**Wiki:** Continued to learn about JavaScript. Team was assigned their final onboarding project where they were given a few pages from past iGEM teams to redesign, implementing the following features: create text over an image upon hovering over it, implement showing and hiding a div upon clicking on it, implement a feature when hovering over text/link, button changes color upon clicking on it

**Business:** Our team analyzed our 2017 BSE winning business plan and focused on preparing our Engineering Innovation competition slide deck using those insights

# April 2023

March 26th to April 1st:

**Wet Lab:**
With spring break approaching, no lab activity was performed. The focus of the subteam was finalizing brainstorming details to choose Team Cornell’s 2023 project.

**Product Development:**
Subteam members learned about their role in helping the team meet all medal criteria, and each individually researched a different criteria.

**Policy and Practices:**
Members continued to reach out to stakeholders in both agricultural and medical fields to gain a better understanding of its applications. Interview with Dr. Duplais indicated that agriculture would be a possible application; however, current pesticides are far more effective and that 7-methylxanthines would not be the appropriate class of methylxanthines to produce. Recommended we explore medical and dietary supplement applications instead.

**Wiki:** Subteam members continued to work on their final onboarding assignment, which would be due the week after spring break.

**Business:** Subteam members completed an assignment defining their understanding of ENERGEM and listed potential grant funding sources unique to ENERGEM

April 2nd to April 8th:

SPRING BREAK

April 9th to April 15th:

**Wet Lab:**
A joint meeting with product development was conducted to discuss the finer details of the 2023 project, ENERGEM, after it was voted on in group meeting. Encapsulation, ADE cycle, modeling, and reactor design/testing aspects were discussed and improved upon.

**Product Development:**
Individual medal criteria research was presented at the team meeting. All new and old project materials were moved into the new lab in Ward Hall. The subteam met and discussed potential uses for seven-methylxanthines, as well as where the caffeine reactant can be sourced from.

**Policy and Practices:** Conducted interview with Dr. DeLisa, who recommended that we make purity a focus of our project if we wanted to choose a medical application. We had an interview later with Dr. Alibi, who confirmed what Dr. DeLisa said. Members brainstormed a list of teams that we could possibly reach out to for collaboration based on their previous projects and experiences, as well as our relationship with teams from previous years. Members began to plan out the rest of the season based on medal criteria and the information from this year’s project.

**Wiki:** Subteam members continue to work on their JavaScript/JQuery project. With ENERGEM selected as our project, each subteam member was tasked to design a logo for the project, to be voted on by the entire team.

**Business:** Subteam members were assigned specific business plan aspects for ENERGEM to focus on and were responsible for brainstorming what their sections could like for ENERGEM

April 16th to April 22nd:

**Wet Lab:** Due to high conflicts, lab work was canceled this week; instead, literature review was the primary focus for Wet Lab.

**Product Development:**
Wet Lab and Product Development met to discuss the reaction process and the logistics behind future collaboration. Each member was assigned research surrounding either the bioreactor housing material or enzyme encapsulation methods.

**Policy and Practices:** Members began developing infographics for social media and publicity. The first one was on methylxanthines and chocolate to align with the timing of the team’s chocolate fundraiser. Members attended Splash! where we taught a class of bioethics, teaching students about synthetic biology and iGEM and hosting mini debates on different ethical issues in the field.

**Wiki:** Final logo design was selected by voting from the entire team and will be the ENERGEM logo moving forward. Each subteam member showcased their third assignment to the rest of the subteam. Each member gave their feedback for each project.

**Business:** With the entire business plan sectioned out, members drafted a few sentences under each header.

April 23rd to April 29th:

**Wet Lab:**
Tested alginate encapsulation procedure with brilliant blue dye to help product development begin their encapsulation work. Establishing protocols for this summer’s lab work. Protocols were written for adapted and directed evolution, enzyme immobilization via alginate bead encapsulation. Theory established for epPCR and his-tag affinity chromatography.

**Product Development:**
Members presented their individual encapsulation methods and housing material ideas at the subteam meeting. Members determined that some form of beading (likely sodium alginate beads) would be most effective in encapsulating enzymes.
Members began developing CAD models that would immobilize these beads.

**Policy and Practices:**
Conducted an interview with Dr. Bishara, a physician who specializes in neonatal pulmonary disease. She said that 7-methylxanthines are no longer treated for asthma and COPD on the pediatric level. She recommended that we research pharmaceutical supply chains and how the COVID-19 pandemic impacted them as a possible avenue of exploration. Members continued to brainstorm possible educational and outreach events for the summer and fall.

**Wiki:** With onboarding completed, we started planning for our Wiki. Started creating design options for our wiki (theme, color scheme, etc.) to be voted on by the entire team. Subteam members were each teasked to create a homepage banner for our wiki.

**Business:** An initial rough draft of the respiratory illness based ENERGEM was compiled.

# May 2023

April 30th to May 6th:

**Wet Lab:** Wet Lab reviewed the summer plan during the final week of the spring session. No in-lab work was performed.

**Product Development:** Members finalized their CAD models from the previous week and presented them to the entire team at the meeting. Members brainstormed improvements for the aforementioned CAD models.

**Policy and Practices:** Members began work on brainstorming ideas for an ENERGEM specific game. Finally decided on a game where players would be given different starting materials to come up with a final product of their own; teaching them the different components and the workflow of an iGEM project. Conducted an interview with Dr. Langer, a clinical pharmacist. She recommended we look at specific drugs with 7-methylxanthines listed as an ingredient. She supported the fact that we should look into COVID-19 supply chains. Conducted an interview with Dr. Milner, who confirmed that our current technical plan was feasible and provided some advice on our questions.

**Wiki:** Homepage banner chosen by a vote from the entire team. This will be the base of our design theme for the entire website.

**Business:** Peer review of drafted business plan was conducted, other subteams were contacted for feedback

May 7th to May 13th:

**Wet Lab:** Wet Lab was not in session until summer.

**Product Development:** Product Development did not meet this week.

**Policy and Practices:** PnP discussed starting up the summer class information including brainstorming possible activities. Since we were still deciding about whether we would host the class in person or online, we were primarily focused on how to navigate a hybrid environment while being as engaging as possible.

**Wiki:** Created and went over our work timeline for summer break. This included who was going to work on game development (for PnP) as well as those working on the base of the wiki.

**Business:** A spring 2023 ENERGEM rough draft was completed

May 14th to May 20th:

FINALS WEEK

May 21st to May 27th:

BREAK

# June 2023

May 28th to June 3rd:

BREAK

June 4th to June 10th:

BREAK

June 11th to June 17th:

**Wet Lab:** Wet Lab was not in session.

**Product Development:** Product Development did not meet this week.

**Policy and Practices:** PnP did not meet this week.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Business team is on break during the summer session

June 18th to June 24th:

**Wet Lab:**
With the start of the summer, preparatory work was conducted which included inventory, autoclaving materials, and defrosting the freezers.

**Product Development:** Members inventoried all available materials in the Ward lab.

**Policy and Practices:** First priority of the summer was to look into marketing for the summer class, which included reaching out to various local middle and high schools as well as schools throughout upstate New York. Given that we were worried about responses as many schools were already out for the summer, we began planning how we would adapt our content to a completely virtual mode. Members began to reach out more interviewers for possible stakeholder interviews to understand more about other avenues for applications of 7-methylxanthines given the interviews at the end of the semester that informed us that they were not as readily used for respiratory diseases any longer.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Business team is on break during the summer session

# July 2023

June 25th to July 1st:

**Wet Lab:** Wet Lab began work on PCRing newly ordered parts and attempting Gibson Assembly to acquire our full plasmid. The PCR resulted in low concentrations and the Gibson failed.

**Product Development:** Jonathan began modeling the bioreactor in ANSYS. Jatin and Sonia began working on the bioreactor. They developed an experimental method for testing residence time. They purchased PVC to house the reactor.They refined CAD models from the spring.Rita and Rachel began working on enzyme encapsulation.They practiced forming beads by dropping sodium alginate solution into calcium chloride solution.They created beads from incrementally increasing concentrations of sodium alginate to test bead properties.

**Policy and Practices:** Members worked on finalizing the pathways that we would include in the game. They were all based on former iGEM projects. Outline was finalized and sent to the Wiki subteam to start work early on. Given that we received minimal responses to our class, we decided that we would develop lesson plans to post online for people to access if they would like. Members all focused on one specific subteam and their work and came up with possible projects that children could do at home, aligning with the lesson plan.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Business team is on break during the summer session

July 2nd to July 8th:

**Wet Lab:**
Wet Lab conducted some troubleshooting and continued to work on getting the Gibson Assembly to work. Highlighted some potential design issues and re-ordered primers to rectify the issue and potentially raise concentrations. We modeled a basic Poiseuille’s Tube in ANSYS to gain a better understanding of the pressure and velocity gradients of a basic tube. Modeling plan was revised and refined slightly to better accommodate for any hardware changes.

**Product Development:**
Jonathan continued working on ANSYS modeling. Jatin and Sonia continued bioreactor work: They created a mock-up sketch of the reactor design. They began slicing CAD models. They began the process of building a mock reactor. Rita and Rachel continued encapsulation work: They wrapped up analysis of the sodium alginate concentration experiments. They created calcium chloride solutions of different concentrations and began creating beads with each solution. They began brainstorming a test enzyme to use, but Wet Lab’s stock of enzymes that are both cheap and easy to implement is running low. They may wait until Wet Lab produces the actual lysate.

**Policy and Practices:** Members began reaching out to ethics experts to gain a better understanding of the policy work needed for ENERGEM. Given that the interviews at the end of Spring semester were not very enthusiastic about an application for respiratory disease, we decided to incorporate this expert advice and continue to explore other avenues. Our research indicated myopia, specifically childhood myopia, as a possible area where 7-methylxanthines would be effective, so members began reaching out to ophthalmologists and researchers in the field.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Business team is on break during the summer session.

July 9th to July 15th:

**Wet Lab:**  With new primers, Wet Lab performed PCR amplifications of inserts and backbones and re-attempted Gibson Assembly.Worked on documentation for an in-house documentation deadline. We added a sphere of finite volume to our ANSYS model, mimicking one encapsulated bead in the tube.

**Product Development:** Continuing build process of engineering cycle for the reactor: locating 3D printer options for immobilization design. Testing varying calcium chloride concentrations for encapsulation method to determine ratio best suited for our purposes. ANSYS modeling difficulties with lab access

**Policy and Practices:** Members continued to reach out to stakeholders for possible applications in myopia. Members began working on researching policy aspects to the project, including understanding important legislation, bioethical issues, and safety and risk assessment requirements. Members began evaluating our project using different human practices frameworks, building upon the work of Exeter iGEM. We used ELSA, AREA, and SEG. Members reached out to ethics experts to gain feedback on the framework we developed last year: EUDI. Once we gain advice on our framework, we will apply it to our project.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Business team is on break during the summer session.

July 16th to July 22th:

**Wet Lab:** Continued Gibson Assembly attempts with transformation and gel proof of concept. Shifted focus to troubleshooting the protocol - highlighted an issue with our backbone.

**Product Development:** Completed single bead model in laminar flow conditions, results are confusing and will continue testing other options. Encapsulation method finished testing and 2 combinations of concentrations were determined as most viable, waiting for enzymes to fully test encapsulation constraints. Reactor built with PVC and plastic beads to model future produce encapsulated beads with enzyme.

**Policy and Practices:** Members brainstormed ideas for our summer experiment video series, which would consist of how-to videos to show experiments to younger kids related to important iGEM concepts. Members began writing scripts for the videos. Members began researching and writing for our methylxanthines application handbook that will include information on all the methylxanthines involved in the caffeine degradation pathway, not just 7-methylxanthines as our work can be applied to producing other chemical products in the pathway, not just 7-methylxanthines.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development. Base design and formatting for wiki was complete.

**Business:** Business subteam met with Joe Price, CEO of Evolutor, who offered perspectives of how to scale a start-up from an iGEM team (Sheffield) and offered a knowledge exchange of increasing the metabolic rate of our proteins with our sponsorship package.

July 23th to July 29th:

**Wet Lab:** Continued troubleshooting with extra positive and negative controls to pinpoint the issue with our backbone.

**Product Development:** Starting documentation process for all sub-projects. Moving forward with ANSYS model and incorporating turbulent flow dynamics. Encapsulation methods are lengthy: brainstorming potential auto-bead maker options. Finalizing 3D printed immobilization method and scaling as well as procurement of a printer.

**Policy and Practices:** Members got together to film the videos for the Summer Experiment Series. Team finalized 5 different videos which will cover areas ranging from DNA to polymer chemistry. Members began editing videos to be ready for distribution in mid-August. Members continued to work on our applications handbook.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Business subteam met with Charlie Maddox, CEO of Enzymatic Inc, who advised our team on taking steps to protect our IP and scale our commercial processes.

# August 2023

July 30th to August 5th:

**Wet Lab:** PCR amplified a distribution kit backbone source and performed a gel of Gibson Assembly, old, and distribution kit backbone sources to confirm or repute backbone presence. Discovered a potential issue with the DNA cleanup step. Ordered a new pSB1C3 backbone for current and future team use. GenScript free plasmid was ordered after winning the giveaway of the fully proposed plasmid created by CUGEM.

**Product Development:** Turbulent model is being worked on, one layer meshing needs work as well. Have to rescale immobilization method due to printer constraints - moving forward with testing in a 10 mL syringe tube to model future scale-up designs. Dimensional analysis training for potential reactor calculations
Starting large scale encapsulation to produce more beads for testing

**Policy and Practices:** On August 5th, we hosted our second Sciencenter event of the competition season. We continued with the same events from the last session, just adding an extra station that was specific to ENERGEM. Event at Kendal @ Ithaca was finalized this week after emailing with their event coordinators and sharing an event advertisement to include in their September newsletter.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Business subteam met with PacBio to discuss sponsorship opportunities.

August 6th to August 12th:

**Wet Lab:** DNA cleanup troubleshooting to observe the problem of backbone disappearance during Gibson Assembly. Liquid cultures of new pSB1C3 backbone plasmid were created

**Product Development:** ANSYS one bead model in the works and continuing turbulent flow model. Testing auto bread maker ideas for the encapsulation protocol - most likely using a Harvard Apparatus syringe infusion pump. Waiting for materials to assemble new mock reactor.

**Policy and Practices:** Continued to finish up editing videos for the summer science experiment video series. Members finished their assigned informational graphics on 7-methylxanthines, the applications of methylxanthines, synthetic biology, and more in preparation for our upcoming event at the Farmer’s Market. Event at Longview Senior Living Community was finalized this week after Zoom meetings with their event coordinators, which included sharing plans for content and discussing scheduling.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** Subteam met with representatives from the Office of Technology Licensing to learn more about specific protections that we will need for our project.

August 13th to August 19th:

**Wet Lab:**  Wet lab on break between summer and fall session.

**Product Development:** Product Development on break between summer and fall session.

**Policy and Practices:** Conducted interviews with various stakeholders including ophthalmologists and drug development companies

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development. Wiki Mockup complete

**Business:** Our subteam met with Telesis Bio to discuss sponsorship opportunities and struck a tentative agreement for a BioXP system to be installed on campus.

August 20th to August 26th:

**Wet Lab:** Wet lab on break between summer and fall session.

**Product Development:** Product Development on break between summer and fall session

**Policy and Practices:** This week we took a break before we hosted our event at the Ithaca Farmers Market on August 20 where we learned a lot from those we spoke to there.

**Wiki:** Subteam continued to work on assigned summer deliverables including game and website development.

**Business:** The subteam discussed a new vision for the project given the summer updates of the project. A new table of contents was established and members assigned to it

# September 2023

August 27th to September 2nd:

**Wet Lab:** Performed Gel Electrophoresis on old NdmAB parts, problem still persists. As troubleshooting, re-PCRed old and new part of NdmABD, performed DNA cleanup, and digested pSB1C3 backbone

**Product Development:** Finalizing turbulent flow ANSYS model. Syringe infusion pump testing was positive. Attempting to procure mock enzyme for bead encapsulation testing. Building new reactor model with 10 mL syringe replacing existing PVC tube

**Policy and Practices:** We continued to work on organizing and planning for our upcoming outreach events. Worked with Wiki to finalize what we wanted for the game. Continued to reach out to possible government officials

**Wiki:** First in person subteam meeting of the semester. Wiki and Game dev both updated on summer progress. Game development was slightly behind schedule but no pressing issues. Constant communication with other subteams about documentation deadlines

**Business:** Subteam met with Management Library to learn how to effectively leverage Cornell’s resources for more strategic research. Team further outlined vision for 2023 fall business plan

September 3rd to September 9th:

**Wet Lab:** Nano Dropped all new PCR products. Performed Gibson Assembly of old products. Received new pSB1C3 backbone and began to transform, miniprep, and PCR it.

**Product Development:** Reactor with syringe was constructed. Immobilization method was scaled down and printing this week. ANSYS model finalized

**Policy and Practices:** Continued to work on applications handbook. Finalized slideshows for the nursing home events. Started to reach out to the elementary school to organize event at the beginning of October

**Wiki:** Continued to work on Wiki. Started designing page banners. Created new templates to go with these banners. Worked on promotional video.

**Business:** Subteam prepared to present our project to the Cornell eLabs accelerator program on 9/5/2023. Business model section was renovated.

September 10th to September 16th:

**Wet Lab:** Performed Gibson with new backbone and NdmABD parts

**Product Development:** Met with Professor Bhaskaran to talk about future ANSYS considerations. Reactor with syringe tested with immobilization method and produced encapsulated beads - need a new testing procedure due to error in size of molecules.

**Policy and Practices:** We hosted our event at Kendal at Ithaca - used the feedback from this event to work on improving our event at Longview

**Wiki:** Each subteam member was assigned tasks regarding the website. From design to feature implementation, as well as continue to work with PnP and game dev.

**Business:** Subteam met with Dr. Zatrenau of Rochester Regional Health to understand how to leverage physcian to patient relationships to boost drug sales; understood FDA strategy and regulation

September 17th to September 23rd:

**Wet Lab:** As Gibson is worked on, Wet Lab shifted focus to help PD with reactor testing via transforming bacteria with pink chromoproteins. Created glycerol stocks of approximately 15 tubes of chromoprotein expressed E. Coli. Performed Gel Electrophoresis of new parts’ Gibson → failed

**Product Development:** Discussed with Wet Lab future steps for model of proof of concept of the encapsulation beads. Updating documentation of overall parts of the project. Working on finalizing new ANSYS model after discussing with Professor Bhaskaran how to improve it

**Policy and Practices:** Hosted our event at Longview Senior Living Community.

**Wiki:** Found issues with game development. Reverted much of what was created and almost completely started over. Constant communication with other subteams about documentation.

**Business:** Subteam met with Dr. David Putnam, Dean of Engineering Innovation who taught us the mechanisms of Series A-C funding and how to leverage Cornell’s incubators to turn our project team into a full fledged company

September 24th to September 30th:

**Wet Lab:** Updated documentation of wet lab work so far in preparation for Wiki Freeze

**Product Development:** Waiting for ANSYS model to complete computation. Started testing proof of concept for encapsulation methods using chromoproteins. Cleaning up reactor design to final model for easiest operation.

**Policy and Practices:** Planned out the remainder of the elementary school. Worked on documentation in preparation for wiki freeze

**Wiki:** Each member assigned multiple pages to complete implementation of. Continued to have constant communication with other subteams to discuss wiki design and implementation.

**Business:** Finalized business plan.

# October 2023

October 1st to October 7th:

**Wet Lab:** Continued documentation for wiki freeze. Began encapsulation preparations for the bacteria with chromoproteins for PD’s reactor testing

**Product Development:** Finalized ANSYS model and writing up associated documentation. Continued various encapsulation proof of concept testing

**Policy and Practices:** Hosted event at the elementary school.

**Wiki:** Continuous work up until WikiFreeze.

**Business:** Finished business plan.

October 8th to October 14th: WIKIFREEZE!!!

**Wet Lab:** Finalized documentation for wiki freeze. Created plan to continue working on ENERGEM post-wiki freeze.

**Product Development:** Finalized documentation for wiki freeze. Continued working on scaled-up version of reactor.

**Policy and Practices:** Finalized documentation for wiki freeze.

**Wiki:** Finalizing Wiki and Game for WikiFreeze.

**Business:** Finalized documentation for wikifreeze.

{% endblock %}

{% extends "sidenav-layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block header %}

# Engineering Success

{% endblock %}
{% block sidenavvalues %}
[Wet Lab](#wl)
[The Overview](#wl-ov)
[Cycle 1](#wl-cycle-1)
[Cycle 2](#wl-cycle-2)
[Cycle 3](#wl-cycle-3)
[Cycle 4](#wl-cycle-4)
[Cycle 5](#wl-cycle-5)
[Product Development](#pd)
[Encapsulation](#pd-enc)
[Cycle 1](#pd-cycle-1)
[Cycle 2](#pd-cycle-2)
[Reactor Design](#pd-rd)
[References](#references)
{% endblock %}
{% block page\_content %}

# Wet Lab

## The Overview

The adaptive and direct evolution cycles are the machinery that produce mutations in the proteins produced by NdmA and NdmB in order to produce variants of interest. NdmA catalyzes the N1-demethylation of caffeine to produce theobromine and formaldehyde, while NdmB catalyzes the N3-demethylation of theobromine to produce 7-methylxanthine and formaldehyde.

Directed evolution induces mutagenesis in each generation by first conducting EP-PCR on NdmB and PCR on the backbone, then performing Gibson Assembly on the pSB1C3 backbone with GFP, NdmA, and NdmB. Through EP-PCR, random mutations are induced, while the selectivity of primers specifies the region of DNA. After a successful Gibson Assembly, the NdmAB plasmid is transformed into the BL21 strain of *E. coli* and is cultured for 2-3 days. Next, Miniprep is conducted on the samples; then, the samples are put through EP-PCR and the process is repeated. Preparation for the ADE cycle is now complete.

Adaptive evolution induces mutagenesis one time, then allows natural mutagenesis to take over. First, caffeine cultures will be filled in 24-well plates, and the media will be changed every couple days. Then, 100 µL of the culture will be recultured in a new 24-well plate to allow natural mutagenesis to occur. This process allows each well to be more uniform, ensuring that a sample taken and sequenced will be representative of the majority of bacteria present. However, with adaptive evolution, there is no guarantee of a successful mutation. Thus, our combined approach of adaptive/directed evolution (ADE) ensures the generation of a large library of mutants while ensuring consistency and uniformity. Both aspects are very important to our project.

The directed evolution process can begin after 2 or 3 recultures. Using the top 24 cultures from the 5 plates, take 100 µL of each sample, and miniprep the samples. EP-PCR is done on the samples, using different concentrations of salt depending on the intended amount of variation. The samples are then cultured and recultured in the same method as was done for AE. With directed evolution, more variations are created in each cycle, increasing variation. This in turn increases the chances of a mutation occurring that would suit the purpose of this project. However, this excessive variation can become uncontrollable. To counter this, heavily controlled EP-PCR is needed.

After the mutants are isolated, enzymes will be produced by the bacteria, which will be immobilized within encapsulated calcium alginate beads to protect enzyme activity throughout the bioreactor process. The enzymes are dissolved in a sodium alginate solution to a concentration of 2 mg/mL concentration, then dropped into a calcium chloride solution via a syringe to form droplets, which are obtained after 30 minutes of incubation through filtration.

## DBTL Cycle 1: Gibson Assembly

The design of the plasmid used a His-tagged NdmAB-GFP Plasmid. In the first trial, while building this plasmid, the His-tagged NdmAB inserts were unavailable, so they were substituted with NdmAB inserts. While waiting for the His-tagged parts to come in, we moved forward with creating a vector with NdmA and NdmB+GFP via Gibson Assembly to validate the Gibson Assembly procedure. The confirmation of the first trial was done using gel electrophoresis, which displayed that the assembly was not successful. A potential reason for this was due to the NdmA and NdmB parts we ordered not having the sticky ends needed at the time of assembly. The design remained the same, but we PCR amplified the parts to add the sticky ends and performed DNA cleanup on NdmA and NdmB+GFP prior to use. After testing the results of the DNA cleanup using the nanodrop, it was noted that the concentrations of the NdmA and NdmB+GFP were low: 19.9 ng/µl and 14.7 ng/µl respectively.

Through troubleshooting, it came to our attention that the DNA backbone used in our first trial was not an empty plasmid and therefore did not match the designed primers. We switched to a new backbone for trials using the traditional pSB1C3 from previous years. To prepare the vector, we performed a T4 DNA Ligase Protocol, without adding any inserts, to confirm a circular plasmid. The plasmid was then linearized through an EcoRI-HF digest protocol to get it ready for PCR amplification. Since the pSB1C3 had already been cut in a previous year, the backbone ligation and digest were needed to get a backbone DNA cut at the desired site. To test, DNA cleanup and gel electrophoresis were used and ultimately did not work. Upon reflection, we realized that the T4 DNA Ligase was not added last, as specified by the protocol. The ligation was repeated with the steps in the correct order. EcoRI cutting was also repeated, but with the materials being diluted to scale down by a factor of 20 to both conserve materials and better match the ratios of NEBioLabs protocol.

While performing DNA cleanup in this trial, the NdmA was diluted to 80 µL total so each µL had 10 mM instead of 100 mM. After using the nanodrop machine to test the DNA cleanup and PCR, we noted an over 10-fold higher concentration: 161.2ng/µL for NdmA, 104.7ng/µL for NdmB+GFP, and 140.6ng/µL for the backbone pSB1C3. We ran gel electrophoresis to confirm the DNA parts. Wells B and C roughly match up with the expected length of the respective sequences, and there are no other bands, suggesting that we obtained a pure sample of both target sequences NdmA and NdmB+GFP. Since we were able to obtain great samples of our DNA parts, we proceeded to move onto combining the parts into the pSB1C3 vector backbone.

![](https://static.igem.wiki/teams/4580/wiki/es-fig1.png)

Figure 1: From left to right, well 1 is NEB 1kb DNA ladder, well 2 is PCR-amplified NdmA (1.2kb), well 3 is PCR-amplified NdmB+GFP (1.9kb), and well 4 is the negative control.

Wells B and C roughly match up with the expected length of the respective sequences, and there are no other bands, suggesting that we obtained a pure sample of both target sequences NdmA and NdmB+GFP. Since we were able to obtain great samples of our DNA parts, we proceeded to move onto combining the parts into the pSB1C3 vector backbone.

To continue building, we did a Gibson Assembly, followed by a transformation. To test the success of this, we observed both a negative control of just BL21 *E. coli* cells and NdmAB Full Plasmid in the same strain plates. Neither of the plates showed any growth. We concluded the lack of growth was due to the Gibson Assembly failing as the negative control did not grow colonies as expected. A positive control of competent cells transforming a known plasmid would have confirmed that the BL21 cells were working and that the transformation protocol is adequate. However, we avoided the positive control as the gel electrophoresis revealed gel bands in the wrong place, suggesting that the Gibson Assembly failed. Through more troubleshooting, we repeated the Gibson assembly with adjusted times for annealing. In one assembly, we ran for 45 minutes, and in the other assembly, we ran for an hour. We then ran both products of the assembly in a gel electrophoresis. Both products ran the same in the gel, with separate NdmA and NdmB, but no backbone vector visible.

The first part of the project focused on getting NdmA and NdmB. However, the importance of NdmD in the pathway was overlooked as it is not directly involved in the demethylation pathway. By doing further literature review, it was revealed that NdmD plays an important role in proton transfer that would allow NdmA and NdmB to work more effectively.

## DBTL Cycle 2: Backbone Attack

Once we received the his-tagged NdmA, NdmB, and NdmD parts, we ran a Gibson Assembly on these constructs, this time using a newly ordered redesigned pSB1C3+RFP backbone in a *E. coli* agar stab. The reason behind this was to increase the cohesion between pSB1C3 and the DNA parts in order to raise the efficiency of the Gibson Assembly. The culture was plated on LB with a 1000:1 ratio of chloramphenicol, which helped confirm the presence of RFP.

![](https://static.igem.wiki/teams/4580/wiki/es-fig-2.png)

Figure 2: pSB1C3 culture on LB+chloramphenicol (1000:1) confirms presence of RFP and the target plasmid

Two cell cultures (pSB1C3 A and pSB1C3 B) were inoculated into a liquid culture before the cells were lysed and cleaned. PCR was performed on the lysate and the newly obtained 6x his NdmA/B/D parts to obtain a good purity of the backbone for the Gibson Assembly. The concentrations and purity of the products are listed below.

| **DNA Part** | **Concentration (ng/µL)** | **260/280 Ratio** |
| --- | --- | --- |
| NdmA | 462.5 | 1.84 |
| NdmB | 700.9 | 1.84 |
| NdmD | 515 | 1.82 |
| pSB1C3 A | 567 | 1.81 |
| pSB1C3 B | 483.8 | 1.83 |

Figure 3: Concentrations of all DNA parts

The PCR amplification seems to have gone well for all the DNA parts. We then proceeded with a Gibson Assembly attempting to insert the 6x His-NdmA/B/D into the pSB1C3+RFP backbone. We performed three Gibson Assemblies, one using pSB1C3 A and the other using pSB1C3 B. The last one was a negative control with just the backbone and no additional parts. We performed gel electrophoresis to confirm success shown below.

![](https://static.igem.wiki/teams/4580/wiki/es-gel-ndmd.png)

Figure 4: Gel Confirmation of PCR amplified NdmA/B/D parts, new backbone trials A and B, and newly attempted Gibson Assembly trials A and B in that order of the wells.

Oddly enough, only NdmD showed a visible band revealing there may be an issue with the primer design or the PCR protocol. The issues of NdmA/B and the pSB1C3 backbone not showing up were reflected downstream in the Gibson trials in wells 7 and 8 not showing up either. This was noted for future troubleshooting of the Gibson Assembly protocol.

## DBTL Cycle 3: Product Development is Calling!

After continuous difficulty utilizing the backbone, the focus shifted to assisting the product development subteam with enzyme encapsulation. The new backbone including mRFP1 was used to e test our sodium alginate beads. The red fluorescence proteins were transformed via liquid culture. 20 falcon tubes containing 15mL of liquid culture with 1:1000 ratio of chloramphenicol (15 µL) were centrifuged at 1200 rpm for five minutes using a Beckman Coulter Allegra X-22R. A couple of the tubes were re-centrifuged for an extra 3 minutes in order to form a visible pellet. Out of the 20 tubes used for the liquid culture, 19 were able to develop large *E. coli* pellets visibly showing a pink tinge to confirm expression of RFP. The one liquid culture that was unable to grow pellets was kept in the same tube to incubate longer. For the rest of the tubes, the old LB was thrown out and replaced with a new LB. After two days of incubation, there was sufficient growth of the pellets in all 20 tubes. A nanodrop was performed to ensure the concentration of the pellets was sufficient. After a high concentration was confirmed, glycerol stock was used to store the bacteria. This was done by first spinning down the cells and removing the excess LB. 1 mL of LB was then used to resuspend the liquid culture followed by adding 1 mL of 50% glycerol. This was stored in the -80℃ freezer for future uses. The liquid cultures of pSCB1C3-mRPF1 are shown below.

![](https://static.igem.wiki/teams/4580/wiki/es-liquid-culture.png)

Figure 5: Successful liquid culture of pSB1C3 with high expression of mRFP1

During this process, it was also discovered that a plasmid backbone construct had been delivered from GenScript a few weeks prior. As a result, attempts to create our own plasmid were momentarily paused. Instead, we shifted focus onto using the plasmid already containing the NdmA/B/D from GenScript for transformations.

## DBTL Cycle 4: GenScript to the Rescue

With the GenScript raffle, Team Cornell was able to order our first iteration of pENGM42, the finalized plasmid design for our team. While the final design includes mRFP, the first iteration includes GFP but both fluorescence proteins serve the same qualitative purpose of viewing expression of NdmA/B/D. To confirm the plasmid received was pENGM42, Team Cornell did a transformation and gel electrophoresis in parallel. The transformation confirmed chloramphenicol resistance within the plasmid while the gel confirmed the plasmid was the proper size. The transformation was done in quadruplicate. A picture of the gel is shown below where well 1 is pENGM42 while well 8 is the ladder. Other parts were also tested within this gel as told below.

![](https://static.igem.wiki/teams/4580/wiki/es-success-gel.png)

Figure 6: Successful gel electrophoresis of pENGM42 along with pSB1C3 backbone in well 5

| Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| pENGM42 | pSB1C3 Gibson B | NdmD | pSB1C3 Gibson A | pSB1C3 + mRFP1 A | NdmB | NdmA | 1 kb ladder |

Figure 7: Wells of the gel electrophoresis of Figure 6

While there is a smear, the general size of pENGM42 correlates to what was expected. The same issue of NdmB and NdmA not appearing in the gel can be seen and well 4 shows multiple parts but were not combined into the desired full plasmid. A couple issues could have been the voltage (ran at 135 V) was too high causing smears and the same Gibson Assembly issues as before. To retest pENGM42, we performed liquid 12 cultures (3 from each plate) of the successful transformation of pENGM42 to qualitatively see if GFP conjugated with NdmB would properly express. Successfully, bright green from each plate was shown from the cell cultures when placed under UV light as shown below. If GFP was able to keep its function, it can be assumed that the conjugation would not affect the function of NdmB, the protein of interest, as well.

![](https://static.igem.wiki/teams/4580/wiki/es-liquid-culture-glow.png)

Figure 8: Liquid cultures expressing GFP from pENGM42 done in quadruplicate

These cultures were then mini prepped to collect pENGM42 and had a DNA gel performed once again with a well per trial. After 50 mins at a lower voltage (120 V), the gel confirmed each plate successfully expressed the proper plasmid and solidified that the design received from GenScript was the proper design from Team Cornell.

![](https://static.igem.wiki/teams/4580/wiki/es-gel-confirmation.png)

Figure 9: Gel Confirmation of pENGM42 being expressed in all 4 transformations

## DBTL Cycle 5: Isolating Enzymes and Chasing the Clock

With wiki freeze approaching, our web lab became even more determined to obtain the enzymes of interest (NdmA/B/D) to be encapsulated for the next phase of testing enzyme encapsulation and the reactor. As time closes in, each plate’s liquid cultures were combined and spun down to prepare them for lysing. To save materials, all the cells were only suspended in 5 mL of LB per plate and had the optical density of them taken in order to properly add the correct amount of lysis buffer. The OD600 values are shown below.

|  | pENGM42: A trials | pENGM42: B trials | pENGM42: C trials |
| --- | --- | --- | --- |
| OD600 | 0.428 | 0.348 | 0.353 |

Figure 10: OD600 values of pENGM42 liquid cultures combined

Following the lysis protocol for NEBio Labs, the lysates of each plate were created and the proteins were spun down for storage at 4C as shown below.

![](https://static.igem.wiki/teams/4580/wiki/es-microtubes.png)

Figure 11: Spun down proteins ready for storage from lysis protocol

Our next steps after wiki freeze will be to follow our designed protocol for Ni-NTA Affinity Chromatography utilizing the 6x his tags of NdmA/B/and D to isolate them from the rest of the proteins expressed by our bacteria. These will then be ready for encapsulation by the product development subteam where they will interact with caffeine flowthrough to convert it into 7-MX. In parallel, epPCR will begin using our circular plasmid method to generate mutations in NdmB and begin the adaptive/ directed evolution (ADE cycle). Once those are prepared, they will follow the same process of enzyme isolation to be encapsulated and run through the reactor by product development for 7-MX generation. More information will be provided during the 2023 iGEM Grand Jamboree presentation of Team Cornell of how far our next steps take us!

# Product Development

## Encapsulation

At its very core, this idea consists of an enzyme and its reactants. We want to flow a solution of caffeine, the caffeine breakdown enzyme, or both of these molecules through our bioreactor such that the product (or a solution that consists primarily of product) flows out.

The goal of this design process was to determine the superior way to allow the enzymes and reactants to interact inside our bioreactor. The frequency and speed of reactions, as well as the cost and ease of use of materials were all considered while determining the structure of the bioreactor.

We know that enzyme immobilization while we flow reactants into and products out of the bioreactor is the most conceptually and mathematically simple approach to this bioreactor [4]. Immobilizing enzymes while reactants flow through at a regular rate allows us to calculate the residence time that reactants spend in the reactor interacting with an enzyme, the amount of reactants that each enzyme encounters on average, and more.

## Cycle 1

Several methods of enzyme immobilization were considered during the design process. Utilizing magnetic nanoparticles, hydrogel bead encapsulation, and entrapment in a porous hydrogen-bonded organic framework were all researched. We spoke to an expert in reaction mechanisms, Dr. Phillip Milner, an assistant professor in the department of Chemistry and Chemical Biology at Cornell University, to help us determine which encapsulation method to use. Dr. Milner emphasized that more complex methods of enzyme immobilization are only ever used as a last resort in most products. If a cheaper, simpler way of immobilizing enzymes is possible, that is the method we should pursue. With this in mind, we decided to move forward with hydrogel bead encapsulation.

More specifically, we know that sodium alginate, when dropped into calcium chloride, will form a porous bead that allows reactants to flow in, interact with the encapsulated enzyme, and flow back out [4]. Calcium chloride and sodium alginate are both relatively inexpensive materials, and relatively stock materials in most laboratories. As a result, we concluded that sodium alginate beads would be the best method to encapsulate enzymes.

Before the sodium alginate beads can be formed, we have to make the calcium chloride and sodium alginate solutions.

The calcium chloride solution is made by dissolving a set amount of calcium chloride powder in water depending on the desired concentration of the solution. For the purpose of testing, we created five different calcium chloride solutions ranging in even increments of 0.5 M from 0.5M to 2.5 M.

Sodium alginate solution is also created by dissolving a set amount of sodium alginate powder in water. Once again, the amount of powder used depends on the desired concentration of the solution. For testing purposes, we created five different sodium alginate solutions ranging in even increments of 0.5% from 1.5% weight by volume to 3.5% weight by volume.

When dissolved in water, sodium alginate has a tendency to form lumps that are very difficult to break apart and add time onto the already long dissolving process. In initial testing procedures, sodium alginate was added entirely to the distilled water solution, resulting in the formation of large, undissolved particles. Further research found that despite the hydrophilic nature of alginate salts, the high hydratability led to the formation of lumps and non-uniform dissolution of the powder [1]. Thus in later batches, the protocol was altered and sodium alginate power added very slowly, allowing the powder already in the solution to dissolve almost entirely before adding any more powder. This increased production efficiency by quartering the amount of time required to make the solution.

Before conducting tests about bead formation, batches of sodium alginate and calcium chloride solutions at several different concentrations needed to be made. After being fully dissolved, the calcium chloride solutions exhibited almost no distinct properties. In fact, there were no physical properties that made it possible to distinguish between the different concentrations.

Unlike calcium chloride, the sodium alginate solutions began exhibiting different properties based on concentration immediately. Solutions with a higher weight by volume concentration of sodium alginate demonstrated increased viscosity and more air bubbles, most likely due to the increased amount of time it takes for said bubbles to travel through the solution. Furthermore, the more viscous sodium alginate solutions - more specifically those at a 2.5% weight by volume concentration or higher - were much more difficult to handle. Frequently, the solution would adhere to the sides of pipettes and syringes, and not all of the solution would drain into the desired receptacles.

Originally, a serological pipette was used to transfer the sodium alginate solution between containers and to add the sodium alginate solution dropwise into the calcium chloride solution for the formation of encapsulated beads. However, when testing solutions above a 2.5% weight by volume concentration, the viscosity of the solutions made it almost impossible to allow the sodium alginate to drip out of the pipette one bead at a time. The solutions exhibited an extreme surface tension that proceeded to only allow large globs to be pushed out of the pipette. Even with the minimal extrusion of droplets, the pipette would remain lined with excess solution that would cling to the sides and cause waste, resulting in lower production of beads.

Upon discovering these difficulties, the decision was made to switch from a serological pipette to a twenty milliliter syringe with a four inch needle. The twenty milliliter syringe could hold just as much solution as the twenty milliliter serological pipette, and the smaller circumference of the output allows us to create a smaller, more uniform bead size. Dr. Phillip Milner also told us that smaller bead size will increase reaction frequency, since there will be more beads at a higher concentration in the reactor, and thus reactants will encounter the beads more often. Thus, the production of smaller, more uniform bead size is desirable.

The sodium alginate solution is added dropwise to the calcium chloride solution by pressing slowly down on the syringe until the bead forming at the tip of the needle gathers enough mass to break away and fall into the calcium chloride. Initially, this was all done over a stir plate. The stir bar in the calcium chloride solution was kept even after it had been fully dissolved, because initial testing demonstrated that the calcium chloride required additional time from the initial addition to fully dissolve, and we wanted to ensure that the calcium chloride stayed dissolved, and that the solution was entirely uniform.

However, upon making a batch of beads in this solution, we noticed that the stir bar was negatively affecting the shape of the beads. The sodium alginate beads would enter the solution almost perfectly spherical, but would later be knocked around by the bar, pinned between the bar and the container, and, generally, deformed. As a result of these observations, the protocol was adjusted with the stir bar removed to create the rest of the beads in a calcium chloride solution at rest.

When creating different concentrations of sodium alginate solutions, we added different amounts of coomassie blue dye. When the dye and alginate was fully dissolved, it became clear that the 3.0% weight by volume sodium alginate contained enough dye to make the beads visible in clear solution, but little enough dye to allow us to see through the solution, and thus into the beads. As a result, the varying calcium chloride concentrations were all tested with 3.0% weight by volume sodium alginate. In addition to taking qualitative observations of the beads formed in each calcium chloride solution, we measured the average bead diameter of each batch. These findings are listed below:

| **Calcium chloride concentration (M)** | **Bead diameter (mm)** | | | | | | | | **Mean bead diameter (mm)** |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **0.5** | 3.44 | 3.21 | 3.47 | 3.91 | 3.79 | 3.67 | 3.84 | 3.71 | 3.63 |
| **1.0** | 3.48 | 3.65 | 3.97 | 3.63 | 3.75 | 3.40 | 3.45 | 3.53 | 3.61 |
| **1.5** | 3.46 | 3.55 | 3.62 | 3.65 | 3.34 | 3.41 | 3.48 | 3.41 | 3.49 |
| **2.0** | 3.60 | 4.11 | 3.95 | 3.95 | 4.07 | 3.81 | 3.61 | 3.94 | 3.88 |
| **2.5** | 4.29 | 4.30 | 4.12 | 3.87 | 4.27 | 3.63 | 3.93 | 4.00 | 4.05 |

Figure 12: A table displaying the average alginate bead diameter at varying calcium chloride concentrations.

As seen above in Figure 12, the concentration of the calcium chloride solution had no noticeable effect on the average diameter of each batch of beads. Furthermore, there were no significant qualitative differences between concentrations of sodium alginate.

Beads formed using different sodium alginate concentrations yielded much more interesting results.

| **Sodium alginate concentration (M)** | **Bead diameter (mm)** | | | | | **Mean bead diameter (mm)** |
| --- | --- | --- | --- | --- | --- | --- |
| **1.5** | 3.02 | 3.20 | 2.79 | 3.18 | 3.07 | 3.05 |
| **2.0** | 3.76 | 4.09 | 4.00 | 2.99 | 3.21 | 3.61 |
| **2.5** | 3.93 | 3.69 | 3.96 | 3.40 | 3.41 | 3.68 |
| **3.0** | 3.91 | 4.12 | 3.98 | 3.65 | 3.54 | 3.84 |
| **3.5** | 4.25 | 3.69 | 3.32 | 3.69 | 3.46 | 3.68 |

Figure 13: A table displaying the average alginate bead diameter at varying sodium alginate concentrations.

As can be seen above in Figure 13, sodium alginate concentration actually had an effect on the average diameter of the beads in different batches. The greater the sodium alginate concentration, the greater the diameter of the bead. One noticeable outlier in this trend is the 3.5% weight by volume sodium alginate concentration, which actually yielded a lower average bead diameter than the 3.0% weight by volume concentration. This can likely be attributed to experimental error, including the transition from use of a serological pipette to a syringe and needle.

There were also significant quantitative differences between each batch of beads. A demonstration of some of these qualitative differences is pictured below.

![](https://static.igem.wiki/teams/4580/wiki/es-fig-3.png)

Figure 14: An image containing three rows and 5 columns of beads. By row: split beads, intact beads, and beads upon which pressure was applied. By column: beads made with 1.5%, 2.0%, 2.5%, 3.0%, and 3.5% weight by volume sodium alginate concentrations.

The top row contains beads split with a sharp tool, the second contains untouched beads, and the lowest row shows beads upon which a uniform pressure was applied. One important thing to notice is that beads made with 1.5% and 2% weight by volume sodium alginate concentration split under this pressure, whereas the greater concentrations could only be split with the sharp tool. Below is another photo that demonstrates what these beads looked like before experimentation.

![](https://static.igem.wiki/teams/4580/wiki/es-fig-4.png)

Figure 15: An image displaying 5 groups of beads. From right to left, they were formed with 1.5%, 2.0%, 2.5%, 3.0%, and 3.5% weight by volume sodium alginate concentrations.

When the sodium alginate concentration exceeded 2.5% weight by volume, the beads demonstrated an incredibly heightened amount of “tailing”, the property we used to describe the issue where beads form a teardrop shape rather than a perfect sphere.

No physical or chemical differences were noticed between the beads formed at different calcium chloride concentrations in these experiments. As a result, future production of the beads will use a standard 1 M concentration of calcium chloride.

The benefits of each concentration of sodium alginate were much more difficult to weigh. Beads of a higher concentration are more durable and split less easily, but also are larger and the solution itself is more difficult to work with. We concluded that the best concentration to utilize in further bead making is 2.5% weight by volume. This is because, for present purposes, it is the lowest concentration that will not split under pressure. As a result, it is the smallest bead at the concentration that is easiest to work with out of the most durable beads.

## Cycle 2

With an established uniform procedure for making beads, optimization and speed of the process was the next goal. An estimated fifty to one hundred beads would be required for each bioreactor test session, and our current bead-making procedures were very slow. In order to optimize our use of crucial lab time, we needed to create an automated bead-making machine.

Several designs were considered during the first drafts of this machine. We initially considered using a syringe attached to the bottom of a container of the sodium alginate solution, and allowing gravity to drip the sodium alginate out of the solution. However, the viscosity of the sodium alginate solution proved to be much too high to allow gravity alone to push the solution out of the syringe in a timely manner. As a result, reducing the amount of solution to only that in the syringe and then adding pressure in the form of weight on top of the syringe plunger. However, there were still concerns that this would not produce beads reliably in a short amount of time.

Finally, the idea to use a syringe infusion pump was suggested. Utilizing one of the syringe infusion pumps in our lab, except instead of connecting the syringe to a larger system, the sodium alginate would be allowed to drip out of the needle, similar to the process of making the beads by hand.

Constructing this system was quite simple; the same syringe that was utilized when making the beads by hand would be put into the syringe infusion pump, and a container of 10 mL of calcium chloride would be put underneath the tip of the needle attached to the syringe so that the sodium alginate will easily drip directly into the solution. A video of the finalized setup is shown below.

Figure 16: A video demonstrating the automatic bead making process using a syringe infusion pump.

Testing the success of this automated bead maker was also relatively simple. To determine whether or not it was effective in increasing our rate of bead making, the approximate rate at which beads are produced, both by hand and by the automated bead maker, needed to be compared.

By hand, approximately forty beads could be made in two minutes. That resulted in bead-making by hand exhibiting a rate of twenty beads per minute.

Alternatively, the automated bead maker was observed to produce about seven beads in ten seconds, putting it at a bead-making rate of approximately seventy beads per minute.

Additionally, by simple qualitative analysis, it was observed that the beads made by the automatic bead maker were more consistently sized, had less tailing, and had less air bubbles. Consistent sizing and lack of tailing allows the beads to be loaded into the reactor more easily, and lack of air bubbles ensures that reactions between the reactants and enzymes will not be disturbed by gaseous molecules in the way.

It is evident that the automated bead maker is more successful in efficiently making beads. The uniformity and speed with which these beads are made allow us to use time more efficiently in the lab, and will speed up processing by tenfold when scaleup occurs. As a result, we will move forward with making beads with the automated bead maker only.

## Reactor Design

Our goal was to design and create a fixed-bed bioreactor in which our selected enzyme would be immobilized in alginate beads and those beads then immobilized within the bioreactor [2][3]. Going into this project, there were a few properties we wanted our design to have: modularity, ease of scaling, and cost effectiveness. These were all necessary because we were seeking to replace an existing process with our project: if our project was more costly or difficult to scale or maintain, what improvement would we truly be making?

The first step in our design was determining the structure we would use to immobilize the alginate beads. With an assumed bead diameter of a millimeter, each team member created a prototype CAD model to present to the rest of the product development team. We determined several desirable properties in these alginate bead immobilization systems; for maximum efficiency, our reactor required maximum exposed bead surface area, individual bead separation, and relative ease of bead replacement.

Several rounds of this brainstorming process were completed to fully flesh out the various strengths and weaknesses of each design including covalent adhesion, adsorption, entrapment, as well as dynamic bead swelling. Ultimately, an adsorption method was selected where encapsulated enzyme beads would be pushed against a mesh-like spiral matrix to produce separation of beads and allow fluid flow, while keeping beads immobilized in a relative space of the reactor. The mesh-like properties of this immobilization system allowed for maximum exposed bead surface area, and the attachment of immobilization matrix to central supports theoretically provided easy removal of the entire mesh system should beads need to be replaced or examined due to its simple design of simply sliding into the reactor column. The CAD model is pictured below in Figure 5.

![](https://static.igem.wiki/teams/4580/wiki/es-fig-5.png)

Figure 17: CAD model of immobilization matrix scaled for a 10 mL syringe tube

In parallel, we also came up with a rough idea of what we wanted the bioreactor assembly to look like. To model our tube, we researched various materials and landed on PVC piping for its affordability, lack of reactivity, and ease of use. The relatively large size of our piping (1½ inches in diameter) allowed us to test more parameters as well.

From this, we came up with a mock-up of the bioreactor using easily-acquired materials to determine the necessary parameters for optimal synthesis. With the preliminary immobilization CAD models completed, the prototype immobilization methods were 3D printed using PLA on a Prusa i3 MKS3+ printer. PVC pipe was used to model the reactor chamber, and two mason jars were placed at each end of the pipe to store water. A peristaltic fluid motor pump was fixed to the jar that contained water, and was programmed to flow water through the mock reactor at a constant rate. Plastic beads were used to simulate free-flowing alginate beads in the reactor for initial testing for ease-of-testing. Later, encapsulated beads were exchanged for the plastic beads, proving modularity of the system. Burlap cloth was fixed to either end of the reactor to hold the beads and immobilization matrix inside.

The mockup reactor was tested by measuring the amount of time it took for this simple motor setup to move a known volume of water through the tube. By altering various elements of the setup, such as the incline of the tube (or lack thereof), the power being delivered to the motor, and the amount of “beads” in the tube, we gained valuable insight into the way that our final product would have to operate. An example of these trials is shown below (*Figure 18*).

|  |  |  |  |
| --- | --- | --- | --- |
| **Diameter (in)** | 1.25 | 1 | 0.75 |
| **Volume (cm3)** | 482.63 | 308.9 | 173.7 |
| **Outflow Rate (mL/sec)** | 4.73 | 4.73 | 4.73 |
| **Residence Time** | 102.0359408 | 65.30655391 | 36.7230444 |

Figure 18: Measurements taken to qualify residence time of various reactor tube sizing

After testing, we moved forward with creating a more finalized version of the mock reactor. Our new design consists of a similar immobilization design scaled to our updated testing column dimensions. This design was 3D printed and put inside of a 10mL syringe with a stopper made from the syringe plunger connected to a tube.

![](https://static.igem.wiki/teams/4580/wiki/es-fig-6.png)

Figure 19: CAD model of updated immobilization method scaled for a 10 mL syringe

## References

[1] How to dissolve Alginate. KIMICA Corporation. (n.d.). <https://www.kimica-algin.com/alginate/usage/>

[2] Making gel beads - science & math investigative learning experiences. (n.d.). <https://smile.oregonstate.edu/sites/smile.oregonstate.edu/files/gel_beads_1.pdf>

[3] The ultimate guide to sodium alginate beads - gino gums. Gino Gums & Stabilizers. (2023, April 20). <https://gumstabilizer.com/guide-to-sodium-alginate-beads/>

[4] Urrea, D. A. M., Gimenez, A. V. F., Rodriguez, Y. E., & Contreras, E. M. (2021, October 20). Immobilization of horseradish peroxidase in CA-alginate beads: Evaluation of the enzyme leakage on the overall removal of an azo-dye and mathematical modeling. ScienceDirect. <https://www.sciencedirect.com/science/article/pii/S0957582021005279?via%3Dihub>

{% 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](http://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](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

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

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

## 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 %}