iGEM 2021 — Team Cornell

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Cornell/Notebook

Team:Cornell - 2021.igem.org

[![logo](https://static.igem.org/mediawiki/2021/5/51/T--Cornell--cornell_logo.png)

TEAM

[BIOS](/Team%3ACornell/Team)
[SPONSORS](/Team%3ACornell/Sponsers)
[ATTRIBUTIONS](/Team%3ACornell/Attributions)

OUTREACH

[EDUCATION & ENGAGEMENT](/Team%3ACornell/Education)
[COMMUNICATION](/Team%3ACornell/Communication)
[COLLABORATIONS](/Team%3ACornell/Collaborations)
[PARTNERSHIP](/Team%3ACornell/Partnership)

HUMAN PRACTICES

[INTEGRATED PRACTICES](/Team%3ACornell/Human_Practices)
[POLICIES](/Team%3ACornell/Policies)
[ENTREPRENEURSHIP](/Team%3ACornell/Entrepreneurship)

PRODUCT DEVELOPMENT

[MODELING](/Team%3ACornell/Model)
[ENGINEERING SUCCESS](/Team%3ACornell/Engineering)
[PROOF OF CONCEPT](/Team%3ACornell/Proof_Of_Concept)
[IMPLEMENTATION](/Team%3ACornell/Implementation)
[HARDWARE](/Team%3ACornell/Hardware)

TOOLKIT

* **WET LAB**
* [FOUNDATIONS](/Team%3ACornell/Foundations)
* [DEMONSTRATE](/Team%3ACornell/Demonstrate)
* [PARTS](/Team%3ACornell/Parts)
* [BASIC PARTS](/Team%3ACornell/Basic_Parts)
* [COMPOSITE PARTS](/Team%3ACornell/Composite_Parts)

* **DOCUMENTATION**
* [NOTEBOOK](/Team%3ACornell/Notebook)
* [SAFETY](/Team%3ACornell/Safety)
* [CONTRIBUTION](/Team%3ACornell/Contribution)

DESCRIPTION

HOME](/Team%3ACornell)

![banner](https://static.igem.org/mediawiki/2021/a/a7/T--Cornell--notebook.png)

* [February 2021](#pageheading1)
* [March 2021](#pageheading2)
* [April 2021](#pageheading3)
* [May 2021](#pageheading4)
* [June 2021](#pageheading5)
* [July 2021](#pageheading6)
* [August 2021](#pageheading7)
* [September 2021](#pageheading8)
* [October 2021](#pageheading9)

February 2021

---

February 21st to February 27th

**Wet Lab-**
Wet Lab members met to begin the brainstorming process and discussed the training plan for new members, which includes learning various lab techniques such as PCR, gel electrophoresis, miniprep, gibson assembly, and transformation.
**Product Development**
Product development members met with each other for the first time to begin brainstorming. Subteam members also began new member training. This week we completed our basic biology fundamentals training, introducing the concepts of biobricks and synthetic biology.
**P&P-**
P&P members met to begin the brainstorming process and discuss new member training. The focus was on training members about interviewing and developing professional skills associated with human practices.
**Wiki-**
Wiki team members took part in the brainstorming process. They also began new member training. This week we started learning about html/css basics.
**Business-**
Business team began brainstorming entrepreneurial ideas to implement into our project for the semester. We also began preparing for Cornell University Giving Day, which involves reaching out to past alumni and other organizations.

March 2021

---

February 28th to March 6th

**Wet Lab-**
Continued with training and discussed possible project ideas. Members completed subteam assignments to get familiarized with designing sequences and assembling BioBricks together.
**Product Development-**
Subteam members met briefly to discuss potential product development roles within our existing project ideas. PD members began Python and Modeling / Simulation training. We practiced using the numPy and Pandas libraries in Python, as well as data visualization methods.
**P&P-**
P&P met to work on assignments identifying key stakeholders in the scientific community and how their perspectives would be valuable to our team. The members continued to hone interviewing and communication skills.
**Wiki-**
Wiki team members took part in the brainstorming process. They continued member training. This week we continued learning about html/css basics.
**Business-**
The business team member received a workshop in Excel. They learned basic formulas, formatting, and other functions specific to outreach. They practiced mail merges, which is used for sending out mass mailings.

March 7th to March 13th

**Wet Lab-**
Continued with training and introduced modeling using MatLab. Members completed training assignments that involved using differential equations and Michaelis Menten kinetics to model production and degradation. Worked on practicing PCR.
**Product Development-**
PD continued Modeling and Simulation training. We walked through an example simulation using the sciPy library, and new members were assigned to design and develop a simulation of their own to promote teamwork and in anticipation of modeling being an important aspect of our future project.
**P&P-**
P&P introduced policy categories associated with iGEM. Safety and risk analysis, bioethics, economic analysis, and policy were all covered. Previous project topics were used as exercises for new members to practice with.
**Wiki-**
Wiki team members took part in the brainstorming process. They also continued wiki member training. This week we continued learning about html/css basics.
**Business-**
Sent out emails to past sponsors and alumni for giving day asking for donations.

March 14th to March 20th

**Wet Lab-**
Wet Lab members continued with training, learning how to run a gel. Also continued with the MATLAB training assignments.
**Product Development-**
PD members submitted their modeling and simulation assignment, having designed a simulation of bacterial growth under limited resources and competition between multiple strains. This week we began 3D modeling training, teaching members how to use Fusion360 to design basic products such as a customized stamp.

**P&P-**
P&P started interviewing scientific experts for the final few project ideas. This gave members practice with interviewing and helped narrow down the choices to viable options. The members also began to think of outreach and education events for the coming months.

**Wiki-**
Wiki team members took part in the brainstorming process. They also continued wiki member training. This week we presented the assignments that we had been working on with html and css.

**Business-**
Sourced emails for different contacts from people working in the biomedical industry for future outreach projects.

March 21th to March 27th

**Wet Lab-**
Wet lab concluded training by practicing Gibson Assembly and discussing the theory behind it. We also practiced using SnapGene for plasmid design. This was the last week of training.

**Product Development-**
This week we began the electrical component of our training program. We introduced basic circuitry concepts, such as logic gates and components of a circuit. Members learned to use LTSpice software to design high and low pass filters as a practice assignment. This week concluded our training program.

**P&P-**
P&P continued with interviewing scientific experts and identifying key project criteria from these perspectives. Each member was tasked with designing a theoretical outreach event for a project idea and pitching it to the rest of the group.

**Wiki-**
Wiki team members took part in the brainstorming process. They also continued wiki member training. This week we began learning about design fundamentals.

**Business-**
Brought in guest speaker to talk about business and entrepreneurship within the biomedical space and how Cornell iGEM can overcome the challenges it faces.

April 2021

---

March 28th to April 3rd

**Wet Lab-**
Wet Lab members participated in the team-wide brainstorming process.

**Product Development-**
PD members continued with the team-wide brainstorming process.

**P&P-**
P&P continued interviewing scientific experts and took part in the brainstorming process.

**Wiki-**
Wiki team members took part in the brainstorming process. They also continued wiki member training. This week we continued learning about design fundamentals.

**Business-**
Business members hopped on interviews with P&P as part of the brainstorming process to learn more about the potential of our project ideas.

April 4th to April 10th

**Wet Lab-**
Wet Lab members participated in the team-wide brainstorming process.

**Product Development-**
PD members continued with the team-wide brainstorming process.

**P&P-**
P&P members participated in the team-wide brainstorming process.

**Wiki-**
Wiki team members took part in the brainstorming process. They also continued wiki member training. This week we continued learning about design fundamentals.

**Business-**
Completed the Cornell Engineering Alumni Association grant application. Participated in the team-wide brainstorming process

April 11th to April 17th

**Wet Lab-**
Wet Lab members participated in the team-wide brainstorming process.

**Product Development-**
PD members continued with the team-wide brainstorming process.

**P&P-**
P&P continued taking part in the brainstorming process.

**Wiki-**
Wiki members participated in the team-wide brainstorming process. They also continued wiki member training. This week we began learning how to use ADobe illustrator and Figma.

**Business-**
Looked for external sources of funding. Participated in the team-wide brainstorming process

April 18th to April 24th

**Wet Lab-**
This week, Wet Lab began working on finding sequences to be used in our genetic circuit. We worked with the PD team to research collagen crosslinking and extraction protocols.

**Product Development-**
This week PD developed a collagen printer design and created a preliminary list of hardware parts needed to implement our printer. We also researched collagen crosslinking methods and cell lysis methods for our printer. These included ultrasonication, UV polymerization and dehydration.

**P&P-**
P&P participated in brainstorming. The new members were given more assignments dealing with policy topics. We also continued to reach out to professors who dealt with collagen or polymer production.

**Wiki-**
Wiki members participated in the team-wide brainstorming process. They also continued wiki member training. This week we began learning how to use ADobe illustrator and Figma.

**Business-**
Participated in the team-wide brainstorming process. Received workshop on startup planning and pitch decks

May 2021

---

April 25th to May 1st

**Wet Lab-**
Continued with the design of our genetic circuit and began assembling all the components together. Discussed the summer work plan and schedule.

**Product Development-**
PD scheduled in-person work times, and drafted a project overview containing our goals for the project, which at this stage included a bioprinter to create collagen structures.

**P&P-**
P&P participated in team-wide brainstorming. The team also worked on educational presentation materials focused on collagen production. These materials were planned to be used for a variety of outreach activities.

**Wiki-**
Wiki members participated in the team-wide brainstorming process. They also continued wiki member training. This week we continued learning how to use ADobe illustrator and Figma.

**Business-**
Conducted customer discovery and industry research about our chosen project, which has to do with collagen.

May 2nd to May 8th

**Wet Lab-**
Researched the yeast cell types that we planned to use for our project and explored the ways that collagen production would be induced. Contacted professors to interview to get more information about our project idea.

**Product Development-**
PD researched our collagen production, extraction, and printing process. We looked into different ways of crosslinking collagen and extracting it from Wetlab’s cells.

**P&P-**
P&P coordinated with wet lab to receive project updates for further interview progress. The team also worked on defining key criteria for human practices and education and outreach. Past examples of outstanding outreach were examined for inspiration.

**Wiki-**
Wiki members participated in the team-wide brainstorming process. They also continued wiki member training. This week we continued learning how to use ADobe illustrator and Figma.

**Business-**
Came up with lists of questions to ask potential customers of our developed product.

May 9nd to May 15th

**Wet Lab-**
Developed a list of summer goals and responsibilities. Finalized the design of our genetic circuit and produced a list of materials to order for summer work. Spoke to professors about optimizing our project idea and whether it seemed feasible.

**Product Development-**
PD finalized our collagen production, extraction, and printing process. We decided to build a bioreactor to culture our collagen, incorporating a light to accommodate Wetlab’s plan to utilize blue light to prompt collagen production. We also chose UV light crosslinking for our collagen. This week we also drafted a schedule for summer work.

**P&P-**
P&P finished up some interviews and created a list of people to contact over the summer. The team also worked on generating additional outreach event ideas.

**Wiki-**
Wiki members participated in the team-wide brainstorming process. They also continued wiki member training. This week we presented our final projects that used design as well as html and css.

**Business-**
Summer Break

May 16nd to May 22th

**Wet Lab-**
Break before summer work.

**Product Development-**
Break before summer work.

**P&P-**
Summer break

**Wiki-**
Summer Break

**Business-**
Summer Break

May 23nd to May 29th

**Wet Lab-**
Break before summer work.

**Product Development-**
Break before summer work.

**P&P-**
Summer break

**Wiki-**
Summer Break

**Business-**
Summer Break

June 2021

---

May 30th to June 5th

**Wet Lab-**
Break before summer work.

**Product Development-**
Break before summer work.

**P&P-**
Summer break

**Wiki-**
Summer Break

**Business-**
Summer Break

June 6th to June 12th

**Wet Lab-**
Break before summer work.

**Product Development-**
Break before summer work.

**P&P-**
Summer break

**Wiki-**
Summer Break

**Business-**
Summer Break

June 13th to June 19th

**Wet Lab-**
Wet lab began summer work! Reviewed our plan and mapped out weekly goals for experiments. Collected yeast materials from Dr. Gibney and checked concentrations. Made YPD medium for yeast growth. Worked on general project description.

**Product Development-**
PD began our summer work. We recapped the research done in previous weeks and mapped out our summer work plan.

**P&P-**
P&P outlined a summer work schedule. The project progress was outlined and more key stakeholders were outlined. These stakeholders were assigned to members to contact for interviews.

**Wiki-**
Summer Break

**Business**
Summer Break

June 20th to June 26th

**Wet Lab-**
Met with PD to discuss some of the issues with the project and how they could be solved. Decided to pivot to working with prokaryotic collagen produced by bacteria, instead of eukaryotic collagen and yeast. Discussed what changes need to be made to the materials and

**Product Development-**
After meeting with wetlab, PD began looking into prokaryotic collagen as opposed to mammalic, and how this might change our process.

**P&P-**
P&P focused on interviewing biotech companies and medical professionals for project applications. The team also worked on expanding outreach ideas for summer work.

**Wiki**
Summer Break

**Business**
Summer Break

July 2021

---

June 27th to July 3rd

**Wet Lab-**
Starting working on a new design for the genetic circuit. Found different sequences and started looking into possible binding sites for modifying prokaryotic collagen, such as fibronectin, heparin, and integrin.

**Product Development-**
PD developed a plan to meet iGEM medal criteria for bronze, silver and gold.

**P&P-**
P&P continued to attend interviews and write up notes for the project. Members also worked on social media content and reached out to collaborate with other teams on Instagram. People discussed outreach ideas and started to outline plans for them.

**Wiki-**
Summer Break

**Business-**
Summer Break

July 4th to July 10th

**Wet Lab-**
Continued reworking project ideas by editing the genetic circuit design. Drafted list of new materials to order and did research about which binding sites would be the most useful.

**Product Development-**
After significantly reworking our project idea into a gradient hydrogel, PD began to design a gradient machine to produce a mixed hydrogel containing collagen-mimetic proteins with different binding sites.

**P&P-**
P&P continued to work on planning outreach events and social media posts. The team worked on interviewing additional science experts. We also worked on helping the Hong Kong Human Practices team with outreach to professors and thinking of further ways to help.

**Wiki-**
Summer Break

**Business-**
Summer Break

July 11th to July 17th

**Wet Lab-**
Designed primers for Gibson Assembly and continued with literature research about our project plan.

**Product Development-**
PD continued development of our bioreactor design and gradient machine design. We began to order materials.

**P&P-**
P&P continued to interview experts and expand outreach ideas. The team also collaborated with GTC Hong Kong iGEM Human Practices to assist with outreach.

**Wiki-**
Summer Break

**Business-**
Summer Break

July 18th to July 24th

**Wet Lab-**
Ordered integrin and fibronectin gBlocks. Modified general project description to fit new project idea. Began working on modeling of Scl2 production and degradation.

**Product Development-**
PD began modeling bioreactor growth with Wetlab and created initial CAD models of our bioreactor and gradient machine designs.

**P&P-**
P&P continued to attend interviews with collagen experts and take notes.

**Wiki-**
Summer Break

**Business-**
Summer Break

July 25th to July 31th

**Wet Lab-**
Continued with Scl2 modeling. Ordered cells containing the backbone plasmid that would be used for Gibson Assembly.

**Product Development-**
PD continued working on both of our machines. After several iterations, we finalized designs and began ordering hardware materials.

**P&P-**
P&P attended several meetings and interviews with professors and biotech companies. Plans were made for outreach events with the Ithaca Sciencecenter and high school science club seminars.

**Wiki-**
Summer Break

**Business-**
Summer Break

August 2021

---

August 1st to August 7th

**Wet Lab-**
Finalized Scl2 modeling and began looking into modeling crosslinking using PyMol. Discussed options for improved part. Outlined how we were planning on hitting all the medal criteria for Bronze, Silver, and Gold.

**Product Development-**
PD began physically building our bioreactor and gradient maker.

**P&P-**
P&P finalized plans for the Sciencecenter outreach event. The group also concluded summer interviews with biotech companies and worked on analyzing the interview content for wet lab and PD.

**Wiki-**
Summer Break

**Business-**
Summer Break

August 8th to August 14th

**Wet Lab-**
Break after summer session.

**Product Development-**
Break after summer session.

**P&P-**
Summer Break

**Wiki-**
Summer Break

**Business-**
Summer Break

August 15th to August 21st

**Wet Lab-**
Break after summer session.

**Product Development-**
Break after summer session.

**P&P-**
P&P collaborated with the Ithaca Sciencecenter to design synthetic biology and collagen activities for children.

**Wiki-**
Summer Break

**Business-**
Summer Break

August 22nd to August 28th

**Wet Lab-**
Break after summer session.

**Product Development-**
Break after summer session.

**P&P-**
Summer Break

**Wiki-**
Summer Break

**Business-**Summer Break****

September 2021

---

August 29th to September 4th

**Wet Lab-**
Wet lab resuspended gBlocks and made stock primers. Organized materials from summer work.

**Product Development-**
PD began making hydrogel tests and reaching out to professors for input on our process. We also troubleshooted our bioreactor and gradient machine builds.

**P&P-**
P&P reviewed summer progress and composed a list of ideas to talk with science professionals about the project. The team also continued to meet with GTC Hong Kong iGEM to collaborate on outreach.

**Wiki-**
Summer Break

**Business-**
Completed iGEM Impact Grant application

September 5th to September 11th

**Wet Lab-**
Performed PCR and ran a gel on fibronectin gblock. Unfortunately, it seemed that at first the DNA was either not present or was not present in a high enough concentration to actually show up on the gel. Did 2 PCR reactions with two different sets of primers. Scl2\_Fibronectin DNA was used for both reactions. PCR ran with no issues at all according to the thermocycler, but plan to confirm later in the week with gel electrophoresis.

**Product Development-**
PD continued modeling our bioreactor and made finishing touches on the physical machine. We began developing our photoreactive crosslinking hydrogel procedure.

**P&P-**
P&P started policy analysis for the collagen project. The group engaged in open discussion about safety concerns, risk analysis, ethical considerations, and economic considerations.

**Wiki-**
Wiki team begins brainstorming themes and colors for the wiki website.

**Business-**
With the project mostly completed, the business team started planning the deliverables that would be posted on our team’s website

September 12th to September 18th

**Wet Lab-**
Ran the same 2 PCRs with adjusted annealing temperature of 65C and 40 sec extension time and were able to see bands on the gel. Made more LB agar. Continued working on part improvement ideas.

**Product Development-**
PD continued work on our photoreactive hydrogels and began modeling our gradient machine’s mixer head in preparation for creating gradient hydrogels.

**P&P-**
P&P worked on reaching out to high school HOSA chapters and science clubs to schedule presentations about iGEM and synthetic biology. The team also brainstormed people to reach out for the final phase of the project. The team also started working on write-ups.

**Wiki-**
Wiki team members take turns sharing themes and voting on their favorite one .

**Business-**
Worked on business plan deliverable.

September 19th to September 25th

**Wet Lab-**
Performed gel extraction to purify fibronectin gBlock, but contraction was too low based on the results from the nanodrop. Received and resuspended integrin gBlock.

**Product Development-**
PD began creating chemically crosslinked hydrogels and writing code to implement our bioreactor. We also planned out our wiki content and looked into various testing methods for our hydrogels.

**P&P-**
P&P worked on designing the presentation materials for the HOSA and science club outreach seminars. The team continued to correspond with science professionals to improve our collagen project. P&P also continued working on write-ups.

**Wiki-**
Wiki team works on creating a home page banner/background with the voted theme.

**Business-**
Worked on business plan deliverable.

October 2021

---

September 26th to October 2nd

**Wet Lab-**
Continued working on amplifying and purifying integrin and fibronectin gBlocks using PCR and gel electrophoresis. Worked on culturing and imaging mammalian cells on hydrogels.

**Product Development-**
PD prepared chemicals and fluids to run our bioreactor and continued writing code to implement it. We also continued creating hydrogels for imaging and testing.

**P&P-**
P&P continued documenting past work. The team also hosted outreach events for the local Ithaca high school science club and virtual seminars for California and Michigan HOSA clubs.

**Wiki-**
Wiki team takes turns sharing their banners and sharing ideas between the banners and voting for the best banner.

**Business-**
Worked on business plan deliverable and wiki design layout. Put an emphasis on lots of visuals this year.

October 3rd to October 9th

**Wet Lab-**
Continued working on amplifying and purifying integrin and fibronectin gBlocks using PCR and gel electrophoresis. Plated bacteria containing plasmid backbone. Began working and project write-ups for wiki and finalized ideas for improved parts. Worked with PD to test bioreactor. Worked on culturing and imaging mammalian cells on hydrogels.

**Product Development-**
PD continued making test runs of hydrogel, and troubleshooting issues with lack of gelling and nHAP clumping. We began creating photoreactive hydrogels.

**P&P-**
P&P continued working on wiki content.

**Wiki**
Wiki team begins adding content to the wiki page.

**Business-**
Worked on business plan deliverable and wiki design layout. Put an emphasis on lots of visuals this year.

October 10th to October 16th

**Wet Lab-**
Finalized wiki writeups and worked on bioreactor testing. Finished culturing and imaging mammalian cells on hydrogels.

**Product Development-**
PD finalized our wiki writeups, ran final tests on our hydrogels and tested our gradient machine.

**P&P-**
P&P finalized wiki content.

**Wiki-**
Wiki team finishes adding content to the wiki pages.

**Business-**
Finalized business plan and wiki design layout.

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# Team:Cornell/Engineering

Team:Cornell - 2021.igem.org

[![logo](https://static.igem.org/mediawiki/2021/5/51/T--Cornell--cornell_logo.png)

TEAM

[BIOS](/Team%3ACornell/Team)
[SPONSORS](/Team%3ACornell/Sponsers)
[ATTRIBUTIONS](/Team%3ACornell/Attributions)

OUTREACH

[EDUCATION & ENGAGEMENT](/Team%3ACornell/Education)
[COMMUNICATION](/Team%3ACornell/Communication)
[COLLABORATIONS](/Team%3ACornell/Collaborations)
[PARTNERSHIP](/Team%3ACornell/Partnership)

HUMAN PRACTICES

[INTEGRATED PRACTICES](/Team%3ACornell/Human_Practices)
[POLICIES](/Team%3ACornell/Policies)
[ENTREPRENEURSHIP](/Team%3ACornell/Entrepreneurship)

PRODUCT DEVELOPMENT

[MODELING](/Team%3ACornell/Model)
[ENGINEERING SUCCESS](/Team%3ACornell/Engineering)
[PROOF OF CONCEPT](/Team%3ACornell/Proof_Of_Concept)
[IMPLEMENTATION](/Team%3ACornell/Implementation)
[HARDWARE](/Team%3ACornell/Hardware)

TOOLKIT

* **WET LAB**
* [FOUNDATIONS](/Team%3ACornell/Foundations)
* [DEMONSTRATE](/Team%3ACornell/Demonstrate)
* [PARTS](/Team%3ACornell/Parts)
* [BASIC PARTS](/Team%3ACornell/Basic_Parts)
* [COMPOSITE PARTS](/Team%3ACornell/Composite_Parts)

* **DOCUMENTATION**
* [NOTEBOOK](/Team%3ACornell/Notebook)
* [SAFETY](/Team%3ACornell/Safety)
* [CONTRIBUTION](/Team%3ACornell/Contribution)

DESCRIPTION

HOME](/Team%3ACornell)

![banner](https://static.igem.org/mediawiki/2021/0/07/T--Cornell--engineering.png)

* [Overview](#pageheading1)
* [Creation Process](#pageheading2)
* [Design Process](#pageheading3)

Overview

---

Hydrogels have widely used applications in biomedical fields because of their biocompatibility and close resemblance to natural tissue. Drawing inspiration from the designer collagens [1] and gradient collagens [2], we focused on an orthopedic approach by constructing a hydrogel that would mimic tissue interfaces, such as bone-cartilage, tendon-bone, and ligament-bone interfaces.

In the lab we produced two different types of Scl2 proteins, one of which we combined with nHAP nanoparticles, particles used to replicate the physical gradient of tissue interfaces, to create our first gradient. We then used two different types of collagen (differentiated by their own binding sites) hydrogels to create a second gradient produced through our gradient maker (further explained in the gradient maker section).

After meeting with many experts in the field, including Dr. Lawrence Bonassar, whose research focuses on biomaterials and tissue engineering, and Jos Olijve, a member of the Scientific Development Team at Rousselot, we decided our best options for crosslinking would either be chemical crosslinking, which is a widely used method that has a high degree of crosslinking but has potentially cytotoxic effects, and UV photo-crosslinking, which enhances the mechanical properties of hydrogels but has a lower crosslinking density. Through trial and error, we decided to use UV photo-crosslinking for reasons described in later sections.

From our successful production of our gradient-mimetic-collagen hydrogels we hope that our hydrogels can potentially be used for injuries requiring bone-tissue healing.

Creation Process

---

We now describe the process of making Scl2 hydrogels. Although we were unable to obtain Scl2 proteins, we followed this process with rat-tail collagen and used the same process. First the Scl2 proteins are functionalized for photoreactive crosslinking by conjugation with acrylate-PEG-N-hydroxysuccinimide inside a buffer solution, then placed on a shaker for 48 hours at 4°C. Next, the proteins were diluted with acetic acid, and PEGDA power was added to each solution to 5 wt.%. For our bone-mimicking gel, we add nHAP power to 5wt.% for imaging purposes. Next, we create a UV photoinitiator solution of 2,2-dimethoxy-2-phenyl-acetophenone in N-vinylpyrrolidone, which is added to the Scl2 solution.

To create the final gels, we used our gradient machine to mix the viscous solutions into silicone molds, with a gradient generated over the distance of 1cm to 1mm depending on the machines settings. To crosslink the hydrogels, we exposed them for 5 minutes to longwave UV light (6 mW cm-2 , Spectroline). This UV irradiation time was based off of our rheological testing, with a longer exposure time due to the use of a lower power source. The two sources were balanced for total exposure energy. The hydrogels were then immersed in PBS for 24 hours.

Design Process

---

Previous hydrogels designed as cell scaffolds lack specificity in catering to joint tissues, where cell types do not grow in even layers but in a gradient, such as with the bone-cartilage or bone-tendon interfaces [2]. Papers such as *Bioactive hydrogels based on Designer Collagens* [1] focus on the creation of hydrogels able to grow a single type of cell. Our gradient hydrogels encourage growth of the multiple cell types required for injuries in the knee and elbow joints. In addition to the previous papers mentioned, we used the results from Nanocomposite hydrogels for biomedical applications [3] and *Biomimetic gradient hydrogels for tissue engineering* [4] to create our procedure for the hydrogels.

We first created hydrogels out of gelatin to gain familiarity with the process and test our hardware devices. The process for gelatin hydrogels started with modeling the gels based off of a scientific paper describing designer collagen hydrogels [1]. This involved a two-step creation process in which the Scl2 proteins were functionalized with photoreactive crosslinking sites followed by collagen conjugation to polyethylene glycol (PEG) using photoreactive crosslinking as well. This procedure was then modified in a specialized manner according to the details of our project. We started off using chemical crosslinking and then made modifications from this. The gelatin hydrogels underwent multiple rounds of iteration and trials, but we finally made hydrogels using water, gelatin, nHAP particles, and a 2% glutaraldehyde solution.

We observed from our first trials that the nHAP nanoparticles were prone to clumping in the gelatin-glutaraldehyde solution, and after 10 hours of crosslinking, the hydrogels had a low viscosity with the nHAP particles settling at the bottom of the liquid. In contrast, the gelatin solution without the nHAP particles had a thicker consistency resembling that of a hydrogel. We used part of both of the hydrogels to attempt making a gradient hydrogel.
![](https://static.igem.org/mediawiki/2021/2/29/T--Cornell--engineering1.png)
Figure 1 3% wv gelatin with nHAP particles made at a 1:10 ratio of glutaraldehyde to gelatin solution (left without flash, right with flash)
![](https://static.igem.org/mediawiki/2021/6/62/T--Cornell--engineering2.png)
Figure 2 3% wv gelatin made at a 1:10 ratio of glutaraldehyde to gelatin solution (left without flash, right with flash)
![](https://static.igem.org/mediawiki/2021/2/29/T--Cornell--engineering3.png)
Figure 3. Gradient hydrogels

With the remaining gels we froze them for 12 hours at 4°C and then lyophilized them for 48 hours. These gelatin hydrogels served as the basis for the experimenting done for the collagen hydrogels as these were a more general solution while we waited for the wet-lab subteam to engineer the collagen-secreting *E. Coli*.

In anticipation of our Scl2 proteins, we first created rat tail collagen hydrogels. Rat tail collagen, compared to gelatin, has the advantage of requiring the same procedure as Scl2 to create hydrogels. First, we separately created our two scaffold types - with and without nHAP, to encourage growth of bone and cartilage tissue, respectively. For the hydrogel with the nHAP, we first dissolved the rat tail collagen in 20 mM acetic acid, and then combined this with the PEGDA powder and nHAP powder. For the crosslinking, we added glutaraldehyde at a 1:1 ratio to the PEGDA. The hydrogel without nHAP followed the same procedure, excluding the nHAP.
![](https://static.igem.org/mediawiki/2021/0/07/T--Cornell--engineering4.png)
Figure 4 First trial of making rat tail collagen hydrogels with nHAP particles (right without flash, left with flash)
![](https://static.igem.org/mediawiki/2021/8/83/T--Cornell--engineering5.png)
Figure 5 Second trial of making collagen hydrogels (left side has nHAP, right side does not)
![](https://static.igem.org/mediawiki/2021/0/08/T--Cornell--engineering6.gif)
Figure 6 Slow motion video showing consistency of the collagen hydrogels pictured above.

Early on, our major issues were that the consistency of our hydrogels were not firm enough. Through testing, we determined that this resulted from issues within our procedure, specifically our crosslinking methods. We decided to test two different crosslinking methods: chemical and photocrosslinking.

During the design process, we tested multiple different amounts of each of our crosslinking chemicals and UV light time exposure, and ultimately chose the procedure yielding the firmest hydrogel, detailed above (refer to “Creation Process”). When choosing between different crosslinking methods, our team selected our photocrosslinking procedure for our final product, as it yielded firmer hydrogels. In addition, after doing more research and receiving feedback from Jos Olijve, a member of the Scientific Development Team at Rousselot, we learned that consideration of the cytotoxic effects of glutaraldehyde present in the chemically crosslinked hydrogels should be taken into consideration.

Once we decided on photocrosslinking, we further refined our procedure for this method. After imaging, we discovered that our nHAP particles still tended to clump to each other, as opposed to the even distribution we had intended. To solve this, we added the additional step of sonicating our nHAP solution before adding the collagen, which yielded improved distribution and imaging results. We then evaluated the quality of our hydrogels through imaging, mechanical testing, and cell cultures, described in our Proof of Concept section.

Resources

---

* 1. E. Cosgriff-Hernandez, M.S. Hahn, B. Russell, T. Wilems, D. Munoz-Pinto, M.B. Browning, J. Rivera, M. Höök, Bioactive hydrogels based on Designer Collagens, Acta Biomaterialia, Volume 6, Issue 10, 2010, Pages 3969-3977, ISSN 1742-7061, https://doi.org/10.1016/j.actbio.2010.05.002.
* 2. Lauren M. Cross, Ashish Thakur, Nima A. Jalili, Michael Detamore, Akhilesh K. Gaharwar, Nanoengineered biomaterials for repair and regeneration of orthopedic tissue interfaces, Acta Biomaterialia, Volume 42, 2016, Pages 2-17, ISSN 1742-7061, https://doi.org/10.1016/j.actbio.2016.06.023.
* 3. Gaharwar, Akhilesh K et al. “Nanocomposite hydrogels for biomedical applications.” Biotechnology and bioengineering vol. 111,3 (2014): 441-53. doi:10.1002/bit.25160
* 4. Sant S, Hancock MJ, Donnelly JP, Iyer D, Khademhosseini A. “Biomimetic Gradient Hydrogels for Tissue Engineering.” Can J Chem Eng, 2010 Dec, 88(6):899-911, doi: 10.1002/cjce.20411. PMID: 21874065; PMCID: PMC3160739.

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# Team:Cornell/Experiments

# Experiments

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

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

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [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)

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# Team:Cornell/Results

# Results

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

### What should this page contain?

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

### Describe what your results mean

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

### Project Achievements

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

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

### Inspiration

See how other teams presented their results.

* [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)