iGEM 2022 — Team cornell

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block lead %}{% endblock %} {% block page\_content %}

[* February 2022](#feb-22)
[* March 2022](#mar-22)
[* April 2022](#apr-22)
[* May 2022](#may-22)
[* June 2022](#jun-22)
[* July 2022](#july-22)
[* August 2022](#aug-22)
[* September 2022](#sept-22)
[* October 2022](#oct-22)

## February 2022

Jan. 30th - Feb 5th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The subteam began to brainstorm for the new project season. They learned about the role of WL within the iGEM team and got oriented with the lab space.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam met for the first time and began brainstorming. We also went over basic biological engineering concepts as a introduction to the Wet Lab aspect of iGEM. For brainstorming,
the subteam looked into previous winners and assessed what those teams did well.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: PnP subteam members met for the first time, briefly discussing new member training and education. Through brainstorming, members analyzed the integrated human practices section of previous winners.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: Wiki had their first subteam meeting. They went over the basic skills of HTML, CSS for web development. Then, the members had their first assignment, which was to redesign one of the old Cornell iGem’s wikis. They also had to work in groups for their brainstorming assignment, researching another team’s project and identifying their strengths and medals.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business: The business subteam held our first meeting. In addition to working on our general team brainstorming process we start reviewing business plans that were nominated for best supporting entrepreneurship awards and on finding applicable grants for our team to apply for.

Feb. 6th - 12th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: Wet Lab had its first modeling training and learned to use MATLAB to model biological processes. We continued to brainstorm. In lab, new members learned how to use PCR to amplify DNA and perform gel electrophoresis to analyze the results of their PCR.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: This week the subteam worked on developing their own project ideas as well as further training. For this week, the training project was on learning ECE components and creating low and
highpass circuits.

![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: PnP subteam members conducted research to inform their own project ideas for brainstorming purposes. After establishing these proposals, they analyzed how the subteam’s roles and responsibilities would
apply to these projects.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: This week, wiki members came up with their own ideas for the brainstorming assignment to present to the team. They also continued discussing their progress on the assignment as they created a different color scheme and organization for the 2018 Cornell Wiki page.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business: This week the business subteam continued brainstorming ideas for potential team-wide projects. Additionally, business team members were compiling lists of alumnus at different companies to reach out for fundraising goals.

Feb. 13th - 19th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The subteam continued with the brainstorming process, as well as learning to use MATLAB to model biochemical processes.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam continued with the brainstorming process. Additionally, PD worked on wiring the circuit schematics from the previous week on an actual breadboard.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: This week, the subteam continued brainstorming. Furthermore, all subteam members conducted mock interviews in preparation for effectively communicating with potential stakeholders.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: This week, the members had a group meeting with our brainstorming groups, narrowing down our individual ideas to a total of five project ideas per group. They also presented their completed Wiki pages in the Wiki subteam meetings, sharing their changes and reasoning.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business: The business subteam continued work on brainstorming. The business subteam finalized a list of alumnus to reach out for fundraising efforts.

Feb. 20th - 26th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team continued brainstorming while working on practicing MATLAB skills.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam continued with the brainstorming process and began Computer Science training. For this training, the members had to model cell growth within a bioreactor.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: PnP continued with the brainstorming process and started exploring potential scientific contacts to reach out to. Each member contacted two individuals, applicable to the brainstorming ideas, for informational interviews this week.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: Wiki split up subteam responsibilities for the slides and also worked on researching ethics, feasibility and uniqueness for the project idea. In the Wiki subteam meeting, we talked about the second assignment, which was a Figma design project to recreate an existing webpage and then also come up with an improved design.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business:  Business subteam members continued working on brainstorming efforts. Business subteam members also conducted presentations on the critiques and flaws on the prior years business plan.

## March 2022

Feb. 27th - Mar. 5th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team continued with brainstorming assignments. They worked on MATLAB assignments to model different biochemical kinetics and produce powerful visuals. They began to learn how to create genetic sequences in SnapGene.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam continued with brainstorming assignments. The subteam also began working on a group Matlab assignment in which they had to model the growth rate of two bacteria that were
in competition.

![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp discussed the difference between interviewing professionals for technical interviews, and interviewing stakeholders. Pnp also ran practice interviews with the Wet Lab and PD leads. Also continued to reach out for technical interviews.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: Wiki continued working on their brainstorming, as well as discussing their designs for the Figma assignment.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business:  Members continue working on their brainstorming, as well as analyzing past business plans for strengths and weaknesses.

Mar. 6th - 12th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team continued to brainstorm while working with SnapGene to create genetic sequences for projects in the brainstorming process.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam continued with the brainstorming process. The subteam also completed their group assignment in Matlab and learned about Mechanical Engineering concepts. The members began
working on a CAD training assignment.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp worked on reaching out for technical interviews for each of the brainstorming projects as well as on making a general list of stakeholders for this upcoming year.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: This week, members had to further analyze and provide research on the same 2 ideas from the last brainstorming assignment. Wiki discussed the projects and raised any questions or concerns that they had.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: This week the members continue to work on and expand their brainstorming assignment from the previous week. Members run swot analysis and stakeholder analysis as well as competitor analysis on the brainstorm product of their choice. Members also create presentations detailing strengths and weaknesses from past business plans.

Mar. 13th - 19th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team continues to use SnapGene and MATLAB to create models and constructs for the projects remaining in the brainstorming process.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam worked on brainstorming assignments and finished their CAD assignment from the previous week. The members also began working on sketches of the hardware aspects for each
of the project ideas left in the running.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Working on reaching out to stakeholders and conducting technical interviews for our final two brainstorming projects.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: This time, members were assigned to new groups and we had to work on one project idea. For wiki, members came up with design and color scheme ideas as well as features for accessibility. They presented their redesigns for the Wiki assignment in the subteam meeting.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business: Members had multiple activities to cultivate skills essential for business analysis. Members spent time learning how to design infographics and look out for essential details to maintain consistency. Team members also worked on furthering brainstorming ideas and performed data analysis and competitor analysis for their respective brainstorming teams.

Mar. 20th - 26th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team decided on a final project idea. They got to work designing the genetic circuit and looking into metabolic pathways that would be useful during the design process.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam worked on decided a final project idea for this year. The subteam also got an introduction to using Arduinos and worked on a training assignment in which they had to wire
an LCD Display and code it to say “Cornell iGEM”.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: We continued to reach out to stakeholders and conduct technical interviews. We discussed pros and cons of each final project to decide which one to go with. We also started discussing potential outreach opportunities for both projects.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: For the project’s wiki ideas, members came up with several logo ideas for the web page on Figma. They drew up sketches for a mascot idea, which was included in the presentation for wiki ideas. For our wiki subteam meeting, members participated in a mini workshop on JS, learning about several features about JavaScript and were introduced to the next assignment. The next assignment involves enhancing another iGem team’s wiki page with Javascript.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business: This week, business members came up with fundraising ideas to gain money for travel to the Jamboree as well as started grant research. Members also continued working on their respective brainstorming ideas.

Mar. 27th - Apr. 2nd

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team continues with the construct work from last week. They found potential parts that the team could use to create plasmids to transform into our bacteria.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam began working on the final project idea, MicroMurals. The subteam worked on further brainstorming the hardware and software aspects of the project.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp continued conducting interviews for the final project, MicroMurals. The subteam also continued to brainstorm outreach events to set up for the summer.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: Members continued working with Javascript features on a webpage, working with text and image animations.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Members continued working on designing fundraisers and worked on specific grant applications as well as research for more. Members also learned how to send mass emails using Mail Merge. Members also continued to work on brainstorming ideas.

## April 2022

Apr. 3rd - 9th

SPRING BREAK

Apr. 10th - 16th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team wrote a progress report for the rest of Cornell iGEM and worked to prepare slides for the role of WL in this year’s project.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam wrote up a progress report for PD so far. Within this report, plans for revamping the bioreactor, converting the 3D printer to a 3D bioprinter, and the MicroMurals App were
mentioned.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp started getting ready for the first outreach event for the project at Splash! Cornell. Worked on presentation and different activities for the high school students to do that related to synthetic biology.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The members finished up with some ideas and mockups for the project, MicroMurals and continued learning about Javascript features.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Planned a trivia fundraising event. Additionally drafted up a list of grants that are applicable to MicroMurals to raise funds.

Apr. 17th - 23rd

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team met and continued to find parts that would be useful in CuGEM’s final construct, as well as working with MATLAB.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam worked on a timeline for our project and went through the various special awards that the subteam should aim for. The members continued developing the hardware and software
aspects of the project.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp went over medal criteria for bronze, silver, and goal and discussed our strategy to implement our interviews, human practices, and outreach events to best fit medal criteria. Subteam also continued to reach out to stakeholders and for technical interviews.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: This week, wiki worked on color scheme, accessibility, and mascot designs. The members finalized some designs and made mockups for the logo and banner, as well as prototypes for the app. They used Figma for the designs and through deliberation and voting with the rest of the team, the logo was decided.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business:  Continued grant research and identified multiple finding opportunities that apply to MicroMurals. Worked on completing and submitting grant applications.

Apr. 24th - 30th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team met with our product development team to answer questions about the design of the bioreactor and the 3D printer.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam began looking into designing our own Handheld Bioprinter and had a meeting with Wetlab. During our meeting, we fleshed out a printing walkthrough to guide the development
of our printing system from the cell culture to the final product.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp collaborated with iGEM Patras by sending a photo of a member standing as a T to represent the nucleotide. Continued talking about outreach and reaching out to stakeholders. Also started making educational infographics.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The members worked on training and the assignment for Github this week. They learned how to work on a repository with other team members, and update the CSS and HTML code on the team page and push those changes to the GitHub repository.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Continued sourcing and applying for grants applicable to MicroMurals.

## May 2022

May 1st - 7th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team created the actual plasmid that would be used in the final project, reliant on a certain number of important accessory parts. We discussed summer work expectations.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: Members did further research on the hardware aspects, modeling, and testing of our project. We discussed what changes we needed to make to the 3D printer, how the bioreactor will operate,
and what testing needs to be done to the handheld bioprinter.

![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Subteam members reached out to two contacts in the areas of biology and art, in addition to conducting interviews. Furthermore, PnP-related medal criteria was analyzed and important points were outlined.
In smaller subgroups, the subteam collaborated on potential outreach ideas in the areas of media, Cornell Botanical Gardens, art outreach, and a collaboration event with other Cornell student organizations.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The members finalized designs, and looked at the timeline for Wiki for the coming semester. They heard updates from the other team members and subteams to get a better understanding of the project, and the timeline for the coming semester.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business:  Reviewed past business plans to determine the best structure and most important elements. Used these findings to draft the outline of our entrepreneurial plan.

May 8th - 14th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team finalized the schedule for summer.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam finalized all plans for the summer. This included making a GANTT Chart in which everyone could visualize our timeline for the summer session. The subteam also placed orders
for any materials needed for the summer.

![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Subteam members continued analyzing the PnP-related medal criteria, specifying potential ideas to achieve the criteria. Potential outreach ideas were discussed in a subteam setting and revised to better
effectively reach our target audiences. Additionally, potential stakeholders were contacted for interviews and interviews were conducted.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The members planned out all design-related content and software for the upcoming fall semester.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Created final iterations of the business plan outline, reflected on work to do for the next semester and planning the timeline and tools required to complete the business plan.

May 15th - 21st

FINALS WEEK

May 22nd - 28th

BREAK

## June 2022

May 29th - Jun. 4th

BREAK

Jun. 5th - 11th

BREAK

Jun. 12th - 18th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: Recorded B07 Lab iGEM inventory
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam began the summer session. The members began designing the new bioreactor, working on a hydrogel making procedure to follow, and developing the new 3D printer carriage.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Went over the goals for the summer with outreach plans and interviews as well as went over slight updates in the project to catch everyone up.

Jun. 19th - 25th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The subteam continued lab preparation through autoclaving materials such as pipette tips and learning how to utilize an alcohol lamp. The first transformation of chromoproteins within DH5 alpha *E. Coli* competent cells were also performed through resuspending chromoproteins (purple, red, yellow, and blue) from the 2021 iGEM DNA distribution kit. The plasmid being used is pSB1C3.
![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam ordered and purchased some more materials for our project. Members began developing a plan for extrusion testing, CAD for the printer carriage, and sketches of our Handheld
Bioprinter.

![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Scheduled outreach events and reached out to stakeholders.

Jun. 26th - Jul. 2nd

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The subteam continued with additional transformations after analyzing the previous transformation complications such as the antibiotic used not dissolving. Discovered contamination issues within the
lab.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam made hydrogels, finished the first iteration of the CAD for the handheld bioprinter, began working on fluids modeling, finalized the new bioreactor mixing fan, walked through
the Arduino code for the bioreactor.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Interviews were conducted, planned outreach events, and continued scheduling interviews with stakeholders.

## July 2022

Jul. 3rd - 9th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The subteam decided to work on fixing the contamination issue within the lab through improving sterile technique. Transformations were also performed with newly shipped competent cells (DH5 alpha
strain). Finally, wet lab also focused on documentation for the project.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam had a joint meeting with Wetlab to discuss the best way of optimizing the bioreactor for them. Additionally, the subteam made more hydrogels, began wiring the electrical
components of the bioreactor, and cleaned up all sketches and CAD for the 3D printer and handheld bioprinter. We also began working on documentation for our project.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: PnP did a medal criteria check in to ensure we are meeting gold medal criteria and continued with interviews and outreach and education. Also worked on infographics for the website.

Jul. 10th - 16th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The subteam continued with improving sterile technique by replacing ethanol. Transformations were also re-analyzed and concentrations of all relevant parts from the 2021 and 2022 iGEM DNA kit were
measured using a spectrophotometer (nano-drop).

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: Members finished the Handheld Bioprinter CAD, took measurements of the 3D printer carriage, worked on testing the bioreactor components, and began fluid modeling on ANSYS. The subteam
also continued working on the electrical wiring of the bioreactor and planning out the electrical component for the handheld bioprinter.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Reached out to policy stakeholders to begin the process of policy analysis for MicroMurals.

Jul. 17th - 23rd

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: After not seeing chromoprotein expression in the last few transformations, a new protocol was adopted and tested against the original protocol to see if the new protocol would be more effective.
The new protocol showed promise during analysis.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam had a zoom meeting with CCA iGEM for our collaboration with them. Members continued working on ANSYS fluid modeling, the CAD for the handheld bioprinter, creating an extrusion
method testing procedure, and creating the CAD for the bioprinter head.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Continued working on infographics for pnp content and continued to schedule and plan an outreach event with Seniors at Longview Community Center and reaching out for other events.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Brainstormed fundraising ideas for start of semester, assigned a iGEM Business Manual as required reading for subteam members, and to write a reflection on it due in August.

Jul. 24th - 30th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: This week, the subteam revisited the protocols being used for transformations and further tested new protocols using the New England Biolabs (NEB) protocol for transformation which is the protocol
that came with the cells ordered in early July. Liquid cell cultures were created of a transformation that showed promise of color but after 36 hours, no significant color was seen.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: This week, the subteam focused on completing the electrical wiring of the bioreactor, completing ANSYS modeling, shrinking the dimensions of the handheld bioprinter, and looking into
any code adjustments for the 3D printer. Additionally, we performed confined compression testing to test our hydrogels.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Started policy analysis and reviewing policy framework from previous iGEM teams and worked on starting an educational game for outreach.

## August 2022

Jul. 31st - Aug. 6th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: This week the subteam focused on writing documentation, working on literature for our improved part, and ordering parts for the fall session to continue work from the summer.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: This week the subteam completed hardware assembly of the bioreactor, the handheld bioprinter, and the 3D bioprinter head. Additionally, more hydrogels were made for further extrusion
and confined compression testing. We also focused on writing our documentation this week.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: PnP prepared and conducted the Sciencenter outreach event. Furthermore, documentation was solidified and further analysis was conducted on our progress on medal criteria.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business: Created a large social media fundraiser to help development of our project, with a bingo board and grand challenges based on amounts raised. The subteam also edited and finalized grant applications.

Aug. 7th - 13th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: This is the last week of the summer session! Wet Lab focused on continuing documentation and answering feedback from leads for final touches.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: This week the subteam completed hardware assembly of the bioreactor, the handheld bioprinter, and the 3D bioprinter head. Additionally, more hydrogels were made for further extrusion
and confined compression testing. We also focused on writing our documentation this week.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: PnP continued to focus on documentation, editing ethics write-ups, and developing the ethics handbook.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Created an automated budget sheet for the team to use for any inflows/outflows of money, submitted a few grants, and members submitted a reflection on the business manual.

Aug. 14th - 20th

BREAK

Aug. 21st - 27th

BREAK

Aug. 28th - Sept. 3rd

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team came back from summer session and got straight to work transforming bacterial colonies with pieces of the final construct. They also created liquid cell cultures that would be used to test fermentations in the coming weeks.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: First week back from summer session and Product Development worked on troubleshooting the 3D printhead design, upgraded the Handheld bioprinter casing, and worked on recruitment for next year’s team.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp continued to work on documentation and ethics handbook. Also started reaching out and checking in with stakeholders from the last few months. Pnp also started making plans to set up and host a
bioart exhibit here at Cornell.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: Wiki met with Product Development to discuss the implementation of the MicroMurals app, discussing frameworks and programming languages. The team reached out to various sources to inquire about how to best implement this mobile application.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Re-analyze award winning business plans and understand aspects where they stood out/areas where they could improve and edit our business plan outline to reflect these changes and incorporate the strengths from prior award winning plans.

## September 2022

Sept. 4th - 10th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: WL continues to perform bacterial transformations. We also worked on getting set up to perform a Gibson Assembly to finish off our construct build.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: This week, PD made some more hydrogels, designed a new 3D printhead, and looked through feedback on developing the MicroMurals app. We also continued working on our bioreactor.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp continued to work on documentation, writing up the ethics handbook, and checking in with stakeholders. Pnp also prepared for an upcoming outreach event with Big Brothers Big Sisters of Ithaca.
Pnp also started reaching out about the bioart exhibit.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: Wiki met with Product Development to discuss the implementation of the MicroMurals app, discussing frameworks and programming languages. The team reached out to various sources to inquire about how to best implement this mobile application.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Re-analyze award winning business plans and understand aspects where they stood out/areas where they could improve and edit our business plan outline to reflect these changes and incorporate the strengths from prior award winning plans.

Sept. 11th - 17th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: WL continues to do work amplifying parts to be used in the Gibson assembly. We calibrated and tested the performance of certain machines in the lab.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam continued working on our bioreactor, testing the 3D printhead with hydrogels we produced, testing the handheld bioprinter, and continued updating documentation.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp continued to check in with stakeholders and also reached out to science students to probe interest about a potential biolab here at Cornell. Also, prepped for the outreach event that is happening
on Sept 18th. Finished up ethics handbook and continued completing documentation.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The members started working on background designs for Cornell’s collaboration with McGill for the bacteria book. The members decided to use React Native to create our mobile application.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Provided feedback and incorporated changes to worksheets, checked in with members about their respective business plan sections and started compiling information into a rough draft.

Sept. 18th - 24th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team amplified all of our linearized gene fragment parts and did a Gibson Assembly to build out our construct. Liquid cultures of our bacteria were created and solutions to induce production of our proteins of interest were made as well.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: This week, we worked on setting up the livestream for our APP, helping PnP with creating an online game, and we continued testing both bioprinters with hydrogel.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp hosted an outreach event with Big Brothers Big Sisters of Ithaca where kids came and visited different stations that taught them about synthetic biology, the scientific method, and the experimental
process. Pnp also found a location for the bioart exhibit and started collaborating with art students to set it up. Pnp continued to work on documentation and finalizing write ups for the wiki.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The members continued refining the background designs for Cornell’s collaboration and continued working on the implementation of the application, working on features like storing images and profile data. The members started formatting the wiki and brainstorming features for the pages.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Combined all the sections and created a rough draft of the business plan. Talked to WL + PD to understand how our commercialization would work hand in hand with them, ideation session for best business models to use for our product, provided edits and feedback to members. Talked to an IP lawyer to understand regulations involving our product.

Sept. 25th - Oct. 1st

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: Gibson was performed to synthesize our construct and bacteria were transformed using the newly made plasmid. Colony PCR was performed to verify successful transformation.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: This week the PD subteam worked on incorporating CD nanofibers into our hydrogels, finishing up documentation, and printing our hydrogels using our 3D bioprinter. Additionally, the subteam worked on completing the PnP game and the livestream set up for the app.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Pnp continued to work on documentation and finalizing write ups for the wiki. Also worked on editing and improving infographics for the wiki. Pnp continued to reach out and collaborate with artists
to set up the bioart exhibit which will be set up Oct. 2nd.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The members continued implementing the pages into the wiki. Also, the subteam provided graphics and sprite designs for the MicroMurals video game. They continued finalizing the front-end aspect of the mobile application.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
 Business: Final draft with substantial revisions created, created infographics of various data points.

## October 2022

Oct. 2nd - 8th

![](https://static.igem.wiki/teams/4322/wiki/icon-wl.svg)
Wet Lab: The team worked this week to help PD set up the bioreactor, while also creating batch fermentation flasks to compare growth in our proprietary fermentation reactor.

![](https://static.igem.wiki/teams/4322/wiki/icon-pd.svg)
Product Development: The subteam worked on sterilizing the bioreactor, testing the handheld biprinter device, and printing more images with our 3D bioprinter. We also finalized all documentation before wiki freeze.
![](https://static.igem.wiki/teams/4322/wiki/icon-pnp.svg)
PnP: Worked on finalizing documentation and infographics, before wiki freeze. Set up our bio art exhibit which will be running for the week.
![](https://static.igem.wiki/teams/4322/wiki/icon-wiki.svg)
Wiki: The subteam members worked to finalize the wiki, adding in all of the information, graphics, images into the web page. They added additional features, such as a scroll to top button and an audio button for accessibility.
![](https://static.igem.wiki/teams/4322/wiki/icon-business.svg)
Business: Sent business plan to industry stakeholder for feedback, incorporated feedback + worked to quality check and finalize the business plan.

{% endblock %}

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

Wet Lab. .
To create the MicroMurals project, wet lab’s goals have been split into 3 parts: recreate chromoprotein expression from Team Uppsala’s 2012 and 2013 teams, enhance color intensity, and create bio-ink. Part 1 will be completed when we see color and use spectroscopy to confirm. Part 2 will have worked when transformed colonies with the auxochrome treatment are visibly seen to be more vibrant than without the auxochrome treatment. Part 3 consists of having these chromoproteins fused to csg (curli nanofiber) monomers and chemically crosslinked into a hydrogel to create the bio-ink needed for the bioprinters that our product development subteam created.
Product Development. .
Bioreactor. .
Overview: As our primary product is a bacterially produced bioink with expressed chromoproteins, it is essential that we can provide a safe and efficient place for our bacteria to grow. This bacterial home must maintain a precise balance of several variables and react to the changing environment of the bacteria in real-time. In order to produce a large amount of bacteria without needing a giant growth chamber, we have chosen to create a continuous flow bioreactor. In essence, we supply nutrients and dilute basic solutions to meet the needs of the growing E. coli, which produce the csgA subunits necessary for the assembly of the bioink. When existing E. coli have expressed the desired csgA protein, they are pumped out of the growth tank to be replaced by new generations of bacteria to continue the process. This whole process is monitored by a fleet of sensors, which connect to multiple support systems in order to optimize our protein production.

[* Wet Lab](#wl)
[* Product Development](#pd)
[* Sources](#sources)

## Wet Lab

To create the MicroMurals project, wet lab’s goals have been split into 3 parts: recreate chromoprotein expression from Team Uppsala’s 2012 and 2013 teams, enhance color intensity, and create bio-ink.
Part 1 will be completed when we see color and use spectroscopy to confirm. Part 2 will have worked when transformed colonies with the auxochrome treatment are visibly seen to be more vibrant than without the auxochrome treatment. Part 3 consists of having these chromoproteins fused to csg (curli nanofiber) monomers and chemically crosslinked into a hydrogel to create the bio-ink needed for the bioprinters that our product development subteam created.

#### Part 1: Recreate Chromoprotein Expression

##### Cycle 1:

**Design:** We will use chromoprotein parts created by Team Upsalla to express chromoproteins in *E. Coli*. In this first cycle, we attempted to transform parts 7J (BBa\_K1033922, meffRed), 9L (BBa\_K1033930, amilCP blue/purple), and 9N (BBa\_K1033931, amilGFP yellow) from plate 6 of the 2021 DNA distribution kit into HMS174 (DE3) chemically competent cells. We also tested cells that were transformed in the previous cycle to see if they were still viable.  Since chromoproteins are visible to the naked eye, the transformation will have worked when we see color qualitatively.

**Build:** As Team Cornell was sent the plasmids for the chromoproteins via the distribution kit, the first build process is simply to successfully transform the plasmid into E. Coli and view color expression. If testing goes correctly, the part 2 build will be planned to create the plasmids needed to enhance color.

**Test:** To test the chromoproteins, the parts that were detailed above from the iGEM 2021 DNA distribution kit were used within a transformation on plates with chloramphenicol antibiotic. After 48 hours, colonies grew but no color could be seen. Furthermore, the colonies grew abnormally with highly concentrated sections of cells and contamination.

![](https://static.igem.wiki/teams/4322/wiki/engineering-ecoli-1.jpg)

Figure 1: Results of Transformation after 48 hours showing highly concentrated E. Coli colonies.

![](https://static.igem.wiki/teams/4322/wiki/figure2wetlab.jpg)

Figure 2: Continued results of transformation with abnormal E. Coli growth and contamination.

**Learn:** After viewing the transformation, Team Cornell reviewed what happened during the transformation and came to conclusions that lead to a new design.

1. Glass Beads used for spread plates were not sterile leading to contamination
2. Petri dishes were also not sterile and could cause contamination
3. Chloramphenicol antibiotic was made using water which later was learned to be insoluble allowing for non-transformed colonies to grow

##### Cycle 2: Stopping Contamination Issues

**Redesigning Cycle 1 Design:** After discovering more issues within part 1 of the design, Team Cornell implemented the following changes to design part 1:

1. Created separate chloramphenicol solution with 100% dimethyl sulfoxide (dmso) and 95% ethanol in a 25mg/mL ratio, both to be tested to see which antibiotic solution works best.

1. 100% ethanol was sterile filtered to ensure zero contamination
2. DMSO was not sterile filtered as it was an unopened container

2. New DH5 alpha E. Coli cells were ordered to perform the transformation due to viability concerns
3. New chromoproteins were selected: Parts 7N (BBa\_K1033925, spisPink) and 7P (BBa\_K1033927, asPink)
4. Control plates were added with no antibiotic or plasmid within the E. Coli, it will now be a standard addition
5. **Sterility considerations**:

1. The glass beads were placed in ethanol for 30 minutes and autoclaved immediately after.
2. Petri dish packages are now opened inside a biosafety cabinet (BSC) to ensure no contaminants come into contact with plates.
3. Any process done outside of a BSC will be performed near an alcohol lamp

**The New Build:** The new experiment would be to identify which solution of chloramphenicol, dmso or ethanol, would lead to the best transformation results. Five 100 mm plates were made: two containing chloramphenicol dissolved in sterile-filtered 100% ethanol, two containing chloramphenicol dissolved in 95% dmso, and one with no antibiotic. Standard transformation protocol was followed.

**Testing the new build:** Parts 7N (BBa\_K1033925, spisPink) and 7P (BBa\_K1033927, asPink) from plate 6 of the iGEM 2021 DNA distribution kit were used in this round of chemical transformation experiments. The plates were prepared as described in the previous section. After 18 hours, team members observed that one plate with chloramphenicol dissolved in dmso seemed to show better growth, while the other dmso plate had relatively little growth. A similar result was observed for the plates with ethanol, while the plate with no-antibiotic had many colonies growing.

![](https://static.igem.wiki/teams/4322/wiki/figure3wetlab.png)

Figure 3: Transformations done with chloramphenicol dissolved in ethanol (top right, bottom left), DMSO (bottom right, top), and no chloramphenicol (top left).

**Learn \*continued\*:** After this round of transformation, conferring with subteam and team leads, and additional literature review, team members reviewed the results and came to these conclusions/changes about this experiment:

1. Sterilizing the glass beads and petri dishes before use seemed to provide good results
2. Chloramphenicol dissolved in both sterile-filtered 95% ethanol and 100% dmso provided better results than water-dissolved antibiotic. From this result, we will no longer use water-dissolved chloramphenicol.
3. New information learned:

1. The container of ethanol used was not actual ethanol, but instead a mislabeled waste beaker. We identified pure 95% ethanol that was for general lab use and used it to prepare a new stock chloramphenicol solution for the next cycle. A new 25 gram bottle of chloramphenicol was ordered to ensure sterility and viability would not be a concern.

##### Cycle 3:

**Redesign, again:** After research and discussion with team leads, we decided to only dissolve chloramphenicol in ethanol instead of DMSO. This development was informed by the realization that the ethanol we have been using for the past transformations was contaminated (explained in cycle 3). Any perceived *E. Coli* growth from the previous transformation was therefore not considered.

A concern was raised among team members that there may have been too much DNA in our resuspensions. We found the resuspended double stranded DNA concentrations via a nanodrop spectrophotometer and decided to perform this transformation with lower concentrations of resuspensions in the hope that this would result in a greater transformation efficiency [1].

|  |  |
| --- | --- |
| 2021 Plate 4 Well | DNA Concentration (ug/ML) |
| K9 | 113 |
| K11 | 94 |
| I15 | 32 |
| G17 | 37 |
| C21 | 34 |

|  |  |
| --- | --- |
| 2022 Plate 4 Well | DNA Concentration (ug/ML) |
| B2 | 5 |
| J6 | 11 |
| P8 | 12 |
| D11 | 11 |
| F12 | 13 |

Lastly, a new shipment of chloramphenicol and true 95% anhydrous ethanol allowed us to have a proper stock solution of antibiotic when making plates.

**Build:** Following these preparation steps, a transformation was performed to test the new antibiotic stock, as well as to see how plasmid concentrations impacted transformation ability. Both control plates were plated with untransformed cells (blanks). One control plate contained chloramphenicol antibiotic to test if the antibiotic is effective while the other contained no antibiotic to test if the cells were viable.

**A proper test:** Parts K9 (BBa\_K1033919, gfasPurple), K11 (BBa\_K1033932, spisPink) , and C21 (BBa\_K1033933, asPink) from plate 4 of the 2021 DNA distribution kit were selected for this transformation due to these resuspensions having a high concentration; they were 113 μg/mL, 94 μg/mL, and 34 μg/mL, respectively.

**Results:**

![](https://static.igem.wiki/teams/4322/wiki/figure4wetlab.png)

Figure 4: An attempt at a streak plate (control plate with antibiotic) damaged the plate and resulted in no growth

![](https://static.igem.wiki/teams/4322/wiki/figure5wetlab.png)

Figure 5: Plate without chloramphenicol had growth, which signifies viable cells

![](https://static.igem.wiki/teams/4322/wiki/figure6wetlab.png)

Figure 6: Bottom three plates contain cells that underwent the transformation protocol. A lack of growth suggests that the cells did not acquire the plasmid.

**Learn:**

1. Further research indicated that the optimal concentration of DNA for transformations is 10-20 μg/mL. A new standard step of our transformation protocol, now, is to check the concentration of our DNA via nano-drop to ensure we only use parts with this concentration.
2. Since sterility issues were fully addressed, our current transformation protocol may require reconsideration.

##### Cycle 4:

**Procedural Shift:** Under the impression that our protocol was faulty, we decided to try a different procedure. Courtesy of DeLisa Lab at Cornell University, we tried their protocol which includes a second ice bath following the initial heat shock. Sterility is no longer a major concern as we have made every last effort to ensure all processes regarding transformations are done near an alcohol lamp or in a biosafety cabinet.

Another concern regarding the state of the plasmid samples from the 2021 iGem distribution kit was brought to light. Given their age and our lack of information concerning its previous handling, the DNA could be damaged. Therefore, we decided to try to take our samples from the 2022 distribution kits instead. Interestingly, when rehydrating these parts, there was noticeably less color in the solution. This is most likely because of the smaller concentration of DNA as tested with the nano-drop.

**Comparing Methods:** To ensure experimental control, we performed two transformations – one using the new, DeLisa Lab protocol (Experiment 1) and one using the old protocol (Experiment 2). Experiment 1 bacteria were transformed with DNA from well I15 (BBa\_K592012, eforRed) in plate 4 of the 2021 DNA distribution kit. Experiment 2 bacteria were transformed with DNA from well F12 (BBF10K\_003463, T4T5\_AmilCP) in plate 2 of the 2022 DNA distribution kit. The resuspension of these parts had concentrations of 32 ug/mL and 13 ug/mL, respectively. We also included a chloramphenicol plate with untransformed cells as a control.

**Testing:** The original and new protocols can be found on our Experiments page. After 12 hours, the Experiment 2 plate showed no bacteria growth (bottom right):

![](https://static.igem.wiki/teams/4322/wiki/figure7wetlab.png)

Figure 7: Final Product of Transformation of Original and DeLisa Lab Protocols

Experiment 1 showed plenty of growth – which was clear after 12 hours – as well as clear red chromoprotein expression –  clear after 24 hours.

![](https://static.igem.wiki/teams/4322/wiki/eng-fig8.jpg)

Figure 8: Appearance of a reddish-tinge on one of the plates from the DeLisa Lab protocol

To check if each experimental output really did express the plasmid, we created liquid cultures of both Experiment 1 and 2 bacteria to see if we could identify a color change in the LB. This protocol was [recommended by iGEM Team Uppsala](http://parts.igem.org/wiki/index.php?title=Part:BBa_K592012), the original creators of the chromoproteins we are using. After 36 hours, the result appears to be that the bacteria were not transformed as the LB did not change color:

![](https://static.igem.wiki/teams/4322/wiki/eng-fig9.jpg)

Figure 9: Liquid Cultures of colonies taken from the DeLisa lab plates, which had clear cells on the bottom, indicating a lack of chromoproteins.

**Takeaways and Progress:**

1. Sterility is likely no longer an issue, since the cultures now clearly contain *E. Coli* colonies and not contamination.
2. The transformation procedure that we are using required modification. The DeLisa lab protocol successfully did so.
3. There could be an issue with the plasmid we are using.

##### Cycle 5:

**Procedural Shift:** Given that the DH5α EColi strain was the bacteria being transformed, we also decided to try a protocol specific to this strain. Our bacteria were sourced from New England Biolabs (NEB) and they provide a separate, albeit very familiar, transformation protocol for this strain. The NEB protocol included a longer ice bath after the original heat shock, as well as incubating the transformed bacteria at 37C with 250 rpm agitation. However, we edited the original [protocol](https://www.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987) in order to streamline the process while also ensuring that the protocol would still work properly. Furthermore, instead of using LB after the second ice bath, the NEB protocol used SOC outgrowth. SOC has a higher concentration of nutrients compared to LB which makes it a better option for incubation.

**Build:** This experiment was done solely with the NEB protocol. We did not use our original protocol or the DeLisa Lab protocol. Parts from plate 3 of the 2021 DNA distribution kit were used for this experiment. Wells 2J (BBa\_K592011, cjBlue) and 2L (BBa\_K592011, cjBlue) were rehydrated with concentrations of 11 ug/mL and 84 ug/mL, respectively. The concentration of well 2J was within the optimal concentration range of 10-20 ug/mL, so only well 2J was used for this experiment.

**Test Complications:** During the transformation, the bacteria were left in the incubator for about an extra 10 minutes. Moreover, during the plating process, we realized that each eppendorf tube required two plates:1 with 20 uL of bacteria and 1 with 200 uL of bacteria. Due to complications with the autoclave, we only had two plates available. Therefore, we decided to plate 20 uL first. After new LB agar was autoclaved, we plated 200 uL of bacteria onto new plates.

The plates containing 20 uL of bacteria had little to no growth. Considering the low volume used, this is not an unexpected result.

![](https://static.igem.wiki/teams/4322/wiki/figure10wetlab.png)

Figure 10: After 24 hours, plates containing 200 uL of bacteria showed some growth, although there was no visible color production

![](https://static.igem.wiki/teams/4322/wiki/figure11wetlab.png)

Figure 11: 48 hours after being plated, there appears to be some visual contamination, the cause of which is uncertain.

**Takeaways:**

1. Sterile technique was not properly followed during this transformation

1. Troubleshooting: Petri dish package may have been left open, LB agar was not fully autoclaved

2. The NEB protocol seems to provide good bacterial growth. A repeat of this experiment but with a control (transformation performed with the DeLisa Lab protocol in comparison with the NEB protocol) could provide more insight

##### Cycle 6: The Color at the End of the Tunnel

**Procedural Shift:** After the multiple cycles of transformations, we decided to make more procedural changes through the next transformation. Instead of using a well with previous resuspended DNA, we opened a new well, 9F, and started from scratch. The well 9F contained the pink chromoprotein BBa\_K1033926 . Furthermore, instead of leaving the NEB 5-alpha Competent *E. coli* cells on ice to thaw until we were ready to transfer, we decided to only thaw the cells for exactly 10 mins before starting the NEB transformation protocol.

**Build:** After changing the thaw time in our protocol, we performed the NEB protocol and plated 2 plates with 20uL and 200 uL of the cells with the chromoprotein inside of it. After approximately 20 hours, *E. Coli* growth with pink color began to show.

![](https://static.igem.wiki/teams/4322/wiki/engineering-12.png)

Figure 12: Successful transformation of 200 uL of BBa\_K1033926 within DH5-alpha E. Coli cells after 20 hours (left) and 44 hours (right) of incubation

**Takeaways:**

1. Thawing for too long may have caused the optical density of competent cells to go above the necessary 0.5-0.7 absorbance (at 600 nm). By shortening the thawing time to 10 mins, it is more likely the optical density stayed within that 0.5-0.7 range.
2. With color successfully seen, the next step was to perform a lysis of the cells to extract color.

#### Part 2: Extracting Color

##### Cycle 1:

**Design and Build:** To start the lysis of the pink chromoprotein colonies, we used the NEB lysis protocol with an arbitrary amount of 200 uL of lysis buffer to break open 1 mL of liquid cultures that were made after color was expressed. This ended up being more buffer than the protocol recommended in order to provide more supernatant for chromoproteins to be inside in case the cells absorbed all the buffer and created too many bubbles.

**Test:** By doing the lysis protocol, this was to test the pink expression seen in the liquid cell cultures were chromoproteins and not pink contamination. If the supernatant after the protocol was pink, it would be known that the pink seen was chromoprotein expression. After the protocol was completed, the supernatant was successfully the same pink shade as the original cell colonies meaning the lysed proteins were the desired chromoproteins.

![](https://static.igem.wiki/teams/4322/wiki/pink-lysis.png)

Figure 13: Process of getting pink chromoproteins from competent cells into workable supernatants.

**Learn:** Through the lysis protocol, we were able to confirm that the chromoprotein BBa\_K1033926 was successfully transformed into DH5-alpha cells and their color could be successfully extracted from those cells. Now that we succeeded with color, the next step was to perform a successful Gibson Assembly.

#### Part 3:

##### Cycle 1: Starting out Gibson Assembly

**Design and Build:** For our Gibson Assembly, we planned on creating a PCR colony with our forward and reverse primers and gene fragments of csgA alpha, gamma, csgC, E, and G. CsgA alpha were also linked with the three chromoproteins of focus (asPink, aeBlue, and amajLime). These biobricks were then used within the NEB Gibson Assembly protocol to combine the backbone pSB1C3 and the gene fragments to create a full plasmid. These plasmids were later transformed into E. Coli cells and placed within liquid cultures with IPTG solution in order to induce color.

**Test:** At a molar concentration of 0.4 pmol of DNA per part, we ran 4 consecutive Gibson assembly reactions. They were csgA-alpha + asPink, csgA-alpha + amajLime, csgA-alpha + aeBlue, and csgA-gamma. Transformation occurred 24 hours after Gibson Assembly and DNA purification. 24 hours after transformation, the following images were taken:

![](https://static.igem.wiki/teams/4322/wiki/wetlab-transformed-plasmid.jpg)

Figure 14: Transformed plasmid within *E. coli* cells with circles indicating isolated colonies.

**Learn:** After the experiment, we discovered possible errors in our protocol such as too high concentration of the plasmid being transformed and not purifying the DNA before beginning the transformation.

##### Cycle 2:

**Procedural Shift:** After the first Gibson Assembly attempt where no color was seen in the liquid cultures, we decided to edit the procedure by adding a DNA purification step before transforming the plasmid and lowering the concentration of the plasmid in order to attempt to get better results. Furthermore, unpurified PCR products have many leftover enzymes, nucleotides, etc, so DNA purification would ensure that only the DNA would be present during Gibson assembly.

**Design & Build:** We used 0.4 pmol of each DNA part, using NEBio’s DNA calculator to calculate the proper volume. Overall, 14 PCR reactions were performed to ensure the 4 necessary Gibson reactions could be performed. Once the Gibson reactions were performed, colony PCRs were performed to ensure the success of the Gibson Assembly.

**Test:** An example of a PCR reaction used in preparation for Gibson assembly is shown below:

|  |  |  |  |
| --- | --- | --- | --- |
| Tube | Primers | DNA | Length of DNA part |
| 1 | CP\_Green\_csgC-F (35) + csgE-csgC\_R(28) | rbs\_csgC | 421 |
| 2 | CP\_Pink-CsgC\_F(33) + CsgE-CsgC\_R(28) | rbs\_csgC | 421 |
| 3 | CP\_Blue-CsgC\_F(31) + CsgE-CsgC\_R(28) | rbs\_csgC | 419 |
| 4 | BCKBN-PRMTR\_F(25) + CsgC-CsgA\_gamma\_R(38) | promoter\_rbs\_csgA\_gamma (39) | 1162 |
| 5 | CsgC-CsgE\_F(27) + CsgG-CsgE\_R(30) | rbs\_csgE (20) | 465 |
| 6 | BCKBN-TRMNTR\_R(24) + CsgE-CsgG\_F(29) | rbs\_csgG\_term (22) | 1098 |
| 7 | CsgC-CP\_Green\_R(36) + Linker-amajLimeF(75) | amajLime(43) | 749 |
| 8 | CsgC-CP\_Blue\_R(32) + Linker-ae\_blueF(73) | ae\_blue(42) | 753 |
| 9 | CsgC-CP\_Pink\_R(34) + Linker-as\_pinkF(71) | as\_pink(41) | 756 |
| 10 | BCKBN-PRMTR\_F(25) + amajLime-LinkerR(76) | prmtr\_rbs\_csgA\_alpha\_linker (40) | 845 |
| 11 | BCKBN-PRMTR\_F(25) + ae\_blue-LinkerR(74) | prmtr\_rbs\_csgA\_alpha\_linker (40) | 843 |
| 12 | BCKBN-PRMTR\_F(25) + as\_pink-LinkerR(72) | prmtr\_rbs\_csgA\_alpha\_linker (40) | 843 |
| 13 | TRMNTR-BCKBN\_F(23) + PRMTR-BCKBN\_R(26) | pSB1C3 | 2125 |
| 14 | CsgA\_gamma-CsgC\_F(37) + CsgE-CsgC\_R(28) | rbs\_csgC(18) | 415 |

Concentrations for the PCR reaction were in the range of 150 - 300 ng/uL. 4 Gibson reactions were run afterwards: csgA-gamma, csgA-alpha-linked-pink, csgA-alpha-linked-lime, and csgA-alpha-linked-blue. 0.2 pmol concentrations of each relevant DNA part were used. An example of a Gibson Assembly reaction can be found below, at 0.4 pmol.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | csgAα + aeBlue | | csgAα + asPink | | csgAα + amajLime | |
| Gibsone Assembly Component | Tube # | Volume (uL) | Tube # | Volume (uL) | Tube # | Volume (uL) |
| csgC | 3 | 0.57 | 2 | 0.50 | 1 | 0.70 |
| csgE | 5 | 0.56 | 5 | 0.56 | 5 | 0.56 |
| csgG | 6 | 0.97 | 6 | 0.97 | 6 | 0.97 |
| csgAα + Linker | 11 | 0.92 | 12 | 0.96 | 10 | 1.13 |
| pSB1C3 | 13 | 3.48 | 13 | 3.48 | 13 | 3.48 |
| Chromoprotein | 8 | 0.93 | 9 | 1.01 | 7 | 0.90 |
| dH2O |  | 2.57 |  | 2.52 |  | 2.26 |
| G.A. Master Mix |  | 10.0 |  | 10.0 |  | 10.0 |

![](https://static.igem.wiki/teams/4322/wiki/pink-lysis.png)

Figure 15. Agarose gel electrophoresis depicting no successful amplification from our colony PCR.

**Learn:** The concentrations of these Gibson reactions were better after using the PCR-cleaned DNA parts. This was incorporated into our future protocol designs for our Gibson Assembly procedure. The colony PCR was performed, but results showed nothing on the electrophoresis gels. Amplifying the colony PCR using chromoprotein primers or csgA-gamma primers was a good decision as they would help troubleshoot issues of color production or lack thereof However, a better decision would be to use primers that amplify the csgA and chromoprotein, or a combination of more fragments. This way, we would have a more confident confirmation that the Gibson Assembly worked. On a gel, the band would be at around 750 bp (for chromoprotein only) but we were unable to see any band. Even better, we could miniprep liquid cultures of our colonies and send the resulting plasmids for sequencing to have further confirmation. This will hopefully be incorporated if time and funds allow.

Further, in our troubleshooting process, we learned from the NEB website that the strain we were using, DH5ɑ *E. coli*, was not as compatible with protein expression as other strains.

##### Cycle 3: Procedural Shift

**Procedural Shift:** After literature review of why our colony PCR might have failed, we ended up learning that BL21(DE3) *E. coli* would be a better candidate for protein expression rather than  DH5ɑ *E. coli*. This is due to proteases produced by the latter strain that may interfere with proper protein expression. DH5ɑ *E. coli* is typically utilized for plasmid preparation, while BL21(DE3) is better for protein expression [2].

**Design & Build:** We utilized the workflow from the 2019 Manchester iGEM team [3] in which they used DH5ɑ to prepare their plasmids, mini prepped liquid cultures of these cultures, then transformed BL21(DE3) with the miniprep product to result in successful protein production.

**Test:** A miniprep was performed on 4 mL of liquid cultures made with pink CP expressing colonies from Figure 12. The NEB Monarch Plasmid Miniprep Kit was used. Though the kit suggested using cultures that had been incubating for 12-16 hours, we used a culture that was incubated for ~24 hours as it takes our transformed colonies with the pink CP longer to grow and express the pink color. After miniprep was completed, a transformation of BL21(DE3) cells was performed using the NEB protocol. 200 uL was plated onto a LB+Chlor plate and incubated for ~18 hours. Figure 16 shows a dense lawn of pink colonies, indicating successful plasmid transfer!

![](https://static.igem.wiki/teams/4322/wiki/wetlab-dense.png)

Figure 16. Dense lawn of transformed BL21(DE3) cells, all showing pink CP expression.

**Learn:** From this cycle we learned that plasmid transfer between DH5ɑ and BL21(DE3) is the proper method to use for protein expression. We also learned that a lower volume of the transformation recovery culture should be plated, since we obtained such a dense lawn during this trial. The success of this experiment suggests that we should be using this process for the expression of our fusion proteins. This method will be implemented next in order to fix the issue of non-expression in our transformed cells that should be expressing our final constructs.

## Product Development

#### Bioreactor

[Bioreactor Manual](https://static.igem.wiki/teams/4322/wiki/bioreactor-manual.pdf).

**Overview:**  As our primary product is a bacterially produced bioink with expressed chromoproteins, it is essential that we can provide a safe and efficient place for our bacteria to grow. This bacterial home must maintain a precise balance of several variables and react to the changing environment of the bacteria in real-time. In order to produce a large amount of bacteria without needing a giant growth chamber, we have chosen to create a continuous flow bioreactor. In essence, we supply nutrients and dilute basic solutions to meet the needs  of the growing *E. coli*, which produce the csgA subunits necessary for the assembly of the bioink. When existing *E. coli* have expressed the desired csgA protein, they are pumped out of the growth tank to be replaced by new generations of bacteria to continue the process. This whole process is monitored by a fleet of sensors, which connect to multiple support systems in order to optimize our protein production.

##### Cycle 1

**Design Process:**  The nature of our project requires a functioning bioreactor, which we were able to implement by building upon the bioreactor implemented last year in Cornell’s 2021 iGEM project, Collatrix [1]. Despite appearing functional, this setup had setbacks that prevented it from performing at its desired capabilities. Thus, the goal of our engineering cycle was to improve and optimize this former setup into a system that suits our new requirements.

![](https://static.igem.wiki/teams/4322/wiki/figure1pd.png)

Figure 1: 2021 iGEM Cornell Collatrix bioreactor implementation [1]

Our primary concern was the scale of our project compared to that of last year. With the need for a larger volume of effluent in order to supply the 3D printer with a proper amount of printing material, we considered increasing from 2 jars to 3 jars. This would also provide 3 compartments for us to be able to grow 3 different types of modified E. coli that expressed unique chromoproteins for a larger range of color when 3D printing. Before finalizing this larger system, we decided to first optimize the existing smaller bioreactor.

As we brainstormed further ways to optimize, we made various sketches of what we wanted the updated version to look like.

![](https://static.igem.wiki/teams/4322/wiki/figure2pd1.png)

![](https://static.igem.wiki/teams/4322/wiki/figure2pd2.png)

Figure 2: Sketches of the updated bioreactor for the first iteration

**Build Process:**  Once we began building the updated version of the bioreactor, we realized that a nozzle shown in Figure 2, while in theory is great for sampling, would cause issues. These issues being that it could lead to potential contamination of the cells within the bioreactor, and how to ensure sterility on the outside nozzle. Additionally, once we put the sensors onto the sides of the bioreactor, we noted that three motor fans would be too many and they would bump into the sensors. Thus, we opted for one motor fan for our next iteration.

##### Cycle 2

![](https://static.igem.wiki/teams/4322/wiki/figure3pd.png)

Figure 3: Sketch of the bioreactor for the second iteration

Our primary area of optimization was the mixer blades. From last year, the mixer blade was harnessed to a threaded rod and connected to the motor via a shaft coupler. Initial considerations wondered if the use of 2-3 total mixer blades per threaded rod would be effective in providing uniform mixing of bacteria. This was considered as we progressed through our CAD design process. Ultimately, this multi-stack mixer blade was not used because of the presence of the sensors in the motor as well as the uncertainty of actual improvement. Due to the sensors, there was a physical limit to how far up the motor blade could be positioned. Additionally, it was skeptical if having more motor blades would actually improve efficiency when the additional blades would not be positioned in the center of the solution, but instead near the top or bottom. Thus, we opted to remain with only one mixer blade per bioreactor.

![](https://static.igem.wiki/teams/4322/wiki/figure4pd.png)

Figure 4: Mixer blade from Collatrix 2021

![](https://static.igem.wiki/teams/4322/wiki/figure5pd.png)

Figure 5: First iteration of new mixer CAD model

![](https://static.igem.wiki/teams/4322/wiki/figure6pd.jpg)

Figure 6: Final design for mixer CAD model

The mixer blades themselves were redesigned to have a total of 6 wings as well as having a larger radius. This was our solution to having more uniform mixing by increasing the surface area of the blades, there would be more volume that was constantly in motion to create even mixing. We maximized the length of the mixer blades to provide clearance for the sensors necessary for the maintenance of the bacteria by gorilla taping the sensors to the walls of the container.

![](https://static.igem.wiki/teams/4322/wiki/figure-7-pd.png)

Figure 7: Electrical box of the bioreactor with working LCD display (left)

In addition to the mixer blades, we made improvements to the electrical circuit of the bioreactor. In Cornell Collatrix 2021, the final system was not tested due to the fact that one of the motors was reversed and inadvertently overflowed the base additive over the electrical wiring and boards. Our solution to this problem was in two steps: firstly, building a modular electrical box to house all our motor driver boards, arduinos, wires, and breadboard, and secondly, improving the standardization of wiring and code such that a miscommunication or misalignment would not result in harming our project.

![](https://static.igem.wiki/teams/4322/wiki/figure8pd.jpg)

Figure 8: View of the electrical box in relation to the existing bioreactor and potential placement of the modular component.

The electrical box was made with recycled plexiglass from the construction of the bioreactor housing. The corners were joined and sealed with waterproof caulk and a thin foam sheet was fitted to the bottom of the casing to allow the parts to stay more static. A physical improvement to the circuit was the addition of a working LCD display that will be used to periodically confirm that the system is functioning properly. Furthermore, we were able to consolidate the circuit down from four motor drivers to three.

The standardization of wiring and code was done in part with the help of multi-colored wires. All sets of wires from motor drivers were consistent colors as seen in figure 8 and when applicable, wires connecting ground and power were black and red respectively. Additionally, all wires running from motor drivers to motors were color coded in pairs to make pin input into the code more systematic along with the addition of marking tape on the wires. The marking tape allowed us to identify which wire of the colored pair would map to the positive or negative terminal of the motor (we selected a marked wire as the positive end), such that if a wire were to come loose or needed adjustment it would be simple to reattach and maintain. These new implementations to our electrical system were in attempts to optimize and improve on what was learned from Cornell Collatrix. A comprehensive guide to our electrical system assembly can be found in [Contribution](https://2022.igem.wiki/cornell/contribution) bioreactor section.

Future plans for the electrical system involve the transfer of the breadboard wiring onto a perforated board. This soldered connection is more secure and reliable as compared to our existing breadboard and would ensure that our system is solid and could be transportable. Ideally, this would evolve into a custom PCB system that would truly solidify our wiring.

![](https://static.igem.wiki/teams/4322/wiki/figure9pd.jpg)

Figure 9: Second iteration of the bioreactor from Cornell Collatrix 2021

Overall, the structure of the bioreactor is similar to that of Cornell Collatrix. Notable changes include the addition of an output in the lid of the bioreactor that allows easy access for pipette to harvest grown cells. When not in use, this output is closed with a plug as seen on the right green lid in figure 9. Additionally, taking note of our previous bioreactor, the base housing (pink lid) can be seen positioned farthest away from the electrical box. An improvement to this base system would be the dual motor system installed on the pink lid. By consolidating two base containers to just one eliminates added risk as well as optimizes the number of motor drivers necessary for the electrical system.

![](https://static.igem.wiki/teams/4322/wiki/figure10pd.jpg)

Figure 10: Completed bioreactor system

![](https://static.igem.wiki/teams/4322/wiki/figure11pd.png)

Figure 11: Completed bioreactor electrical circuit schematic

**Software:**  Similarly to the hardware, we inherited the software for the bioreactor from Cornell’s 2021 iGEM project, Collatrix. The code controls the major components for bacterial growth, such as pH, O2 concentration, temperature, and feed. With the aid of an Arduino, the native packages from the sensors that we utilized were installed and used to monitor these levels. La Chatelier’s principle will be applied in the Arduino such that when either pH, O2 concentration, or temperature are not at the optimal level, this would trigger a motor to input base, O2 bubbles, or adjust the sous vide machine to counteract the change to reestablish equilibrium.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Conditions | pH | | O2 Concentration | | Temperature | |
| Change | drop | rise | drop | rise | drop | rise |
| Effect | Input base | N/A | O2 bubbles | N/A | Sous vide machine | Sous vide machine |

Table 1: Maintenance of optimum condition in the bioreactor

The feed of the system is monitored differently and has to be modeled as the feed has to be supplied accordingly to the bacteria growth rate. It is necessary that the feed for the bacteria also increases exponentially to maintain optimum equilibrium between the feed and the bacteria. With a stepwise feed cycle, we are able to mimic an exponential growth curve by inputting increased amounts of feed at a set time interval, always maintaining more feed than the bacteria need. This implementation allows us to always have excess feed, minimizing the possibility of feed being a limiting factor, therefore resulting in better growth. An excess supply of feed also optimizes process time by minimizing the amount of calculation necessary for the Arduino to perform in the loop.

##### Testing

To test the bioreactor, it was set up in our lab space and tested in comparison to a shaker flask culture. All parts were sterilized according to our  [bioreactor manual](https://static.igem.wiki/teams/4322/wiki/bioreactor-manual.pdf). This test was meant to compare growth in both environments. Since our reactor was optimized for pH, oxygen, and feed to provide a better growth environment, we expected to see greater growth in the reactor versus the shaker flask.

During set up, one of the mason jars for the reactor cracked. Thus, we had to continue the testing with only one jar.

![](https://static.igem.wiki/teams/4322/wiki/pd-crackedmasonjar.png)

Figure 12. Cracked mason jar that was meant to be used in the bioreactor initial setup.

Figure 13. Video of final bioreactor up and running in the lab

The reactor was set up with 1000 mL of LB broth, 1000 uL chloramphenicol solution, and inoculated to a starting OD of 0.04 with *E. coli* DH5ɑ that was transformed with the asPink chromoprotein. Though this was not our fusion protein, we wanted to observe growth that best reflected the transformed cells we would be using for the final product, so we decided the transformed pink colonies would be a better model to test with than un-transformed cells. Sodium hydroxide at pH 11 was used as a base to control for pH. The reactor was temperature controlled in a water bath at 37°C.

The shaker flask was set up with 60 mL of LB broth, 60 uL chloramphenicol solution, and inoculated to a starting OD of 0.04 using the same cells as the reactor. The flask was temperature controlled in a shaking incubator, shaking at 150 rpm. Due to the size of our incubator, we could not go higher than 150 rpm or the flask would tip and spill.

Both the reactor and flask were left to run for 40 hours. Samples were taken at 8 hour intervals for both. At first we began with single aliquots, but starting from hour 24 we took three aliquots so we could obtain average OD values in case of data outliers. Between hours 8 and 16 of the reactor, the tape we used to fasten some tubing had fallen off and we did not have extra to use to refasten them. This was interfering with the impeller as the tubing and wires were becoming tangled without being fastened by tape. Thus, the reactor was left for another 8 hours without stirring or oxygen control to avoid overflowing, but was maintained at 37°C. To maintain consistency, the shaker flask was also removed from shaking and kept at 37°C.

Figure 14 below shows the average OD600 readings for the 40 hour period, with error bars depicting the standard deviation. Both setups showed relative stagnation until between 32 to 40 hours, upon which cell density increased greatly. Based on this data, the flask exhibited a greater rate of growth at later times compared to the bioreactor, which was not what we expected.

![](https://static.igem.wiki/teams/4322/wiki/pd-graph.png)

Figure 14. Bioreactor vs. Flask growth experiment over a 40 hour period to compare cell density over time.

Our team came up with several reasons for this observed pattern:

* Growth rate lowered between 8 and 16 hours due to lack of rotation/spinning
* Our transformed cells have typically taken longer than usual (i.e. compared to wild type *E. coli* DH5ɑ) to grow and exhibit any color, which suggests why the cell density did not increase until later times
* Feed was only periodically pumped into the reactor, and might not have been done so often enough to refresh the cultures

Another run of this experiment will be conducted to gain more data, especially since the standard deviations at later times are so large. Some fixes will also need to be made to the reactor, such as finding a more secure way to fasten tubing, editing the time between pumping feed, and ensuring pH adjustment is operating as intended. With these adjustments, we still hypothesize that the bioreactor will exhibit better growth than the flask.

#### 3D Printer

**Overview:** This year our project is focused on the development of environmentally friendly bioart using engineered bacteria encoded to create a variety of colorful hydrogels that we can arrange in a design, and additionally supplementing this art by modifying the material to uptake carbon dioxide and (volatile organic compounds) VOCs to help the environment, essentially creating a living material that can perform all these functions. The primary method of delivering this hydrogel into our final art product is through a modified 3D bioprinter.

Our project this year will be focused largely on developing 3D bioprinting capabilities through the modification and upgrades to a traditional 3D printer. We will be using the base of a Makerbot Replicator 2 and adapting the printhead and print code to remove the plastic heating elements and become compatible with hydrogel extrusion. Inspired by the 3D printing systems of Dr. Joshi at Northeastern University and with feedback from Dr. Meyer at the University of Rochester, and previous work done by the TU Delft 2015 iGEM project, our team will be developing a new method of bioprinting using shear-thinning hydrogels printed without the use of specialized substrates, meaning printing can be accomplished on a wide range of surfaces to enable the creation of unique art [2, 4].

#### Hardware:

##### Cycle 1

The major hardware component of this project is the development of a biocompatible hydrogel 3D bioprinter. We will be accomplishing this through the modification of a 3D bioprinter to become compatible with hydrogel extrusion. Our base printer is a Makerbot Replicator 2, which will be altered in 2 primary ways: the adjustment of the print head, and the creation of a hydrogel reservoir to replace the traditional plastic extrusion roll.

![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure1pd.jpg)

Figure 1: 3D printer with new syringe-holder printer head

Most household 3D printers print plastic through the use of a heating element designed to melt the extruded material, and quickly cool and resolidify in layers. Heating elements can heat the plastic to over 200 degrees Celsius. Temperatures this extreme would cause damage to our hydrogel and kill live bacteria in the hydrogel (thus eliminating any CO2 uptake capabilities of our art) [1, 3]. In order to avoid this, we will be removing the heating elements from the printing head of the Replicator. The removal of the heating element, which also serves as the extrusion nozzle, will then be replaced with a custom-designed hydrogel extrusion head. The extrusion head will feature a motor connected to a syringe needle through a series of gears, which will serve as the new extrusion head for our 3D printer. The exact diameter and extrusion rate will be determined through testing of model hydrogels [see extrusion testing for more information]. In order to house the needle and syringe, we will design a new 3D printed part to replace the carriage of the Replicator. Since the specialized part will be 3D printed, it is designed based on the existing carriage assembly, and will thus be easily interchangeable with the existing carriage, requiring fewer hardware and software changes to make the printer compatible.

![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure2pd.png)

Figure 2: Sketches for Syringe-Holder Printer Head

An initial 3D printed design was created, however it was found to not fit using the originally determined CAD dimensions (specifically the base dimensions were off by 1 mm, and the syringe holder slot was not properly fitted to allow the syringe to slide in), and as such a second version was printed using the modified dimensions. The modified extrusion head was mated with the syringe, and then installed onto the print head (in place of the heated element) to be used for testing and further printing.

![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure3pd.jpg)

Figure 3: Image of Syringe-Holder Printer Head issues

In order to feed the hydrogel to our extrusion head, we will be creating a new reservoir for our hydrogels, which will help serve as the “ink cartridge” for our printer. Initially, there are 2 proposed designs for the reservoir. The first would be to use a syringe preloaded with hydrogels, which is attached directly to a linear stepper motor that drives the extrusion of the hydrogels [1]. This design would utilize a similar hydrogel extrusion model to the Collatrix hydrogel extrusion form last year, with adjustments made to accommodate for the extrusion through tubing into the printer extrusion head. This design is limited by the size of the syringe, where each ink cartridge can only hold as much as the syringe volume, and would then need to be replaced. The second design would use a small tank, containing a larger volume of hydrogel, with tubing fed through a peristaltic pump into the printer tubing. In this design, the extrusion rate is directly proportional to the pump rate, and the use of a larger reservoir means that fewer ink refills are necessary, however, the use of a peristaltic pump for hydrogel extrusion may lead to complications with 3D printing. After testing and feedback from interviews our team decided to move forward with the syringe model as 20mL syringes hold enough ink for several development scale prints, and can be quickly swapped out to test different inks without needing to clear transport tubes as required with a pump based design.

##### Cycle 2

In order to further incorporate the new printing head with the old printer, a geared syringe holding block was built that could integrate with the previous motor used for plastic extrusion. This allows the former wiring and code used to run the printer to be seamlessly transferred to the syringe based system, removing the need for a new software package to be designed and installed. The initial printed model for this head was off in several dimensions, as tolerances between the new part and the existing hardware had to be tested. The first model was also able to be used and reconfigured in order to determine what modifications would make the system run smoothly.

![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure4pd.jpg)

Figure 4: First 3D printed gears and print head

A new model was designed in an integrated assembly in order to test gear interactions and syringe motions.
![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure5pd.png)

Figure 5: CAD Model of the new print head

![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure6pd.jpg)

Figure 6: Disassembled printer head showing individual parts

Figure 7: Extrusion test video

After installation the new design worked, and was able to extrude prepared hydrogels using existing printer gcode. However, the extrusion rate used for the plastic based system pushed a much smaller diameter feed using a gear with a smaller diameter, two factors that both contributed to an extrusion rate that was initially nearly two orders of magnitude too high. After reducing this speed, the mechanisms behind the printer head were safe from self destruction and extrusion testing could begin.

![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure8pd.jpg)

Figure 8: Video camera connected to printer

Modifications were made to the syringe diameter, nozzle, and racking system in order to improve the print quality. A video camera was also added to the printer in order to record video and allow for a livestream to be connected. Achieving a quality print required honing in on sufficient balance between print speed and volume, nozzle geometry, and ink composition. The bacterial hydrogel mimetic used was gelatin, which presented issues due to forming clumps and forming weak bonds after printing in its hydrated state. With the thixotropic bacterially produced hydrogels, they will have the ability to reconnect and form strong bridges and 3D structures.

Figure 9: Printing iGEM logo

![](https://static.igem.wiki/teams/4322/wiki/hardwarefigure10pd.jpg)

Figure 10: Gelatin iGEM logo printed on plexiglass

**Software:**  There are two major software components relating to the 3D printer. The first part is the conversion of the submitted images into a 3D printer compatible matrix, and the second portion is the adjustment to the 3D printing code itself.

Our project aims to make bioart accessible to large audiences, and part of that goal is allowing publicly submitted art to be created on our 3D bioprinter. In order to make these submitted art pieces compatible with our 3D printer, we need to convert the image format (either .png or .jpg) file into a 3D printer compatible matrix format. We will be accomplishing this through our app and translation into matrices using MATLAB. Our app will accept the image submission, and then feed the image data to a MATLAB script, which will use image analysis to convert the image into a 3D matrix of values, using 4 layers (1 layer for each color channel and the 4th layer corresponding to the 3D height of each pixel). This converted image will then be fed to our 3D printer where it will then be printed.

In addition to the conversion of the image type, we will be adjusting our 3D printing code to make our 3D printer biocompatible by editing gcode in the Cura slicing software. Traditional 3D printers which extrude plastic through the use of a heating element in the head must control the temperature of the heating element in the base code. Since our 3D printer is adjusted to remove the heating element, we must also remove the code controlling heating in order to enable the 3D printer to begin printing without issues, as most 3D printing codes will not run if there is no heating element [3]. In addition, we must also make changes to enable multiple color channels, since most 3D printers are only designed to print in a single color. We will also be adjusting the 3D printing extrusion code to be compatible with our hydrogel extrusion. Since we will be using a combination of stepper motors and pumps to extrude our hydrogels, we must adjust the extrusion code to activate our motor/pump proportional to the layer height of our 3D printed image. All changes will be made directly to the 3D printing gcode made after the processing of our image into an stl, then slicing into the final print code. The changes will be made using Cura slicing software, and gcode changes will be logged for future use.

#### Handheld Bioprinter

**Overview:** As an alternative to a large 3D printing machine, we decided to create a 3D handheld bioprinter to provide a mobile and user-friendly device for artists. Previous IGEM teams such as Warwick and LMU-Munich have developed 3D bioprinters to aid in tissue and/or bone repair, yet none of them have created a handheld device. This handheld bioprinter of 145 mm height x 32.5 mm width x  mm length will contain two 3 mm syringes with extrusion nozzles to allow printing of two colors. This device will allow for easy access to bioprinting in the art industry and will act as a substitute for paint brushes and/or colored pencils.

**Hardware:** The hardware is similar to the gradient machine implemented in Cornell Collatrix and based off other sources [1,3]. It operates using two stepper motors powered by motor drivers connected to an Arduino Uno. The rotation of the stepper motor will push a threaded rod through a syringe pusher into a syringe to initiate the extrusion process. However, we plan on extruding through both syringes as opposed to Collatrix’s gradient mixer, which uses two syringes to extrude from one nozzle. Therefore, a push was integrated into the breadboard to control stepper motors and drivers to alternate between syringes and colors.

![](https://static.igem.wiki/teams/4322/wiki/handheldfigure1pd.png)

Figure 1: CAD Model of Handheld Bioprinter

**Bioprinter Casing:** When designing the bioprinter casing, we prioritized minimizing space while still being able to hold two syringes. The casing includes a base that protrudes from the back to maintain syringe pusher position and prevent rotation along with the threaded rod. The syringe pusher will move along the base to initiate extrusion.

![](https://static.igem.wiki/teams/4322/wiki/handheldfigure2pd.png)

Figure 2: CAD Model of Syringe Pusher

![](https://static.igem.wiki/teams/4322/wiki/handheldfigure3pd.jpg)

Figure 3: Sketches for Bioprinter Casing

![](https://static.igem.wiki/teams/4322/wiki/handheldfigure4pd.jpg)

Figure 4: First Iteration of Casing

**Reloading:** When considering reloading, our initial idea was to use the counterclockwise rotation of the stepper motors and threaded rods to move the syringe pusher upstream of the base. The syringe pusher was designed to reload the syringe as it was moved up the base. However, this mechanism failed since the stepper motor’s maximum speed of 15 rpm was excruciatingly slow and ineffective for reloading. Thus, we came up with an alternative solution by redesigning the casing to include a cover attached to a friction freeze hinge and a sliding rod to fixate the cover when closed. This would allow users to open the casing and remove syringes when printing finished. Second iteration of the casing can be seen here:

Figure 5: Second Iteration of Casing

![](https://static.igem.wiki/teams/4322/wiki/handheldfigure6pd.png)

Figure 6: Syringe Pusher for Reloading

As seen in Figure 5, the new casing for the device will contain a hinge. This iteration of our design will be implemented as our next steps to further providing user-friendly aspects.

**Testing:** We designed the handheld bioprinter based on the comfortability and ease of use. This was tested based on how comfortable it was to hold the bioprinter and by printing an entire syringe of hydrogel. Based on our extrusion tests, the bioprinter could be held comfortably and printed successfully.

Testing was conducted using gelatin hydrogel. We tested extrusion by determining if it could extrude the hydrogel smoothly and at a consistent rate throughout the nozzle. Speed could be adjusted through the code by changing the rate of revolution of the stepper motor. Tested at the fastest setting of 15 rpm, extrusion rate was 0.882 mm/s. However, this also revealed inconsistencies in printing. To fix this, we added a 1 mml syringe tip and tested. Testing failed due to pressure buildup and we concluded it was more effective to maintain the 3 mm syringe tips. Extrusion can be seen here:

Figure 7: Video of Extrusion

Accuracy and precision of the bioprinter was tested by printing sketches of sample bioart and comparing expected results to printed products. From this, resolution, precision, and accuracy were determined through user testing seen in Figure 9.

Figure 8: Video of Smiley Face Drawing

In the above video, one can see that in the middle of the printing process, the hydrogel pauses for a second or two before continuing to print. This is due to the fact that when filling the syringe, there were air bubbles that did not allow for further movement of the gel. All in all, the smiley face drawing showed us that our resolution and accuracy can improve with further iterations of the handheld device, in which we can shrink the syringe size for precision and use a different type of hydrogel that does not contain gelatin.

##### Cyclodextrin (CD) Nanofiber Incorporation

After testing gelatin and glutaraldehyde hydrogel, we attempted to enhance the mechanical properties of the gel by combining CD nanofiber into it, which have also been used for VOC-uptake applications [\*\*]. Professor Tamer Uyar from Cornell Human Ecology Department kindly offered us 0.6 g of electrospun CD nanofiber from his lab to experiment on.

![](https://static.igem.wiki/teams/4322/wiki/engineering-fig9.png)

Figure 9: CD nanofiber in test tube (left) sample in sonicating machine (right)

According to Professor Uyar’s instructions and previous sources [5] we first cut the nanofiber into very small pieces and soaked them in warm water to break them down faster. After a day sitting on the bench, we used a magnetic stirrer to stir them for some time until they were mostly hydrated and were floating in the tubes. We performed sonication with pulse on 3 seconds, pulse off 4 seconds and repeated for 20 mins to further disperse them in water. The process pictures are shown below. Our nanofiber is not breaking down at a speed that we hoped, and currently we are trying to increase the efficiency. Here is the [CD Nanofiber Procedure](https://static.igem.wiki/teams/4322/wiki/cd-nanofiber-procedure.pdf) for our experiment.

![](https://static.igem.wiki/teams/4322/wiki/engineering-fig10.jpg)

Figure 10: Post Sonication - Tube A (left) original size Tube B (right) chopped nanofiber sheet into pieces before sonication

If we successfully make the CD nanofiber hydrogel, we plan to test its properties and perform VOC uptake testing as well.

## Sources

#### Wetlab

[1] *Does the amount of plasmid DNA used in bacterial transformation have an ...* ResearchGate. (n.d.). Retrieved from https://www.researchgate.net/post/Does-the-amount-of-plasmid-DNA-used-in-bacterial-transformation-have-an-effect-on-the-transformation-efficiency

[2] *T7 and DH5 alpha*. Protocol Online. (n.d.). Retrieved from http://www.protocol-online.org/biology-forums-2/posts/12993.html.

[3] *Act II: Experimental and Results.* iGEM Manchester 2019 - Cutiful. (n.d.) Retrieved from https://2019.igem.org/Team:Manchester/Experiments.

#### Product Development

 **Bioreactor**

[1] “Hardware.” 2021 iGEM Team:Cornell, 2021, [https://2021.igem.org/Team:Cornell/Hardware](https://2021.igem.org/Team%3ACornell/Hardware)

[2] Kropp, Christina & Massai, Diana & Zweigerdt, Robert. (2016). Progress and Challenges in Large-Scale Expansion of Human Pluripotent Stem Cells. *Process Biochemistry*. 59. 10.1016/j.procbio.2016.09.032.

 **3D Printer**

[1] Ioannidis, K., Danalatos, R. I., Champeris Tsaniras, S., Kaplani, K., Lokka, G., Kanellou, A., Papachristou, D. J., Bokias, G., Lygerou, Z., & Taraviras, S. (2019). A Custom Ultra-Low-Cost 3D Bioprinter Supports Cell Growth and Differentiation. *Frontiers in Bioengineering and Biotechnology*. https://doi.org/10.3389/fbioe.2020.580889

[2] Gungor-Ozkerim, P. S., , Inci, I., , Zhang, Y. S., , Khademhosseini, A., , & Dokmeci, M. R., (2018). Bioinks for 3D bioprinting: an overview. *Biomaterials science*, 6(5), 915–946. https://doi.org/10.1039/c7bm00765e

[3] Spiesz, E. M., Yu, K., Lehner, B. A. E., Schmieden, D. T., Aubin-Tam, M.-E., & Meyer, A. S. (2019). Three-dimensional patterning of engineered biofilms with a do-it-yourself bioprinter. *Journal of Visualized Experiments*, (147). https://doi.org/10.3791/59477

[4] Balasubramanian, S., Yu, K., Cardenas, D. V., Aubin-Tam, M.-E., & Meyer, A. S. (2021). Emergent biological endurance depends on extracellular matrix composition of three-dimensionally printed *escherichia coli* biofilms. *ACS Synthetic Biology*, *10*(11), 2997–3008. https://doi.org/10.1021/acssynbio.1c00290

**Handheld Bioprinter**

[1] Ying, G., Manríquez, J., Wu, D., Zhang, J., Jiang, N., Maharjan, S., Medina, D. H. H., & Zhang, Y. S. (2020, August 21). An open-source handheld extruder loaded with pore-forming bioink for in situ wound dressing. *Materials Today Bio*. Retrieved September 30, 2022, from https://www.sciencedirect.com/science/article/pii/S259000642030034X

[2] Duraj-Thatte, A. M., Manjula-Basavanna, A., Rutledge, J., Xia, J., Hassan, S., Sourlis, A., Rubio, A. G., Lesha, A., Zenkl, M., Kan, A., Weitz, D. A., Zhang, Y. S., & Joshi, N. S. (2021, November 23). Programmable microbial ink for 3D printing of living materials produced from genetically engineered protein nanofibers. *Nature News*. Retrieved September 30, 2022, from https://www.nature.com/articles/s41467-021-26791-x

[3] Hakimi, N., Cheng, R., Leng, L., Sotoudehfar, M., Ba, P. Q., Bakhtyar, N., Amini-Nik, S., Jeschke, M. G., &amp; Günther, A. (2018, April 11). Handheld skin printer: In situ formation of planar biomaterials and tissues. *Lab on a Chip*. Retrieved October 6, 2022, from https://pubs.rsc.org/en/content/articlelanding/2018/lc/c7lc01236e

[4] O'Connell C;Ren J;Pope L;Li Y;Mohandas A;Blanchard R;Duchi S;Onofrillo C; (no date) Characterizing Bioinks for extrusion bioprinting: Printability and rheology, Methods in molecular biology (Clifton, N.J.). *U.S. National Library of Medicine*. Available at: https://pubmed.ncbi.nlm.nih.gov/32207108/ (Accessed: October 6, 2022).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

[* Wet Lab](#wet-lab)
[* Product Development](#pd)

## Wet Lab

[General Web Lab Protocools](https://static.igem.wiki/teams/4322/wiki/cornell-igem-wet-lab-protocol-manual.pdf)

[Gibson Assembly Protocol](https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510) - Contrust Assembly Method

[High Efficiency Transformation Protocol](https://www.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987) - Transforming E. coli DH5ɑ strain

[Transformation Protocol for BL21(DE3) Competent Cells](https://www.neb.com/protocols/0001/01/01/transformation-protocol-for-bl21-de3-competent-cells-c2527) - Transforming E. coli BL21(DE3) strain

[Protocol for cell lysis using NEBExpress E. coli Lysis Reagent](https://www.neb.com/protocols/2021/10/04/protocol-for-cell-lysis-using-nebexpress-ecoli-lysis-reagent-neb-p8116) - Chromoprotein compatible E. coli lysis protocol

[Preparation of CsgA-α, CsgA-γ and CsgA-αγ hydrogels (Neel S. Joshi)](https://www.nature.com/articles/s41467-021-26791-x#Sec6) - Bacterial hydrogel collection procedure

## Product Development

##### Hardware

[Hydrogel Making Procedure](https://static.igem.wiki/teams/4322/wiki/hydrogel-making-procedure.pdf)

[Extrusion Method Testing](https://static.igem.wiki/teams/4322/wiki/extrusion-method-testing-procedure.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% 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 %}