iGEM 2022 — Team uoregon

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}See the [team section](https://2022.igem.wiki/uoregon/team) for details of who participated on each committee.{% endblock %}
{% block page\_content %}

## General Project Notebook

Week 1 — Week of April 4

## Entire Team

---

* iGEM application submitted
* Slack workspace set up
* Team registration and member recruitment

Week 2 — Week of April 11

## Entire Team

---

* Member recruitment

Week 3 — Week of April 18

## Entire Team

---

* First PCR/Pipette skills training workshop

Week 4 — Week of April 25

## Entire Team

---

* Team roles finalized, see [team section](%E2%80%9Chttps%3A//2022.igem.wiki/uoregon/team%E2%80%9D) for assignments

Week 5 — Week of May 2

## Entire Team

---

* Official iGEM kickoff
* Team roster finalized

Week 6 — Week of May 9

## Entire Team

---

* Team vote & discussion to determine the most promising ideas
* Calin meets with the director of the Eugene Science Center
* Lab Safety training for all members
* Final project ideas evaluated by PIs

### Brainstorming project ideas

* Lactic Acid
* Concussion Detection
* Cyanobacteria Blooms
* Hemostatic Agents
* Drug Delivery

Assigned team members to research the 3 most promising ideas:

* Concussions

+ Meaghan Smith
+ Carmen Resnick
+ Oliver Loreto
+ Arushi Singhal

* Hemostatic Studies

+ Keane Deas
+ Mindy Tran
+ Theo Seah
+ Alonso Cruz

* Drug Delivery for Insulin

+ Tulsi Patel
+ Peter Weisel
+ Rose Hoang
+ Ascensy Perez

Week 7 — Week of May 16

## Entire Team

---

* Voted on the most promising project idea
* moving forward with concussion biosensor

Week 8 — Week of May 23

## Entire Team

---

* Literature research for concussion biosensor
* Set up a schedule for weekly team meetings
Set up one-on-one meetings between each team member and an instructor to determine personal/team goals

Week 9 — Week of May 30

## Entire Team

---

* Identified team values and personal goals
* Set up student keycard access to the iGEM lab

Week 10 — Week of June 6

## Entire Team

---

* Lab safety training

Week 11 — Week of June 13

## Entire Team

---

* Team divided into 6 committees: Wiki, Human Practices, Fundraising, Organization, Modeling, Social Media, and Leadership.
* Established a system to report weekly S.M.A.R.T. goals for each committee

## Committees

---

### Wiki

* Oliver Loreto
* Arushi Singhal

### Human Practices

* Tulsi Patel
* Ava Komons
* Peter Weisel

### Fundraising

* Meaghan Smith
* Adelin Alfred
* Rose Hoang

### Organization

* Theo Seah
* Keane Deas

### Modeling

* Mindy Tran

### Social Media

* Alonso Cruz
* Mindy Tran

### Leadership

* Carmen Resnick

Week 12 — Week of June 20

## Entire Team

---

* Prepared “elevator pitches” for concussion detection mechanisms

+ Complementation system in the cell/split fluorescent proteins
+ Making single chain variable fragment (scFv) from known s100β antibody
+ Bacterial Surface display system using truncated OmpT
+ FRET to detect protein interactions in *E. coli*
+ Histidine kinases for bacterial sensing
+ Capacitive biosensors

* Collected contact info for each team member
* Discussed team values identified in one-on-one meetings

## Committees

---

### Wiki

* Completed first draft of intro page/landing text

### Human Practices

* Sent draft of outreach email to UO professionals for feedback

### Fundraising

* Completed final draft of impact grant application

### Organization

* Investigated clickup for team organization

### Social Media

* Created a posting schedule for instagram and twitter platforms
* Met with Lewis Taylor (Director of Communications at the Phil and Penny Knight Center for Accelerating Scientific Impact) to discuss possibilities for twitter account

Week 13 — Week of June 27

## Entire Team

---

* Finalized concussion detection mechanism; Split complementation systems
* Twitter account @UOregon\_iGEM goes live

## Committees

---

### Wiki

* Outline/direction for all website pages
* Backfilled project notebook from slack

### Human Practices

* Sent outreach emails to athletic team

### Fundraising

* Submitted final version of impact grant & safety and security grant

### Organization

* Set up lab space and sign in/sign out system

### Social Media

* Launched twitter platform: @UOregon\_iGEM

### Leadership

* Put together sign-in sheet
* Started lab space planning and organization
* Arranged lab photo and picnic

Week 14 — Week of July 4

## Entire Team

---

* iGEM team potluck
* Split into two groups to pursue different detection mechanisms; capacitance and protein biosensor

## Research Groups

---

### Protein Biosensor

* Focussed research on early biomarkers with the goal of having a rapid blood (finger poke), or saliva-based test

## Committees

---

### Wiki

* Finalized citations for project description/our story wiki pages
* Created draft of team section with HTML templates
* Updated project description page to reflect the two subprojects

### Human Practices

* Brainstormed ways to get a lead into the athletic department

### Fundraising

* Created a contact list of UO departments to approach
* Created a draft email to send to UO departments
* Researched hands-on fundraising opportunities
* Created a draft letter for individual/corporate contributions

### Organization

* Invited team members to clickup workspace

### Social Media

* Created twitter post about iGEM team potluck
* Collected headshot and bios from team members

Week 15 — Week of July 11

## Research Groups

---

### Protein Biosensor

* Background research on successful protein biosensor implementations

### Capacitive Sensor

* Background research on successful capacitive sensor implementations

## Committees

---

### Wiki

* Transferred wiki google doc to uoregonigem@gmail.com account
* Created documentation for pouring bacterial plates
* Edited team section, project notebook draft wiki pages

### Human Practices

* Sent follow up email to Craig from football department
* Sent emails to other professionals in UO Athletic Medicine Department

### Fundraising

* Set up GoFundMe page
* Edited donations letter draft
* Created sponsorship outreach materials
* Finalized Genscript base-pair giveaway application
* Researched alternative fundraising sources (Track Town Pizza)
* Researched genes/plasmids in iGEM distribution kit (reporters, split proteins, plasmids)
* Finalized UO department contact list
* Finalized UO department email draft
* Investigated ASUO affiliation & funding

### Organization

* Further task organization on Clickup

### Modeling

* Registered for iGEM modeling seminar

### Social Media

* Posted first member spotlight on twitter

Week 16 — Week of July 18

## Entire Team

---

* Each team member is responsible for emailing 3 fundraising contacts
* Discontinued capacitive sensor research
* Team members reassigned to investigate specific aspects of the protein biosensor, see research groups panel of [team page] for assignments

## Research Groups

---

### Protein Biosensor

* Created a model of a control golden gate assembly system with binder controls
* Investigated binder controls for golden gate assembly
* Modeled binding sites for proteins and binders of interest
* Researched complementation systems for biomarker detection readout

### Capacitive Sensor

We discontinued the capacitive sensor because it would take too long to develop, especially with our group’s lack of electrical experience. Attention will be shifted fully to the protein biosensor approach.

## Committees

---

### Wiki

* Transferred wiki google doc to igem gmail
* Documentation for pouring bacterial plates

### Human Practices

* Followed up with UO Athletic Medicine department
* Created a list of medical professionals and researchers at UO and other surrounding institutions
* Sent emails to medical professionals and other researchers in UO

### Fundraising

* Sent fundraising emails to UO departments
* Followed up with ASUO

### Social Media

* Followed other iGEM accounts on Twitter to expand audience
* Posted about fundraising to gather community support

Week 17 — Week of July 25

## Entire Team

---

* Team meeting to follow up on goals
* Keycard access to KC finalized

## Research Groups

---

### Computational

* Presented computational papers for protein sensor design
* Created a list of computational questions for Parissa

### Complementation Systems

* Looked into adding proteins to plasmids identified during literature search

### Combinatorial Golden Gate Assembly

* Created draft golden gate protocol

### Biomarker Detection

* Researched binder controls
* Quantified baseline/elevated biomarker concentrations
* Researched patented solutions
* Researched *E. coli* expression & cell free protein synthesis

## Committees

---

### Wiki

* Collected information for the team page from the entire team
* Polished project description for wiki, social media & DuckFunder

### Human Practices

* Emailed UO soccer trainer
* Reached out to other iGEM teams for collaboration

### Fundraising

* Followed up with UO departments
* Followed up with Track Town Pizza
* Created a draft sponsorship packet

### Modeling

* Set up meeting with Parisa
* Drafted questions for meeting with Parisa

### Social Media

* Worked on DuckFunder website
* Added photos to media folder in google drive
* Wrote draft fundraising twitter post

Week 18 — Week of August 1

## Entire Team

---

* Won iGEM Impact Grant

## Committees

---

### Wiki

* Sent general slack message for the team to evaluate wiki pages
* Published first draft of the team page for the wiki
* Set a goal to have every wiki page populated by the end of the month

### Human Practices

* Created a concussion survey to send to the public
* Researched athletes, organizations, and professionals to provide insight on our project

### Fundraising

* Continued to follow up with UO departments
* Promoted DuckFunder fundraising page to friends and family

### Modeling

* Initial modeling for UCHL1/Ubiquitin complex
* AlphaFold training for computational members
* Presented computational findings to the rest of the team

### Social Media

* Followed other iGEM accounts on Twitter to expand audience
* Posted about fundraising to gather community support

### Leadership

* Launched the DuckFunder fundraising page

Week 19 — Week of August 8

## Research Groups

---

### Computational

* Presented on modeled binding sites for binders of interest

### Complementation Systems

* Presented on the most promising complementation systems & readouts

### Combinatorial Golden Gate Assembly

* Presented the model for control golden gate assembly system with binder controls

### Biomarker Detection

* Presented biomarker baseline levels, cell free systems, and binder controls for golden gate assembly

## Committees

---

### Wiki

* Published final draft of the team page and archival project notebook
* Assigned wiki writing sections to the rest of the team
* Created wet lab notebook template & wiki page draft

### Human Practices

* Created form to collect first-hand experiences from individuals with concussions
* Created a list of medical professionals to contact for feedback on our project

### Fundraising

* Finalized locations for fundraising
* Posted DuckFunder QR code in public areas around campus

### Modeling

* Tested trunctated binders and purification tags

### Leadership

* Assigned hotel rooms for Jamboree attendees
* Researched accommodations in Paris
* Completed designs for DNA orders
* Miniprepped reporters and plasmids from the iGEM distribution

Week 20 — Week of August 15

## Research Groups

---

### Computational

* Modeled S100β as binder for GFAP

### Complementation Systems

* Researched sfCherry2
* Researched split beta-lactamase
* Researched split synthetic & wild-type renilla luciferase
* Researched split firefly luciferase
* Researched split BS2 esterase
* Researched split horseradish peroxidase
* Researched split chorismate mutase
* Researched split fluorescent proteins

### Combinatorial Golden Gate Assembly

* Continued designing primers and plasmids

### Biomarker Detection

* Researched biomarker expression & folding in *E. coli*

## Committees

---

### Wiki

* Published final version of the team page
* Published first draft of the project description page
* Updated the project notebook page
* Finalized the wetlab notebook layout

### Human Practices

* Reached out to other iGEM teams

### Fundraising

* Edited wiki pages
* Research more fundraising opportunities
* Contact LiU iGEM team

### Organization

* Worked on wiki page for golden gate & short description of organization committee’s role

### Modeling

* ZDOCK and HDOCK training for computational members

### Social Media

* Created social media post about DuckFunder fundraising website
* Posted member spotlights
* Posted lab photos
* Reached out to Knight Campus social media for a shoutout

### Leadership

* Completed all DNA sequences to be ordered and started ordering process with IDT
* Miniprepped all distribution necessities

Week 21 — Week of August 22

## Research Groups

---

### Computational

* Simulated binding effectiveness with 6H/FLAG tags
* Simulated binder/biomarker docking with linker & complementation system attached

### Complementation Systems

* Looked into complementation system synthesis
* Researched how to minimize cross-talk and reduce interference from blood serum

### Combinatorial Golden Gate Assembly

* Worked with Carmen to solidify expression system for golden gate

### Biomarker Detection

* Researched biomarker expression & folding in *E. coli*

## Committees

---

### Wiki

* Published project notebook archive through August 1st
* Follow up with team members about wiki writing
* Updated team page with short descriptions of committees and research groups
* Fixed writing style issues in project notebook

### Human Practices

* Contacted HEDCO to see determine their concussion diagnostic and treatment plans
* Wrote human practices wiki page

### Fundraising

* Responded to poster collaboration email
* Scheduled meeting with LiU iGEM team
* Emailed UO business department for funding
* Created a new list of contacts to reach out to for donations

### Organization

* Checked in on lab space
* Informed the team about how to request biohazardous waste pickup

### Modeling

* Presented computational findings and purification tag effectiveness to the rest of the team

### Leadership

* Finalized sequences for center cassette
* Ordered primers for all sequences

Week 22 — Week of August 29

## Research Groups

---

### Computational

* Ran docking simulations taking energy minimization into account & used multiple docking protocols for all GFAP and UCHL1 binders

### Combinatorial Golden Gate Assembly

* Finalized and ordered sequences

## Committees

---

### Wiki

* Put content on every live wiki page
* Ran docking simulations taking energy minimization into account & used multiple docking protocols for all GFAP and UCHL1 binders
* Finished cleaning up project notebook

### Human Practices

* Worked on human practices wiki page
* Looked into iGEM Human Practices ideas and develop a plan to implement new ideas
* Met with Swedish iGEM team LiU

### Fundraising

* Scheduled LiU team meeting
* Emailed UO business department
* Made presentation for LiU team meeting
* Emailed fall term professors about missing class for iGEM Jamboree
* Compiled list of deadlines

### Modeling

* Ran docking simulations for the most promising binders

### Leadership

* Met with Bob Guldberg and Roger Thompson (VP for student services), finalized and ordered sequences, and planned out the Golden Gate process

Week 23 — Week of September 5

## Entire Team

---

The team mainly did wetlab work this week, see the wetlab notebook for more details.

## Committees

---

### Fundraising

* Worked on graphical abstract

Week 24 — Week of September 12

## Entire Team

---

The team mainly did wetlab work this week, see the wetlab notebook for more details.

## Committees

---

### Human Practices

* Met with Dr. Lynch, a local emergency medicine physician

### Leadership

* Sumbitted final project safety forms

Week 25 — Week of September 19

## Entire Team

---

* We cannot express GFAP in *E. coli*, focussing all of our efforts on detecting UCHL1 using split ubiquitin as the binder
* Ordered ubiqutin protein and ubiquitin coding sequence

## Committees

---

### Wiki

* Updated project notebook pages with wetlab findings

Week 26 — Week of September 26

## Entire Team

---

1. Start of fall term
2. The wiki team focussed on getting the wiki up to date, and the rest of the team worked entirely on wetlab.

Week 27 — Week of October 3

## Entire Team

---

1. The wiki committee focussed on getting the wiki up to date, and the rest of the team worked entirely on wetlab.

Week 28 — Week of October 10

## Entire Team

---

1. Week of the iGEM wiki freeze
2. The wiki committee focussed on getting the wiki up to date, and the rest of the team worked entirely on wetlab.

## Wetlab Notebook

Week 19 — Week of August 8

#### Lead: Carmen Resnick

#### Date: August 11

#### Participants:

* Theo Seah
* Keane Deas
* Meaghan Smith
* Ava Komons

#### What did you do?

Resuspended DNA in selected wells from iGEM distribution and transformed to NEB 10-β cells.

#### Why did you do it?

To grow up and miniprep increased concentrations of the DNA in the distribution.

#### How did you do it?

Following the [iGEM kit plate instructions](https://technology.igem.org/distribution/handbook)

Week 20 — Week of August 15

#### Lead: Theo Seah

#### Date: August 15th

#### Participants:

* Arushi Singhal
* Meaghan Smith
* Carmen Resnick
* Oliver Loreto

#### What did you do?

Started overnight cultures, picking out satellite colonies from the plates that were transformed from iGem’s distribution kit.

#### Why did you do it?

#### How did you do it?

Following our [overnight culture procedure](https://2022.igem.wiki/uoregon/experiments)

---

#### Lead: Carmen Resnick

#### Date: August 16th

#### Participants:

* Meaghan Smith
* Keane Deas

#### What did you do?

Mini prepped and made glycerol stocks of the iGEM distribution kit cultures

#### Why did you do it?

#### How did you do it?

1. Added 0.5 mL each of glycerol stock and bacteria culture (from Aug 15th) to a cryotube. Placed the tubes in the -80℃ freezer. 3 of the cultures produced nothing.
2. Transferred the rest of the bacteria colonies to eppendorf tubes and some were [mini prepped](https://2022.igem.wiki/uoregon/experiments).
3. Mini prepped samples were examined using the Implen and then placed in the -80℃ freezer.

---

#### Lead: Keane Deas

#### Date: August 16th

#### Participants:

* Keane Deas

#### What did you do?

Performed a transformation, transforming blac and fluc

#### Why did you do it?

#### How did you do it?

Followed procedure for [well plate extraction](https://2022.igem.wiki/uoregon/experiments), extracting both fluc and blac. Transformed both fluc and blac, and plated in LB+carb broth, then left in the incubator overnight

---

#### Lead: Meaghan Smith

#### Date: August 16th

#### Participants:

* Arushi Singhal

#### What did you do?

Mini prepped the rest of the prepared bacteria cultures samples from 8/15

#### Why did you do it?

#### How did you do it?

1. (earlier in the day) Cultures had already been transferred to eppendorf tubes, centrifuged, and had growth medium removed and stored in the -20℃ freezer.
2. Samples were collected from the freezer and [miniprepped](https://2022.igem.wiki/uoregon/experiments).
3. Miniprepped samples were examined using the Implen and then placed in the -80℃ freezer. \*one sample did not get elution buffer added the first time → needed to be added and recentrifuged. \*\* another sample did not produced very little DNA and had additional elution buffer added and re-centrifuged. \*\*\* two unclean samples marked w/ red stars

---

#### Lead: Andrew Holston

#### Date: August 17th

#### Participants:

* Keane Deas
* Oliver Loreto
* Meaghan Smith

#### What did you do?

Ran gels for the miniprepped samples from 8/16

#### Why did you do it?

To see what was produced from the minipreps

#### How did you do it?

1. Made gels
2. Mixed up 1x TAE solution
3. Prepped mini prep samples from 8/16 and 1kb DNA ladders for gels
4. Ran gels (original images for gels in New Folder (4))

#### Images:

![Photo of Gels from August 17th Experiment](https://static.igem.wiki/teams/4215/wiki/8-17-gels.jpg)

| Position | Contents |
| --- | --- |
| 1 | 1 Kb ladder |
| 2 | B-Gal C1 |
| 3 | B-Gal C2 |
| 4 | R-Luc C1 |
| 5 | R-Luc C2 |
| 6 | HRP C1 |
| 7 | Sup ladder (2Kb) |
| 8 | Blank |
| 9 | 1Kb ladder |
| 10 | GFP1 C1 |
| 11 | GFP2 C2 |
| 12 | GFP2 C1 |
| 13 | GFP1 C2 |
| 14 | AfraGFP C1 |
| 15 | AfraGFP C2 |
| 16 | Sup ladder (2Kb) |

---

#### Lead: Keane Deas

#### Date: August 18th

#### Participants:

* Keane Deas

#### What did you do?

Overnight culture for B-lac and firefly luciferase

#### Why did you do it?

Because it failed last time

#### How did you do it?

1. Performed [overnight culture](https://2022.igem.wiki/uoregon/experiments) protocol, removed two colonies for both blac and fluc, both from the same plate, and added to LB+carb test tubes for overnight culture.
2. Waited ≈16 hours and moved to fridge

---

#### Lead: Keane Deas

#### Date: August 19th

#### Participants:

* Keane Deas
* Peter Weisel

#### What did you do?

Cloned DH5 alpha cells

#### Why did you do it?

#### How did you do it?

1. We first created proper concentrations(10% glycerol) stocks, then combined 50 mL glycerol with DH5a cells, then centrifuged for 15 mins.
2. Poured off supernatant, added 10 mL glycerol, and repeated, with diminishing quantities of glycerol until 1mL, which I then suspended and allocated into 20 epis.

---

#### Lead: Meaghan Smith

#### Date: August 19th

#### Participants:

* Peter Weisel

#### What did you do?

Miniprep samples

#### Why did you do it?

#### How did you do it?

Followed [miniprep protocol](https://2022.igem.wiki/uoregon/experiments) w/ 3 samples (B-lac & firefly luciferase) prepared earlier in the day (prepared by carmen)

Week 21 — Week of August 22

#### Lead: Keane Deas

#### Date: August 19th

#### Participants:

* Tulsi Patel
* Arushi Singhal

#### What did you do?

Transformed DH5a cells recently made electrocompetent with puc-19

#### Why did you do it?

To test whether they were actually made electrocompetent

#### How did you do it?

We followed [transformation](https://2022.igem.wiki/uoregon/experiments) protocols with a non-diluted, 10x, and 20x dilution of DH5a cells transfected with puc-19

Week 22 — Week of August 29

* N/A

Week 23 — Week of September 5

#### Lead: Arushi Singhal

#### Date: September 8th

#### Participants:

* Arushi Singhal

#### What did you do?

Amplify backbone (pSEVA)

#### How did you do it?

Followed protocol provided for amplifying pSEVA backbone.

* For incubation after dpn1 addition, used programed incubation on pcr machine labeled dpn1 digest made by andrew holston for 50 minutes
* for 1st step of PCR clean up kit, did a dilution of 5:1 (used 250 µL of soln)

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| 1 | 27.550 ng/µL | 1.807 | 2.267 |
| 2 | 8.0500 ng/µL | 1.789 | 2.300 |

---

#### Lead: Arushi Singhal

#### Date: September 9th & 12th

#### Participants:

* Arushi Singhal

#### What did you do?

Amplify Chloramphenicol gene (from SMT205)

#### How did you do it?

Followed protocol provided for amplifying Chloramphenicol gene.

* For 1st step of PCR clean up kit, did a dilution of 5:1 (used 500 uL of soln and combined samples into one tube)

#### Images:

![Photo from September 9th Experiment](https://static.igem.wiki/teams/4215/wiki/sep-9-12.jpg)

Week 24 — Week of September 12

#### Lead: Sam Hinton

#### Date: September 9th & 12th

#### Participants:

* Meaghan Smith
* Arushi Singhal

#### What did you do?

Gel extracted fragments CAM and pSEVA backbone

#### Why did you do it?

Wanted to purify CAM and pSEVA fragments for golden gate reactions

#### How did you do it?

1. Made 30 mL of 1% agarose gel (0.3 mg agarose, 30 mL TAE, 3 uL sybersafe)
2. Added 5 uL of dye to pcr samples from 9/8 and 9/9-12 (combined the two separate samples of pSEVA into one)
3. Prepped ladders (1 kb and 100 bp) (4 uL H20, 1 uL ladder, 5 uL dye)
4. Ran gels
5. Pre-weighed eppendorf tube (labeled on tube)
6. Used razor blade to cut out band for CAM fragment, stored in tube, & weighed again (labeled on tube)\*
7. Stored in -20℃ overnight

\*pSEVA fragment was not collected as the pcr sample already started with a very low concentration - not enough to extract successfully (need to rerun pcr for pSEVA)

---

#### Lead: Meaghan Smith & Arushi Singhal

#### Date: September 13th & 14th

#### Participants:

* Meaghan Smith
* Arushi Singhal

#### What did you do?

Amplify pSEVA backbone

#### Why did you do it?

The first pcr did not produce enough - needed to adjust the timing of step 4 for thermocycler

#### How did you do it?

##### September 13th

* Followed pcr protocol
* Ran it with 4 pcr tubes rather than 2
* Let it run at step 7 in thermocycler overnight and continued protocol in the morning

##### September 14th:

* Continued with pcr protocol (for incubation after dpn1 addition, used programmed incubation on pcr machine labeled dpn1 digest made by andrew holston for 50 minutes)

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| 1 | 106.5 ng/µL | 1.678 | 2.32 |

---

#### Lead: Meaghan Smith & Arushi Singhal

#### Date: September 14th

#### Participants:

* Meaghan Smith
* Arushi Singhal

#### What did you do?

Gel electrophoresis on fragment CAM and pSEVA backbone

#### Why did you do it?

Rerunning the gel extract procedure with higher concentration of PSEVA backbone

#### How did you do it?

1. Made 30 mL of 1% agarose gel (0.3 mg agarose, 30 mL TAE, 3 µL sybersafe)
2. Added 5 µL of dye to pcr
3. Samples of PSEVA from 9/14
4. Prepped ladders (1 kb) (4 µL H20, 1 uL ladder, 5 µL dye)
5. Ran gels

\*PSEVA concentration not enough on gel

---

#### Lead: Arushi Singhal

#### Date: September 15th

#### Participants:

* Arushi Singhal

#### What did you do?

Amplified the PSEVA backbone

#### Why did you do it?

To troubleshoot the issue with the dna not showing up on the gel

#### How did you do it?

Ran 3 PCR tubes for troubleshooting purposes

1. PSEVA amplification procedure with 1/4 level of primer
2. PSEVA amplification procedure with Touchdown setting on thermocycler
3. \*PSEVA amplification procedure with no template

\*PSEVA amplification without template had a concentration of 56.8 ng/µl, primer is likely dimerizing

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| 1 | 16.1 ng/µL | 1.695 | 1.952 |
| 2 | 53 ng/µL | 1.776 | 2.074 |
| 3 | 56.8 ng/µL | 1.775 | 1.147 |

---

#### Lead: Arushi Singhal

#### Date: September 15th

#### Participants:

* Arushi Singhal

#### What did you do?

Amplified PEV and KAN

#### Why did you do it?

To amplify PEV and KAN fragments

#### How did you do it?

* Followed PCR protocol
* In 2x PCR tubes for each PEV and KAN

+ 1 ul template
+ 2x2.5 µl primers
+ 25 µl mastermix
+ 19 µl H2O

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| KAN | 14.55 ng/µL | 1.722 | 1.842 |
| PEV | 21.05 ng/µL | 1.855 | 1.733 |

---

#### Lead: Arushi Singhal

#### Date: September 16th

#### Participants:

* Arushi Singhal

#### What did you do?

Amplified pSEVA1411 backbone

#### How did you do it?

Followed the Amplify backbone (pSEVA) protocol

Week 25 — Week of September 19

#### Lead: Arushi Singhal

#### Date: September 19th

#### Participants:

* Arushi Singhal

#### What did you do?

Amplified pSEVA1411 backbone

#### Why did you do it?

Reran the protocol with primer dilution since the gel showed no results last time

#### How did you do it?

Followed the Amplify backbone (pSEVA)
Protocol

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| pSEVA | 36.5 ng/µL | 1.674 | 1.448 |

---

#### Lead: Arushi Singhal

#### Date: September 20th

#### Participants:

* Arushi Singhal

#### What did you do?

Amplified PEV and Kan fragments

#### Why did you do it?

To work on the biomarker control

#### How did you do it?

Followed the amplification protocol for PEV and Kan

---

#### Lead: Arushi Singhal

#### Date: September 20th

#### Participants:

* Arushi Singhal

#### What did you do?

Ran an E-gel on pSEVA1411

#### Why did you do it?

To test if the amplification procedure was successful

#### How did you do it?

1. Added 9 μl H2O into two wells of 1% E-gel
2. Ran the 1-2% setting on E-gel machine for 5 minutes
3. Added 1 μl of 1 kb ladder into one well and 1 μl of sample into the other well
4. Ran the 1-2% setting on E-gel machine for 10 minutes

\*Results not conclusive

---

#### Lead: Oliver Loreto

#### Date: September 20th

#### Participants:

* Keane Deas
* Arushi Singhal

#### What did you do?

Poured bacterial plates & prepared liquid media using LB + Kan

#### Why did you do it?

To have plates for transformation and liquid media

#### How did you do it?

Followed the protocol for pouring bacterials plates, with a final concentration of 50 μg/ml of Kanamycin.

---

#### Lead: Keane Deas

#### Date: September 21st

#### Participants:

* Keane Deas

#### What did you do?

Transformed pSEVA 1141 and pSEVA 244

#### How did you do it?

Followed transformation protocol, up to starting an overnight culture. Used heat shock instead of electroporation

---

#### Lead: Arushi Singhal

#### Date: September 21st

#### Participants:

* Arushi Singhal

#### What did you do?

Amplified pSEVA1411 backbone

#### Why did you do it?

We changed the plasmid template to pSEVA1411 and modified the annealing temperature to get a better result

#### How did you do it?

Followed the Amplify pSEVA1411 backbone protocol

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| pSEVA | 87.3 ng/µL | 1.859 | 2.303 |

#### Images:

![Photo from September 21st Experiment](https://static.igem.wiki/teams/4215/wiki/sep-22.jpg)

---

#### Lead: Arushi Singhal

#### Date: September 21st

#### Participants:

* Arushi Singhal

#### What did you do?

Ran an E-gel on pSEVA1411 amplified backbone

#### Why did you do it?

To test whether the amplification protocol was successful

#### How did you do it?

1. Added 9 μl H2O into two wells of 1% E-gel
2. Ran the 1-2% setting on E-gel machine for 5 minutes
3. Added 1 μl of 1 kb ladder into one well and 1 μl of sample into the other well
4. Ran the 1-2% setting on E-gel machine for 10 minutes

Results look good

#### Images:

![Photo of E-gel from September 21st Experiment](https://static.igem.wiki/teams/4215/wiki/sep-22-egel.jpg)

---

#### Lead: Arushi Singhal

#### Date: September 22nd

#### Participants:

* Arushi Singhal

#### What did you do?

Ran a gel procedure for pSEVA 1411 amplified backbone

#### Why did you do it?

To test the viability of the DNA

#### How did you do it?

1. Used a 1% agarose gel at 120 V and followed the gel electrophoresis protocol

---

#### Lead: Arushi Singhal

#### Date: September 22nd

#### Participants:

* Arushi Singhal

#### What did you do?

Amplified pSEVA1411 backbone and ran an e-gel

#### Why did you do it?

The previous sample had the incorrect DNA ladder length on the gel, procedure had to be redone

#### How did you do it?

1. Followed the amplify pSEVA1411 backbone procedure

---

#### Lead: Arushi Singhal

#### Date: September 23rd

#### Participants:

* Arushi Singhal

#### What did you do?

Ran a gel procedure for pSEVA 1411 amplified backbone

#### Why did you do it?

The previous sample had the incorrect DNA ladder length on the gel, procedure had to be redone with different gel conditions

#### How did you do it?

1. Used a 0.8% agarose gel at 100 V and followed the gel electrophoresis protocol

---

#### Lead: Arushi Singhal

#### Date: September 23rd

#### Participants:

* Arushi Singhal

#### What did you do?

Ran a gel electrophoresis procedure on the pSEVA 1411 template

#### Why did you do it?

The pSEVA backbone amplification sample had the wrong DNA ladder length

#### How did you do it?

1. Prepared samples of the 1 kb DNA ladder, supercoiled ladder, and pSEVA 1411 template with 0.8% agarose gel and 100 V and followed gel electrophoresis procedure.

---

#### Lead: Carmen Resnick

#### Date: September 25th

#### Participants:

* Carmen Resnick

#### What did you do?

Ran a gel electrophoresis procedure on 3 other pSEVA 1411 templates

#### Why did you do it?

The previous pSEVA 1411 template had no DNA in it which is why it didn’t work. Testing new templates.

#### How did you do it?

1. Followed gel electrophoresis procedure

Week 26 — Week of September 26

#### Lead: Carmen Resnick

#### Date: September 26th

#### Participants:

* Carmen Resnick
* Theo Seah
* Meaghan Smith

#### What did you do?

Amplified pSEVA1411 backbone

#### Why did you do it?

Used a new template of pSEVA1411

#### How did you do it?

Followed the amplify pSEVA backbone procedure with an annealing temperature of 61°C and 80 seconds.

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| pSEVA1411 Backbone | 233.85 ng/µL | 1.870 | 1.279 |

---

#### Lead: Theo Seah

#### Date: September 26th

#### Participants:

* Theo Seah

#### What did you do?

Gel extracted CAM gel sample from 9/13/22

#### Why did you do it?

Needed to finish the purification of CAM sample that had been PCRed

#### How did you do it?

1. Followed gel extraction kit protocol
2. Sample mass: 74 mg
3. Dissolving solution: 280 µL
4. Elution buffer: 15 µL
5. Did wash step 3 times instead of 2 due to white tip potentially touching wash buffer that was to be discarded
6. Did a second round of elution buffer addition and centrifuging as no sample was produced the first time.

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| CAM Backbone | 29.000 ng/µL | 1.847 | 2.427 |

---

#### Lead: Meaghan Smith

#### Date: September 27th

#### Participants

* Meaghan Smith

#### What did you do?

Performed a PCR cleanup on pSEVA1411 and PEV

#### Why did you do it?

1. Cleaning the products from amplification of pSEVA1411 and PEV
2. Need pSEVA1411 to run golden gate reactions

#### How did you do it?

1. Followed the PCR cleanup protocol

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| pSEVA1411 Backbone | 66.200 ng/µL | 1.799 | 2.307 |
| PEV Backbone | 118.95 ng/µL | 1.830 | 2.193 |

#### Images:

![Nanophotometer readout of post-cleanup PEV](https://static.igem.wiki/teams/4215/wiki/pev-927.png)

---

#### Lead: Arushi Singhal

#### Date: September 28th

#### Participants

* Arushi Singhal

#### What did you do?

Amplification of Kanamycin gene

#### Why did you do it?

1. To amplify the insert for the biomarker control

#### How did you do it?

1. Followed the amplification procedure for the Kanamycin gene

| Sample | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio |
| --- | --- | --- | --- |
| KAN gene | 99.65 ng/µL | 1.852 | 2.851 |

#### Images:

![Nanophotometer readout of KAN gene](https://static.igem.wiki/teams/4215/wiki-kan928.jpg)

---

#### Lead: Carmen Resnick & Keane Deas

#### Date: September 28th

#### Participants

* Carmen Resnick
* Keane Deas

#### What did you do?

Performed Golden gate with pSEVA 1411 and CAM

#### How did you do it?

1. Followed Golden Gate assembly protocol using a thermocycler
2. Concentration of 23.15 ng/µL

---

#### Lead: Arushi Singhal

#### Date: September 29th

#### Participants

* Arushi Singhal

#### What did you do?

In-Fusion assembly of PEV and KAN fragments

#### Why did you do it?

1. To insert KAN into PEV backbone

#### How did you do it?

1. Followed In-Fusion cloning procedure w/Spin-Column purification procedure

| Component | Positive Control | Negative Control |
| --- | --- | --- |
| PEV Gene | 0.85 µL | 0.85 µL |
| KAN gene | 1.00 µL | - |
| Premix | 2.00 µL | 2.00 µL |
| Water | 6.15 µL | 7.15 µL |

---

#### Lead: Keane Deas

#### Date: October 1st

#### Participants

* Keane Deas

#### What did you do?

Amplified the backbone out of pSEVA3411

#### How did you do it?

1. Followed Golden Gate assembly protocol using a thermocycler
2. Final concentration of 23.15 ng/µL

Week 27 — Week of October 3

#### Lead: Keane Deas

#### Date: October 3rd

#### Participants

* Keane Deas
* Meghan Smith

#### What did you do?

Performed golden gate reactions with the following combinations:

1. A linkers
2. C linkers
3. pSEVA3411

#### How did you do it?

1. Followed Golden Gate assembly protocol using a thermocycler

---

#### Lead: Keane Deas

#### Date: October 3rd

#### Participants

* Keane Deas
* Meghan Smith

#### What did you do?

Performed golden gate reactions with the following combinations:

1. A linkers + UBQ - N
2. C linkers + UBQ - C
3. PSEVA 3411+ D-promoter

#### How did you do it?

1. Followed Golden Gate assembly protocol using a thermocycler

---

#### Lead: Keane Deas

#### Date: October 4th

#### Participants

* Keane Deas

#### What did you do?

Performed golden gate reactions with the following combinations:

1. Complementation fragments
2. Ribo RBS

#### How did you do it?

1. Followed Golden Gate assembly protocol using a thermocycler

---

#### Lead: Meaghan Smith

#### Date: October 6th

#### Participants

* Meaghan Smith

#### What did you do?

Completed pcr cleanup on golden gate samples:

1. A linkers + UBQ - N
2. C linkers + UBQ - C
3. Complementation systems + RiboJ complex

#### How did you do it?

1. Followed pcr cleanup protocol
2. Used 5:1 ratio for binding buffer & 15 μL elution buffer

Combination | Nanodrop Concentration | 260/280 Ratio | 260/230 Ratio || [A linkers + UBQ - C](https://static.igem.wiki/teams/4215/wiki/10-6-c-linkers-ubq-n.png) | 125.10 ng/µL | 1.841 | 2.161 |
| [C linkers + UBQ - N](https://static.igem.wiki/teams/4215/wiki/10-6-tev.png) | 102.05 ng/µL | 1.850 | 2.288 |
| [HRP](https://static.igem.wiki/teams/4215/wiki/10-6-rluc.png) | 130.2 ng/µL | 1.849 | 2.222 |
| [TEV](https://static.igem.wiki/teams/4215/wiki/10-6-blac.png) | 104.00 ng/µL | 1.851 | 2.291 |
| [Blac](https://static.igem.wiki/teams/4215/wiki/10-6-alinkers-ubq-c.png) | 141.45 ng/µL | 1.843 | 2.224 |
| [Fluc](https://static.igem.wiki/teams/4215/wiki/10-6-hrp.png) | 159.80 ng/µL | 1.839 | 2.159 |
| [Rluc](https://static.igem.wiki/teams/4215/wiki/10-6-fluc.png) | 128.9 ng/µL | 1.871 | 2.465 |

---

#### Lead: Keane Deas

#### Date: October 7th

#### Participants

* Keane Deas

#### What did you do?

Performed PCR to amplify Cassette A, B, and C

#### How did you do it?

1. Followed protocol for PCR using IGEM\_\_GG (fwd and rev)

---

#### Lead: Keane Deas

#### Date: October 7th

#### Participants

* Keane Deas
* Meaghan Smith

#### What did you do?

Ran Gel electrophoresis on Complementation system cassette and then performed gel extraction to extract complete cassette.

#### How did you do it?

1. Followed protocol for gel electrophoresis and gel extraction.

#### Images:

![Gel from October 7 experiment compared to ladder](https://static.igem.wiki/teams/4215/wiki/10-7-ladder.png)

---

#### Lead: Keane Deas

#### Date: October 8th

#### Participants

* Keane Deas

#### What did you do?

Amplified TEV gel extract

#### How did you do it?

1. Followed protocol for PCR using IGEM\_\_GG (fwd and rev) on the extracted DNA from the gel ran on October 7th. Only ran PCR on TEV.

---

#### Lead: Keane Deas

#### Date: October 9th

#### Participants

* Keane Deas

#### What did you do?

Cleaned DNA from PCR of TEV

#### How did you do it?

1. Followed protocol for DNA cleanup using NEB PCR clean up kit.

Combination | Sample | 260/280 Ratio | 260/230 Ratio || TEV | 99.2 ng/µL | 1.861 | 2.645 |

Week 28 — Week of October 10

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

#### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

---

Please see the [2022 Medals Page](https://competition.igem.org/judging/medals) for more information.

{% endblock %}

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

# Experiments

Assembling pSEVA3411 (Cassette D)

#### Golden Gate assembly with CAM insert and pSEVA backbone

---

##### Purpose:

To create our pSEVA3411 plasmid, which will be the backbone for the final plasmid coding our system

##### Procedure:

1. We performed golden gate assembly using BsaI-HFv2 and T4 ligase to combine our CAM insert and our pSEVA backbone.
2. Remember that these steps need to be detailed enough that someone can reproduce our work

##### Results:

* Final concentration: 23.15 ng/µL
* pSEVA3411 plasmid backbone is available on the iGEM parts registry as [BBa\_K4215000](http://parts.igem.org/Part%3ABBa_K4215000)

Golden Gate Assembly of Cassettes A&C

#### Golden Gate Assembly with Linkers and Binders

---

##### Purpose:

To create our “A” and “C” cassettes, which functioned as the binder+linker portion of our complex.

##### Procedure:

1. We ran a golden gate reaction with all binders and all linkers using BBSI. We then ran PCR to amplify final product with primers iGEM\_GG\_FWD/REV

##### Results:

* Very low concentrations were achieved, so a repeat of the Golden Gate assembly was warranted.

##### Pictures:

![E-Gel of Several Binder+Linker Combinations](https://static.igem.wiki/teams/4215/wiki/a-c-egel.jpg)

| E-Gel Lane | Sample identity |
| --- | --- |
| M | 100bp ladder |
| 1 | HRP |
| 2 | Blac |
| 3 | Fluc |
| 4 | Rluc |
| 5 | TEV |
| 6 | pSEVA3411 |
| 7 | Binder N |
| 8 | Binder C |
| 9 | Empty |
| 10 | 100bp ladder |

Golden Gate Assembly of Cassette B

#### Golden Gate Assembly with Complementation systems and Ribo J

---

##### Purpose:

To create our “B” cassette, which functioned as the complementing portion of our complex.

##### Procedure:

1. We ran a golden gate reaction with comp system I, Ribo\_complex , comp system II using BBSI. Then we ran PCR to amplify primer sequences in both ends.

##### Results:

* After running the products on a gel, the lengths did not correspond to the expected length of a fully assembled complex. At best, we only got partial assembly, of just the RiboJ and one half of the complementation system.

##### Pictures:

![HRP, 100kb ladder, Rluciferase, Beta lactamase, TEV protease(right to left)](https://static.igem.wiki/teams/4215/wiki/b-ladder.jpg)

HRP, 100kb ladder, Rluciferase, Beta lactamase, TEV protease(left to right). Highest bands all represent lengths that correspond to one fragment of the complementation system. This gel helped us to recognize that an error was made in the initial design of the riboJ complex in which the 5’ BbsI cut site left an incompatible overhang, which prevented the assembly from joining with the N-terminal split proteins.

Golden Gate Assembly with pSEVA3411 and D-promoter

#### Golden Gate Assembly with 3411 and D-promoter

---

##### Purpose:

To assemble the backbone of our final cassette, in which the D-promoter is crucial.

##### Procedure:

1. We performed golden gate assembly using BbsI-HFv2 and T4 ligase to combine our 3411 plasmid with our D-Promoter

##### Pictures:

![D-Promoter E-Gel](https://static.igem.wiki/teams/4215/wiki/d-promoter-egel.jpg)

| E-Gel Lane | Sample identity |
| --- | --- |
| M | 100bp ladder |
| 1 | HRP |
| 2 | Blac |
| 3 | Fluc |
| 4 | Rluc |
| 5 | TEV |
| 6 | pSEVA3411 |
| 7 | Binder N |
| 8 | Binder C |
| 9 | Empty |
| 10 | 100bp ladder |

# Wet Lab Protocols

iGEM Kit Plate Instructions

Adapted from [iGEM Distribution Handbook](https://technology.igem.org/distribution/handbook)

#### Materials

1. iGEM Distribution kit

---

#### Procedure

1. With a pipette tip, punch a hole through the foil cover into the well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross-contamination between the wells.
2. Pipette 10 µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1 µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic, and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic); grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock. We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Preparing Growth Media (Plates & Liquid)

[PDF Version](https://static.igem.wiki/teams/4215/wiki/bacterial-plates.pdf)

#### Materials

1. 1 or 2 1 L bottles with caps
2. 1 or 2 magnetic stir bars
3. Autoclave tape
4. 25 g LB broth powder
5. 15 g Agar powder (omit if making liquid media)
6. Appropriate concentrations of antibiotic(s)
7. Autoclave safe tray (if using 1 bottle)

---

#### Procedure

1. Weigh 25 g of LB Broth in weigh boat

* Folded weigh boat can be used to funnel broth powder into bottle

2. Slowly pour broth powder into bottle
3. Fill bottle to 1000 mL mark with DI water & shake to combine
4. If using two bottles, pour half of the broth solution into each and add half of the agar and
   antibiotic to each in steps 5 and 10

* If only one bottle is used, the mixture will boil over in the autoclave

5. Add stir bar & 15 g agar powder
6. Stir until no dry clumps are present
7. *Loosely* screw on cap & secure lid with autoclave tape

* Autoclave tape will show black stripes after heating

8. Autoclave on cycle 3 (liquid) until complete (≈1 hour)
9. While the solution is being autoclaved, prepare the petri dishes

* Arrange dishes on lab bench with the tops (larger half) covering about 1⁄3 of the plate
  with the rest resting on the bench
* Be careful when opening the petri dish package, you will put your completed plates in
  there

10. When mixture reaches 55–60°C, pipette 100 μL (green box) of antibiotic into bottle

* Liquid media is complete at this point, and can be stored at ambient temperature (covered) or in walk-in fridge

11. Return antibiotic to −80°C freezer
12. Stir to combine on magnetic hotplate
13. Pour solution into petri dishes, each plate should be about 1⁄3 full of media

* A good rule of thumb is to pour enough solution into the plate so that the bottom is 3⁄4
  covered, then swirl the plate
* Rotating the bottle while pouring minimizes dripping
* Clean floor spills as soon as possible, any solution spilled on the lab bench will be easier
  to clean after it has hardened

14. Once the plates have cooled (they will change color to a light yellow), put the lids on and stack the completed plates, *upside-down* in their original packaging
15. Label the package with the name(s) of whoever poured them, iGEM, the date, as well as the
    concentration and identity of antibiotic (“100 μg mL−1 carb”)

Transformation

#### Materials

1. Suspended DNA
2. Cells to be transformed
3. Cuvette
4. Transformation media
5. Glass beads

---

#### Procedure

1. Combine 1 µL of your resuspended DNA with 25 µL of your cells and place on ice
2. LABEL TUBE
3. Mix gently and transfer to cuvette (between metal plates), place in cuvette holder and hit “pulse”
4. Immediately add transformation media (SOB) to the cuvette and transfer recovered cells to a test tube
5. Leave to shake in the shaking incubator for 45 minutes
6. Remove test tubes from incubator and rest on a tube rack
7. Label plates to correspond to the tubes
8. Transfer 100 µL of your transformation to its respective plate, pour 10–15 glass beads onto the plate, close the lid, and shake the plate to cover all the afar with the cells (not the lid, just side to side shaking)
9. Pour beads into a used beads bin
10. Replace lid on plate and place in the incubator upside-down (agar-side up)
11. Leave plates in the incubator for 12–16 hours
12. Bleach tubes after plating cells

Overnight Cultures

#### Materials

1. Transformed E. coli colonies
2. Cellular growth medium

---

#### Procedure

1. Circle colony and label it with a number/letter
2. Transfer 5 ml of media into a test tube
3. Use a pipette tip to very gently tap on selected colony
4. Eject pipette tip into test tube
5. Re-parafilm plate
6. Place tubes in 37°C shaking incubator for 16 hours

MiniPrep

Adapted from [NEB Protocol](https://www.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010)

#### Materials

1. 4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2
2. Culture to be miniprepped

#### Procedure

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
5. Clarify the lysate by spinning for 2–5 minutes at 16,000 RCF.
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
10. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Generic PCR Protocol

#### Materials

1. 2.5 μL Primer 1 (10 μM concentration)
2. 2.5 μL Primer 2 (10 μM concentration)
3. 25 μL Q5 2X Mastermix
4. 0.5 μL pSEVA1411 plasmid template (1 ng/μL concentration)
5. 19.5 μL nuclease free Water
6. 1 μL Dpn1

---

#### Procedure

Do the following in 2 PCR tubes, 50 μL each, 100 μL total

1. Make sure to dilute primers appropriately
2. Mix gently by pipetting up and down, then spin to ensure a homogeneous mixture
3. Place in thermocycler and run PCR cycling

1. 98°C for 1 min
2. 98°C for 10 sec
3. \_\_°C for 20 sec (determine using [NEB Tm calculator](https://tmcalculator.neb.com/#!/main))
4. 72°C for \_\_ sec (depends on length, 10-15sec/kb for simple template like plasmid, for very long amplicons >6kbp use 40– 50 seconds/kb)
5. Go to step 2, 30X
6. 72°C for 5 min
7. 12°C forever

4. Add 0.5 µL DpnI to each reaction
5. Incubate at 37°C for 1 hour
6. Column clean with Monarch PCR clean up kit, follow protocol
7. Elute 15 μL
8. Record Nanodrop concentration, 260/280 ratio, 260/230 ratio from nanophotometer

PCR Cleanup (Column Clean)

Adapted from kit insert of New England Biolabs #T1030, all centrifuging should be carried out at 16,000 RCF

#### Materials

1. 50 μL DNA sample from PCR
2. 100 **or** 250 μL DNA Cleanup Binding Buffer with 0.36 volumes isopropanol added
3. 200 μL DNA Wash Buffer with 4 volumes of 95%+ ethanol added

---

#### Procedure

1. Dilute sample with correct volume of DNA Cleanup Binding Buffer

* The volume of DNA Cleanup Binding Buffer needed depends on the size of your target DNA. Use 100 μL for samples smaller than 2kb (Chloramphenicol & Kanamycin resistance), and 250 μL for samples larger than 2kb (pSEVA & PEV backbones)

2. Mix by flicking the tube or pipetting **do not vortex**
3. Insert column into collection tube and load sample onto column, spin for 1 minute, then discard flow-through
4. Re-insert column into collection tube, add 200 μL DNA Wash Buffer, and spin for 1 minute
5. Repeat previous step, discarding flow through if necessary
6. Transfer column to a clean 1.5 ml microcentrifuge tube, if the tip of the column contacts the flow-through, re-spin for 1 minute
7. Add DNA Elution Buffer to the center of the matrix, wait 1 minute and spin for 1 minute
8. Store at -20°C for later use

Gel Electrophoresis

#### Materials

1. PCR samples
2. 240 mg agarose
3. 30 mL TAEr
4. 3 μL SYBR Safe
5. 5 μL dye for each ladder and sample

---

#### Procedure

1. Combine 300 mg agarose and 30 mL TAE
2. Microwave solution in 20 seconds increments until agarose is fully dissolved, making sure to avoid boiling (a lower intensity setting can help with this)
3. Add 3 μL SYBR Safe into solution
4. Insert comb into tray
5. Cast gel into tray, gel should be less than 1 cm thick
6. Cover gel with tinfoil, wait 10–15 minutes to harden
7. Add 5 μL of dye to each 5 μL DNA sample
8. Prep ladders (1 kb and 100 bp) (4 μL H20, 1 μL ladder, 5 μL dye)
9. Transfer the full 10 μL volume of each dyed dna sample into a lane (if possible, avoid using the edge lanes and adjacent lanes)
10. Run gels at 100V
11. Weigh eppendorf tube and write empty mass on tube
12. Use razor blade to precisely cut out the band of the sample fragment
13. Store in marked eppendorf tube, & weigh again. Record full mass on tube as well.
14. Store in -20℃ overnight

Gel Extraction

Adapted from New England Biolabs kit #T1020L insert, all centrifuging should be carried out at 16,000 RCF

#### Materials

1. Extracted agarose gel containing DNA sample in a 1.5 ml microcentrifuge tube
2. 4 Times the agarose gel’s volume of Gel Dissolving Buffer
3. 200 μL DNA Wash buffer diluted with 4 times its volume of 95%+ ethanol
4. 6+ μL DNA Elution Buffer

---

#### Procedure

1. Add 4 volumes Gel Dissolving Buffer to the gel slice in microcentrifuge tube
2. Incubate sample sample between 37–55℃ (typically 50℃), vortexing periodically until the gel slice is completely dissolved (no visible pieces), about 5–10 minutes
3. Insert column into collection tube and load sample onto column
4. Spin for 1 minute, discard flow-through
5. Re-insert column into collection tube, add 200 μL DNA Wash Buffer, and spin for 1 minute
6. Repeat previous step, discarding flow through if necessary
7. Transfer column to a clean 1.5 ml microcentrifuge tube, if the tip of the column contacts the flow-through, re-spin for 1 minute
8. Add DNA Elution Buffer to the center of the matrix, wait 1 minute and spin for 1 minute
9. Store at -20℃ for later use

Golden Gate with BbsI

Use [online calculator](https://www.promega.com/resources/tools/biomath/) to determine total pmol and pmol/µL

#### Materials

1. 0.06 pmol plasmid backbone (calculate required volume with pmol/µL from above link)
2. 0.12 pmol insert (calculate required volume with pmol/µL from above link). If using multiple inserts, make sure the combined amount is 0.12 pmol
3. 2.5 µL 10X T4 DNA Ligase Buffer
4. 0.25 µL T4 DNA Ligase (NEB #M0202), 2000 U/µL
5. 0.75 µL BbsI-HF (Neb #R3539)
6. to 25 µL nuclease free water

---

#### Procedure

1. Combine all components in PCR tube, mixing gently by pipetting up and down after each addition
2. Program the PCR Cycle

1. 37°C for 5 min
2. 16°C for 5 min
3. go to step 1, 30X
4. 65°C for 20 min
5. 12°C forever

3. Column clean and elute to 13 µL
4. Determine concentration using Qubit fluorometer and record final concentration in ng/µL

Golden Gate with BsaI

Calculate using [link](https://www.promega.com/resources/tools/biomath/) to determine total pmol and pmol/µL

#### Materials

1. 0.06 pmol plasmid backbone (calculate required volume with pmol/µL from above link)
2. 0.12 pmol insert (calculate required volume with pmol/µL from above link)
3. 2.5 µL 10X T4 DNA Ligase Buffer
4. 0.25 µL T4 DNA Ligase (NEB #M0202), 2000 U/µL
5. 0.75 µL BsaI-HFv2 (Neb #R3733)
6. To 25 µL nuclease free water

---

#### Procedure

1. Combine all components in PCR tube on ice (enzyme should be added last), mixing gently by pipetting up and down after each addition
2. Microfuge briefly to combine
3. Program the PCR Cycle

1. 37°C for 5 min
2. 16°C for 5 min
3. go to step 1, 30X
4. 80°C for 20 min
5. 12°C forever

4. PCR sample
5. Column clean and elute to 13 µL
6. Determine concentration using Qubit fluorometer and record final concentration in ng/µL

In-Fusion Assembly

In-Fusion assembly works best with ~50ng of insert and ~200ng of vector. Determine the required volumes of backbone and insert to get a 2:1 molar ratio of backbone to insert with those approximate final masses of DNA. This procedure in adapted from the [manufacturer instructions](%E2%80%9Chttps%3A//www.takarabio.com/documents/User%20Manual/In/In-Fusion%20HD%20Cloning%20Kit%20User%20Manual_102518.pdf%E2%80%9D).

#### Materials

1. 2 μl 5X In-Fusion HD Enzyme Premix
2. ≈200 ng backbone plasmid
3. ≈50 ng vector
4. To 10 μl nuclease free water

#### Procedure

1. Mix the reaction by pipetting up and down
2. Incubate for 15 minutes at 50°C
3. Place on ice
4. Store at -20°C for future use

gBlock Resuspension

Adapted from [IDT protocol](https://www.idtdna.com/pages/education/decoded/article/tips-for-working-with-gblocks-gene-fragments)

#### Materials

1. gBlocks to resuspend (DNA fragment in 25 µL of nuclease-free water at 10 ng/µL)

---

#### Procedure

1. Before opening the tube, spin it down in a microcentrifuge for 3–5 seconds
2. Add nuclease free water to 10 ng/µL
3. Vortex briefly
4. Incubate at 50°C for 15–20 min
5. Briefly vortex and centrifuge
6. Use suspended gBlock fragments for BbsI cassette assembly

Prepare Cassettes

#### Materials

1. Suspended gBlocks

---

#### Procedure

##### Cassettes A&C

Do the following separately for each biomarker, i.e. all GFAP binders together, but UCHL1 and GFAP binders separately

1. Perform Bbs1 golden gate protocol with all binders and linkers
2. Label all reactions appropriately
3. Run PCR to amplify final product with appropriate primers
4. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL

##### Cassette B

Separate these reactions by complementation system, only paired complementation systems will interact with each other (n-term GFP only interacts with c-term GFP, etc.). No need to separate by biomarker, cassette B will contain both parts of a complementation system and the riboJ-RBS complex, so it’s binder-agnostic.

1. Depending on how much RiboJ+RBS complex is needed, it may need to be amplified using appropriate primers
2. Perform Bbs1 golden gate protocol with comp system I, Ribo\_complex, and comp system II
3. Label all reactions appropriately
4. Run PCR to amplify final product with appropriate primers
5. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL

##### Cassette D (backbone)

pSEVA3411 already contains chloramphenicol resistance, this step only adds a single promoter g block (and a BsaI site) to turn it into Cassette D.

1. Perform Bbs1 golden gate protocol with pSEVA3411 and iGEM\_promoter-RBS gBlock
2. Label all reactions appropriately
3. Run PCR to amplify final product with appropriate primers
4. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL

##### Combining Cassettes A, B, C and D

1. Perform *BsaI* golden gate assembly process with all 4 cassettes
2. Label appropriately
3. Clean sample, then measure final concentration with nanophotometer and record concentration in ng/µL
4. Store plasmids at -20°C for later transformation

# Dry Lab Protocols

Analyzing Binder Effectiveness with PyMOL

#### Requirements

1. Working PyMOL installation
2. `.pdb` files
3. [InterfaceResidues.py script](https://gitlab.igem.org/2022/uoregon/-/blob/0081d6bdc367405a47743d6289b8a4aa2e497661/InterfaceResidues.py)

---

#### Procedure

1. `File → open`  all `.pdb` files
2. On the highest ranked file,  `click A → align → all to this`
3. Record executive RMSD values
4. Hide all structures other than rank 1
5. Run the InterfaceResidues.py python script
6. Run: `interfaceResidues <object-name>, chain A, chain B` in the PyMOL console
7. After step 7, the interface residues should be already selected. Then run these two lines to have each chain as a separated object:

   ```
   select mol1, chain A
   select mol2, chain B
   ```
8. Color it with this style:
   `bg_color white`
9. For rank 1 object: `color → gray90`
10. For interface object:

    ```
    show → sticks
    color → spectrum → b factor
    ```
11. You can get the number of residues in the interface with this command:
    `count_atoms byca <interface-object>`
12. Look at the sequence (`set seq_view, 1`)

* [if the color of residues is not quite visible, you can change the color by `set seq_view_color, <your-color>`]

13. Color the first 10 residues (N-terminal) of each “chain” with “marine blue” and the last 10 residues (C-terminal) with “deep salmon red.”
14. Click on your interface selection and then show hydrogen bonds by `A → find → polar contacts → between chains`
15. Count the number of hydrogen bonds manually

AlphaFold Complex Structure Prediction

AlphaFold is heavily biased by examples of natural binding and folding, so its predictions will probably not be accurate for non-native binding (such as nanobodies) or for point mutations. For these applications, (py)Rosetta or docking would be a better choice.

#### Requirements

1. Working Google account
2. Computer with sleep settings disabled

---

#### Procedure

1. Open this [colab notebook](https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb#scrollTo=woIxeCPygt7K) while logged into your google account, it should be named "AlphaFold2\_advanced.ipynb"
2. Make sure you're on your personal account, then click "copy to drive"
3. Complete the following steps on the copied version in your drive (the "copy to drive" option will not be visible)
4. Press the "Play" button at the left of the first cell, this will install required software on the colab GPUs<. This should take around a minute to complete.
5. When the software is installed and the green progress bar at the bottom is full, proceed to the next cell
6. Enter your desired amino acid sequence in the field labelled "sequence"
7. If you're interested in the interactions between multiple sequences (such as a biomarker and a binder), enter both sequences in this field separated by `:`
8. Enter a meaningful name in the "jobname" field
9. Enter the homo-oligomeric assembly state in the "homooligomer" cell. This will very depending on your input in the "sequence" field.

* If you are folding a single protein, leave the value as `1`
* If you are simulating the binding of two monomeric proteins, set the value to `1:1`
* If you are simulating the binding of a dimeric binder and a monomeric biomarker (such as s1000β dimer and GFAP), set the value to `1:2`. The position of these numbers depends on how the original sequences were input, this example assumes that the input was `<GFAP Sequence>:<S100β Sequence>` and tells AlphaFold to calculate the interaction between one GFAP and one s100β dimer.

10. Run the "Search against genetic databases" cell by clicking the play button and leaving the default settings
11. Skip the "filter options", and run the "run AlphaFold" cell with default settings

* The free tier of google colab will discard your data if your computer goes to sleep or the browser tab is closed, which is why your sleep settings should be disabled

12. As the AlphaFold cell runs, it will generate five images with different configurations. The colored picture on the right is colored by pLDDT, the more blue a chain is the more confident AlphaFold is in its prediciton.
13. When the cell completes, skip all the optional cells and jump to "Download predictions" at the bottom
14. This will download a `.zip` file containing several files

* `msa.pickle` & `msa_coverage.png` : These are the results of the MSA (multiple sequence alignment) from the "Search against genetic databases" cell. You can upload these to that cell during future runs to avoid calculating it from scratch again, but it often isn't worth it since the calculation is so quick.
* `settings.txt` These are the settings you chose during the run
* `rank_#_model_#_ptm_seed_#_unrelaxed.pdb` These are the files you want to analyze in pyMOL later. The most important number is the rank (first number), the higher it is the more confident AlphaFold is in that structure.
* `rank_#_model_#_ptm_seed_#_unrelaxed.png` These are the images that the AlphaFold cell produced while working. They're helpful for a quick overview, but the information is redundant with the `.pdb` files.

15. Upload the zip file to the computational team drive in the appropriate subfolder for archiving

Adjusting `.pdb` Chain Identifiers

This is only technically necessary for HDOCK, ZDOCK is smart enough to avoid confusing chains for simple settings. For consistency, we recommend keeping as many variables constant as possible, which means adjusting the chain numbers for all docking software, not just those for which it’s absolutely necessary. Note that `chain [#]` refers to a chain identifier, which is usually a letter, not a number.

#### Requirements

1. Working pyMOL installation
2. `.pdb` file of the receptor protein **or** PDB ID
3. `.pdb` file of the ligand protein **or** PDB ID

---

#### Procedure

1. Enable sequence view by executing `set seq_view, 1` in the pyMOL console
2. Load the receptor and ligand into pyMOL by opening the `.pdb` files or getting the structures from PDB by executing `fetch [PDB ID]` in the console
3. Examine the structures to determine which chains you’re interested in in each object
4. Extract receptor and ligand structures as pyMOL objects `lig` and `rec`

1. `extract rec, chain [#] and [RECEPTOR_OBJECT]`
2. `extract lig, chain [#] and [LIGAND_OBJECT]`
3. `delete [RECEPTOR_OBJECT]`
4. `delete [LIGAND_OBJECT]`
5. To dock dimers (receptor or ligand) in HDOCK or ZDOCK, the dimer must be a single chain, it doesn’t matter if the chains aren’t connected via amino acids
6. The above steps still work for dimer chain(s), but you need to select **all** the chains associated with the dimer, i.e. `extract rec, chain A and chain B and [RECEPTOR_OBJECT]`

5. To avoid chain identifier collisions when docking, change the chain identifier of the ligand to B by executing `alter lig, chain = ‘B’`
6. To avoid residue number collisions when docking, change the ligand residue numbering to start after the last residue of the receptor

* You can do this manually by looking at the sequence in pyMOL’s viewer, but to be sure you can have pymol tell you the residue numbers present in your object by executing `iterate [OBJECT_NAME], resi`. The highest value will be the end of the chain.
* Set the starting residue number of the ligand (chain B), to be one greater than the highest residue number of the receptor by executing `alter lig, resi = int(resi) + [#]` , where `[#]` is one greater than the highest residue number of the receptor

7. Save the receptor object as a `.pdb` file by executing`save rec.pdb, rec`
8. Save the receptor object as a `.pdb` file by executing`save lig.pdb, lig`

Simulating Protein-Protein Docking with HDOCK

HDOCK and ZDOCK work very similarly (for example, they both perform energy minimization), but they use different processes “behind the scenes”. If the outputs of both systems look very similar, that means the protein is more likely to work that way *in vitro* and the reverse is also true.

#### Requirements

1. Working email address
2. `.pdb` files of the proteins you want to dock **or** PDB ID and chain number

---

#### Procedure

1. Navigate to the [HDOCK server website](http://hdock.phys.hust.edu.cn/)
2. Upload your AlphaFold `.pdb` file of the receptor (biomarker). If the receptor is a dimer, make sure to adjust the numbering before proceeding or HDOCK may not work properly.
3. If using a PDB identifier, remember to add the chain separated by a colon, i.e. `3kw5:A` after the identifier so you only dock that specific part of the structure
4. Repeat the above steps for the ligand protein
5. Remember to exclude non-binding residues (such as the insides of a ligand) under “Advanced Options” to get the most accurate model possible
6. Enter a meaningful jobname and your email, and HDOCK will email you a link to your results in a few hours
7. The output from HDOCK is a `.tar.gz` file, all of the predictions in `.pdb` format are in there
8. Upload the output tarball to the computational drive, analyze the docking effectiveness with pyMOL, and compare them to the ZDOCK predictions

Simulating Protein-Protein Docking with ZDOCK

HDOCK and ZDOCK work very similarly (for example, they both perform energy minimization), but they use different processes “behind the scenes”. If the outputs of both systems look very similar, that means the protein is more likely to work that way *in vitro* and the reverse is also true. Unlike HDOCK, ZDOCK is only able to dock the entirety of PDB structures, and there is no option to specify docking with only a particular chain.

#### Requirements

1. Working email address
2. `.pdb` files of the proteins you want to dock **or** PDB ID

---

#### Procedure

1. Navigate to the [ZDOCK server website](https://zdock.umassmed.edu/)
2. Upload your AlphaFold `.pdb` file of the receptor (biomarker). If the receptor is a dimer, make sure to adjust the numbering before proceeding or ZDOCK may not work properly.
3. Repeat the above steps for the ligand protein
4. Enter your email, and ZDOCK will email you a link to your results in a few hours
5. Remember to exclude non-binding residues (such as the insides of a ligand) in the next screen
6. The output from ZDOCK is a `.zip` file, all of the predictions in `.pdb` format are in there
7. Upload the output zip archive to the computational drive, analyze the docking effectiveness with pyMOL, and compare them to the HDOCK predictions

{% endblock %}

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

# Wetlab Summary

During the course of our experimental wet lab phase, we designed a genetic construct to be assembled and produced in *E. coli.* To create this, we utilized Combinatorial Golden Gate assembly to produce four unique genetic cassettes, each encoding a specific portion of our biosensor complex. Each cassette existed as a plasmid, with Cassettes A and C each encoding one half of the binder/linker portion of our construct. Cassette B encoded our split complementation system, which “sandwiched” our RiboJ sequence. Cassette D encoded the backbone of our final plasmid that would be transfected into cells and translated into protein. This cassette included our selected resistances as well as a D-Promoter.

---

## Future Steps

After initial experimentation, we discovered that our RiboJ sequence was not functioning correctly in the Golden Gate assembly, so we needed to fix one end of the riboJ sequence and rerun the Golden Gate assembly. Furthermore, a wise step would be to increase the number of split reporter proteins we are experimenting with, as we gathered many more during the research phase that we never got to test. This pursuit would also adjust split sites and substrate concentrations to maximize signaling.

---

# Computational Summary

There are many biomarkers associated with concussions and mTBI, but their binding interactions with particular proteins are not often studied so some forms of binding (particularly the strength of the biomarker/binder complex) are difficult to characterize using only the literature. After narrowing the field of likely biomarkers and binders from our literature search, we obtained sequences and then used AlphaFold[1](#Ref1) to model complexes between binders and biomarkers. Based on the results from alphafold, we also determined binding effectiveness with two different docking suites, HDOCK and ZDOCK. Based on the results of all these simulations, we decided to use UCHL1 as our biomarker and split ubiquitin as our binder. GFAP had some promising binders as well, but we ultimately decided to focus purely on UCHL1 since we were unable to express GFAP.

---

## Folding with AlphaFold

The four biomarkers we modeled binder complexes for were UCHL1, ENO2, GFAP, and S100β. For UCHL1, wild-type ubiquitin binds to UCHL1, and a particular modified form ubiquitin Ub-VME is used to study UCHL1[2](#Ref2). We didn’t know if reconstituted split ubiquitin would bind similarly, so we used AlphaFold to predict the structure of the complex. As expected from literature research, we found the highest number of hydrogen bonds and binding residues at the biomarker-binder interface and the binding was quite strong. There were over 20 hydrogen bonds and 67 residues at the interface. We calculated binding residues using a python script, this procedure can be found in our [experiments page](https://2022.igem.wiki/uoregon/experiments) under “Dry Lab Protocols”. These generated structures were also fairly consistent based off of the executive RMSD’s (see below) calculated by aligning all 5 generated structures to the best structure in PyMOL[3](#Ref3).

The dimer ENO2 had only 4 binding residues and no hydrogen bonds with bZIP75, and the binding location of bZIP75 blocks the dimerization of ENO2.

![UCH1/Split Ubiquitin Complex](https://static.igem.wiki/teams/4215/wiki/uchl1ubq.png)

Complex of UCHL1 (blue) with split ubiquitin (red and green)

![Interace region of ENO2 and bZIP75, binding residues shown](https://static.igem.wiki/teams/4215/wiki/eno2-interface.png)

Interace region of ENO2 and bZIP75, with binding residues shown

For GFAP, the binders modeled in alphafold were A10, VHHB7, G11, p53, VC1 RAGE, and VC1 w61. A10 and VHHB7 have the least viable modeling as there were no binding residues or hydrogen bonds. We decided to move forward with VHHB7 instead of A10. G11 has one binding residue with GFAP. It is worth noting that, compared to the strength of UCHL1-split ubiquitin complex, all of the GFAP-binder interactions are quite poor.

![Interace region of GFAP and A10, binding residues shown](https://static.igem.wiki/teams/4215/wiki/gfap-a10.png)

Interace region of GFAP and A10

![Interace region of GFAP and VHHB7, binding residues shown](https://static.igem.wiki/teams/4215/wiki/gfap-vhhb7.png)

Interace region of GFAP and VHHB7

![Interace region of GFAP and G11, binding residues shown](https://static.igem.wiki/teams/4215/wiki/gfap-g11.png)

Interace region of GFAP and G11, with binding residues shown

For S100β, binding with the VC1 (RAGE domain) and VC1 (w61) showed 17 and 23 interface residues respectively and no hydrogen bonds. However, using S100β in the experimental system was abandoned since S100β is expressed in the body during exercise and is expressed later in the body than UCHL1 after an mBTI event[4](#Ref4).

![Interace region of S100β and VC1's RAGE domain, binding residues shown](https://static.igem.wiki/teams/4215/wiki/s100b-rage.png)

Interace region of S100β and VC1's RAGE domain, with binding residues shown

![Interace region of S100β and VC1's w61 domain](https://static.igem.wiki/teams/4215/wiki/s100b-w61.png)

Interace region of S100β and VC1's w61 domain

### AlphaFold RMSD Values

| Biomarker | Binder | Binding Residues | Hydrogen Bonds | Rank 2 RMSD | Rank 3 RMSD | Rank 4 RMSD | Rank 5 RMSD |
| --- | --- | --- | --- | --- | --- | --- | --- |
| UCHL1 | Split Ubiquitin | 67 | 10+ | 0.210 | 0.285 | 0.311 | 0.247 |
| ENO2 | bZIP75 | 4 | 0 | 14.452 | 17.952 | 0.246 | 0.328 |
| GFAP | A10 | 0 | 0 | 17.408 | 13.697 | 12.079 | 20.369 |
| GFAP | VHHB7 | 0 | 0 | 19.242 | 12.461 | 18.651 | 18.757 |
| GFAP | G11 | 2 | 0 | 16.186 | 14.927 | 12.463 | 21.550 |
| S100β | VC1 RAGE | 17 | 0 | 0.431 | 12.097 | 25.098 | 0.322 |
| S100β | VC1 W61 | 23 | 0 | 0.251 | 0.161 | 0.240 | 0.208 |

---

## Protein-Protein Docking with HDOCK and ZDOCK

Through protein modeling and docking, we decided to further investigate UCHL1 using split ubiquitin as a binder, as well as GFAP using S100β, G11, and VHHB7 as binders. Following the protocol listed on our [experiments page](https://2022.igem.wiki/uoregon/experiments), we used ZDOCK and HDOCK to dock our biomarkers and binders. Specifically, we modeled UCHL1 with split ubiquitin as a binder, as well as GFAP with S100β, G11, and VHHB7 as binders.

![Several docking predictions of UCHL1 and Split Ubiquitin](https://static.igem.wiki/teams/4215/wiki/uchl1-multiple.png)

Several docking predictions of UCHL1 and Split Ubiquitin

![Several docking predictions of GFAP, S100B, G11, and VHHB7](https://static.igem.wiki/teams/4215/wiki/gfap-multiple.png)

Several docking predictions of GFAP, S100β, G11, and VHHB7

Regardless of docking software, the location of binding between the split ubiquitin and UCHL1 was consistent. Taken together with the results of the AlphaFold folding, which also predict binding in the same site with approximately the same strength we can be confident in the binding location and more confident in the strength of our models *in silico*.

Going forward it would be useful to have a model of the entire biosensor with the biomarker, binder, and linker together, but we were unable to model this due to technical limitations of the computational tools at our disposal.

# References

---

1. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; Bridgland, A.; Meyer, C.; Kohl, S. A. A.; Ballard, A. J.; Cowie, A.; Romera-Paredes, B.; Nikolov, S.; Jain, R.; Adler, J.; Back, T.; Petersen, S.; Reiman, D.; Clancy, E.; Zielinski, M.; Steinegger, M.; Pacholska, M.; Berghammer, T.; Bodenstein, S.; Silver, D.; Vinyals, O.; Senior, A. W.; Kavukcuoglu, K.; Kohli, P.; Hassabis, D. Highly Accurate Protein Structure Prediction with AlphaFold. *Nature* **2021**, *596* (7873), 583–589. <https://doi.org/10.1038/s41586-021-03819-2>.
2. Boudreaux, D. A.; Maiti, T. K.; Davies, C. W.; Das, C. Ubiquitin Vinyl Methyl Ester Binding Orients the Misaligned Active Site of the Ubiquitin Hydrolase UCHL1 into Productive Conformation. *Proc. Natl. Acad. Sci. U. S. A.* **2010**, *107* (20), 9117–9122. <https://doi.org/10.1073/pnas.0910870107>.
3. Schrödinger, LLC. The PyMOL Molecular Graphics System, Version 2.5.0, 2022.
4. Wang, K. K. W.; Kobeissy, F. H.; Shakkour, Z.; Tyndall, J. A. Thorough Overview of Ubiquitin C-Terminal Hydrolase-L1 and Glial Fibrillary Acidic Protein as Tandem Biomarkers Recently Cleared by US Food and Drug Administration for the Evaluation of Intracranial Injuries among Patients with Traumatic Brain Injury. *Acute Med. Surg.* **2021**, *8* (1), e622. <https://doi.org/10.1002/ams2.622>.
{% endblock %}