Notebook

See the team section for details of who participated on each committee.

General Project Notebook

Entire Team

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

Entire Team

  • Member recruitment

Entire Team

  • First PCR/Pipette skills training workshop

Entire Team

Entire Team

  • Official iGEM kickoff
  • Team roster finalized

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

Entire Team

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

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

Entire Team

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

Entire Team

  • Lab safety training

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

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

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

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

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

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

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

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

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

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

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

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

Entire Team

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

Committees

Fundraising

  • Worked on graphical abstract

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

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

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.

Entire Team

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

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

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

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

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.
  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, 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.
  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
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 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 w/ 3 samples (B-lac & firefly luciferase) prepared earlier in the day (prepared by carmen)

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 protocols with a non-diluted, 10x, and 20x dilution of DH5a cells transfected with puc-19

  • N/A

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

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

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

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

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

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

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

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

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 125.10 ng/µL 1.841 2.161
C linkers + UBQ - N 102.05 ng/µL 1.850 2.288
HRP 130.2 ng/µL 1.849 2.222
TEV 104.00 ng/µL 1.851 2.291
Blac 141.45 ng/µL 1.843 2.224
Fluc 159.80 ng/µL 1.839 2.159
Rluc 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

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