iGEM 2018 — Team Stanford

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# Team:Stanford/Notebook

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Home

[![Stanford](https://identity.stanford.edu/img/block-s-2color.png)](https://2018.igem.org/Team%3AStanford)

* [HOME](https://2018.igem.org/Team%3AStanford)
* [PROJECT](https://2018.igem.org/Team%3AStanford/Project)
  + [INTRO](https://2018.igem.org/Team%3AStanford/Project#Introduction)
  + [DNA](https://2018.igem.org/Team%3AStanford/Project#DNA)
  + [PROTEIN](https://2018.igem.org/Team%3AStanford/Project#Protein)
  + [SMALL MOLECULE](https://2018.igem.org/Team%3AStanford/Project#Small_Molecule)
  + [REPORTER](https://2018.igem.org/Team%3AStanford/Project#Reporter)
* [PARTS](https://2018.igem.org/Team%3AStanford/Parts)
* [HUMAN CENTERED DESIGN](https://2018.igem.org/Team%3AStanford/Human_Practices)
  + [INTEGRATED HUMAN PRACTICES](https://2018.igem.org/Team%3AStanford/Human_Practices)
  + [SAFETY](https://2018.igem.org/Team%3AStanford/Safety)
  + [COLLABORATIONS](https://2018.igem.org/Team%3AStanford/Collaborations)
* [TEAM](https://2018.igem.org/Team%3AStanford/Team)
  + [BIOS](https://2018.igem.org/Team%3AStanford/Team)
  + [NOTEBOOK](https://2018.igem.org/Team%3AStanford/Notebook)
  + [ATTRIBUTIONS](https://2018.igem.org/Team%3AStanford/Attributions)
* [MEDALS](https://2018.igem.org/Team%3AStanford/Medal_Eligibility)
  + [MEDAL ELIGIBILITY](https://2018.igem.org/Team%3AStanford/Medal_Eligibility)
  + [MODEL](https://2018.igem.org/Team%3AStanford/Model)
  + [INTERLAB](https://2018.igem.org/Team%3AStanford/InterLab)

Notebook

JUNE WEEK 1

**Wet Lab:** NONE

**Project Development:**- Searched for iGEM aflatoxin projects
- Contacted Tsinghua 2017
- Investigated other potential target pathogens: powdery mildew, salmonella
- Researched Y2H systems, looked for bacterial analogs
- Reached out to Hoschild lab about bacterial two-hybrid system and sequences
- Outlined possible paths & challenges
- Created steps for the summer
- Ordered Addgene plasmids for bacterial two-hybrid system

JUNE WEEK 2

**Wet Lab:** Made chemically competent FW102 cells

**Project Development:**- Gibson assembly review
- Gibson primer design
- Plasmid design protocol
- Submitted safety check-in
- Reached out to Tsinghua 2017 to talk about aflatoxin detection

JULY WEEK 1

**Wet Lab:**- Transformed plasmids from addgene for Bacterial 2-Hybrid system into FW102
- Began PCRs for lambda and alpha backbones and aflatoxin-SCFV1 and aflatoxin-SCFV2

**Project Development:**- Finished primer design for pLacZ+ RFP
- Plate reader tutorial
- Met with advisors

JULY WEEK 2

**Wet Lab:**- Ran calibrations for InterLab
- Attempted to recapitulate the BacterioMatch Two-Hybrid system; we were not successful and saw growth on our non-His plates multiple times
- Third attempt at adding overhangs to scFv1 and backbone for Gibson Assembly

**Project Development:**- Finished design of reporter plasmid
- Finalized project idea: small molecule, DNA, and protein detection
- Met with Stanley Qi about using cas9 for DNA detection
- Looked into possible applications, ex. biomarkers for TB or virus detection

JULY WEEK 3

**Wet Lab:**- Continuation of Interlab study
- Continuation of Hoschild recapitulation
- Another atttempt at Gibson scFv1 and backbone
- Transformed cells for InterLab
- Analyzed alpha+aflatoxin-ScFv1 fusion
- Re-attempted BacterioMatch recapitulation
- Miniprepped, amplified, and analyzed RFP for reporter plasmid
- Cloning to build reporter plasmid

**Project Development:**- Research possible proteins for detection
- Met with Marie La Russa and obtained pd and ad Cas9 sequences
- Talked with Rolando to reach out to possible real-life customers
- Development of promoters for DNA detection system
- Researched previous systems using Cas9 as a transcriptional activator
- Started designing sgRNAs for Cas9 proteins

JULY WEEK 4

**Wet Lab:**- Transformed reporter plasmid
- Continued with Interlab Study
- Transformed pLacZ + RFP plasmid to test functionality
- Miniprep of the scFv fusion proteins and dCas9s
- TRansformed DH5a with pLacZ + RFP, lambda, alpha
- Successfully amplified adCas9

**Project Development:**- Designed experiments for DNA detection testing
- Met with Marie for Gibson troubleshooting
- Discussed recapitulation difficulties with Ann Hochschild

AUGUST WEEK 1

**Wet Lab:**- Re-attempted pdCas9 amplification
- Continued InterLab transformations

**Project Development:**- Finished sgRNA design
- Met with Ritish about liver cancer detection needs

AUGUST WEEK 2

**Wet Lab:**- Plate reader measurements for InterLab
RFP plate reader experiment for assessing reporter

**Project Development:**- NONE - Most of team away on vacation

AUGUST WEEK 3

**Wet Lab:**- Inserted adCas9 and pdCas9 in alpha and lambda backbones
- Inserted anti-IL1B scFv2 into lambda backbone
- Inserted sgRNA into lambda-pdCas9 backbone
- Transformed cells with new assemblies
- Finished InterLab

**Project Development:**- Continued testing reporter plasmid

AUGUST WEEK 4

**Wet Lab:**- Inserted reporter construct into alpha-adCas9 backbone
- Inserted anti-IL1B scFv1 into alpha backbone
- Amplified and sequenced sgRNA designs
- Amplified GFP backbone for testing DNA detection

**Project Development:**- Created diagrams for explaining our detection systems

**Wiki:**TBD

AUGUST WEEK 5

**Wet Lab:**- Gel extracted adCas9 and pdCas9
- Reattempted adCas9+alpha and pdCas9+lambda fusion proteins

**Project Development:**- Analyzed sequencing data for recent assemblies

SEPTEMBER WEEK 1

**Wet Lab:**- Transformed new assemblies into cells
- Added anti-IL1B scFv1 to alpha-RFP backbone

**Project Development:**- Updated safety form
- Worked on poster for student research fair

**Wiki:** TBD

SEPTEMBER WEEK 2

**Wet Lab:**- Gel extracted alpha+adCas9 backbone and opened for RFP insertion
- Transformed cells with alpha+adCas9

**Project Development:**- Submitted samples for sequencing
- Analyzed protein detection assemblies
- Met with Environmental Health & Services to discuss aflatoxin safety
- Assessed results of alpha-adCas9+GFP transformation
- Discussed applications for DNA detection with grad student mentor

SEPTEMBER WEEK 3

**Wet Lab:**- Made liquid cultures from alpha-adCas9+GFP transformants
- Amplified linear GFP fragment for running DNA detection test

**Project Development:**- nalyzed results of lambda+aflatoxin-scFv2 PCR

SEPTEMBER WEEK 4

**Wet Lab:**- Inserted parts into iGEM submission backbone
- Transformed cells with Biobrick-compatible parts
- Made liquid cultures
- Extracted DNA for submission

**Project Development:**- Started Wiki development

OCTOBER WEEK 1

**Wet Lab:**- Attempted to add T7 promoters to DNA detection parts via PCR, but unsuccessful
- Troubleshooted PCR and re-attempted; still unsuccessful, so unable to test DNA detection system

**Project Development:**- Designed primers to adapt DNA detection system for cell free

OCTOBER WEEK 2

**Wet Lab:**- Picked four colonies and grew up cultures of IL1B construct
- Tested for presence of IL1B protein using ELISA
- Validated that our part successfully produces IL1B

**Project Development:**- Final touches to Wiki
- Inserted information into parts registry


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Stanford/Experiments

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[HOME](https://2018.igem.org/Team%3AStanford)

TEAM

[Team Members](https://2018.igem.org/Team%3AStanford/Team)
[Collaborations](https://2018.igem.org/Team%3AStanford/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AStanford/Description)
[Design](https://2018.igem.org/Team%3AStanford/Design)
[Experiments](https://2018.igem.org/Team%3AStanford/Experiments)
[Notebook](https://2018.igem.org/Team%3AStanford/Notebook)
[InterLab](https://2018.igem.org/Team%3AStanford/InterLab)
[Model](https://2018.igem.org/Team%3AStanford/Model)
[Results](https://2018.igem.org/Team%3AStanford/Results)
[Demonstrate](https://2018.igem.org/Team%3AStanford/Demonstrate)
[Improve](https://2018.igem.org/Team%3AStanford/Improve)
[Attributions](https://2018.igem.org/Team%3AStanford/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AStanford/Parts)
[Basic Parts](https://2018.igem.org/Team%3AStanford/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AStanford/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AStanford/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AStanford/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AStanford/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AStanford/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AStanford/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AStanford/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AStanford/Hardware)
[Measurement](https://2018.igem.org/Team%3AStanford/Measurement)
[Model](https://2018.igem.org/Team%3AStanford/Model)
[Plant](https://2018.igem.org/Team%3AStanford/Plant)
[Software](https://2018.igem.org/Team%3AStanford/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Stanford)

# Experiments

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

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Stanford/Results

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Wet Lab

[![Logo](https://identity.stanford.edu/img/block-s-2color.png)](https://2018.igem.org/Team%3AStanford)

* [HOME](https://2018.igem.org/Team%3AStanford)
* [ABOUT](https://2018.igem.org/Team%3AStanford/Description)
* TOOLKIT
  + WET LAB
    - [FOUNDATIONS](https://2018.igem.org/Team%3AStanford/Results)
    - [PARTS](https://2018.igem.org/Team%3AStanford/Parts)
  + PRODUCT DEVELOPMENT
    - [DESIGN PROCESS](https://2018.igem.org/Team%3AStanford/Design)
    - [APPLIED DESIGN](https://2018.igem.org/Team%3AStanford/Applied_Design)
    - [MODELING](https://2018.igem.org/Team%3AStanford/Model)
  + DOCUMENTATION
    - [NOTEBOOK](https://2018.igem.org/Team%3AStanford/Notebook)
    - [SAFETY](https://2018.igem.org/Team%3AStanford/Safety)
* HUMAN CENTERED DESIGN
  + [PRACTICES](https://2018.igem.org/Team%3AStanford/HP/Gold_Integrated)
* OUTREACH
  + [COLLABORATIONS](https://2018.igem.org/Team%3AStanford/Collaborations)
  + [PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AStanford/Engagement)
* TEAM
  + [BIOS](https://2018.igem.org/Team%3AStanford/Team)
  + [SPONSORS](https://2018.igem.org/Team%3AStanford/Sponsors)
  + [ATTRIBUTIONS](https://2018.igem.org/Team%3AStanford/Attributions)

Wet Lab

* [OVERVIEW](#overview)
* [A](#Aid)
* [B](#Bid)
* [FUTURE WORK](#futurework)

OVERVIEW

TBD

A

Introduction

TBD

TBD

TBD

TBD

TBD

B

Introduction

TBD

TBD

B subtitle

TBD

TBD

B subtitle

TBD

TBD

TBD

TBD

FUTURE WORK

TBD