Team:Stanford/Notebook

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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