{% extends "layout.html" %} {% block header_image %}https://static.igem.wiki/teams/4592/wiki/test.png{% endblock %} {% block lead %}Skeleton built{% endblock %} {% block title %}Notebook{% endblock %} {% block page_content %}

June

  1. Fundraising/Planning
    1. Researching arrangements for travel to iGEM Jamboree
    2. Filling out funding applications
  2. Wiki Team
    1. Initial writing of project description, attributions, notebooks, safety page, and human practices
    2. Research on iGEM criteria
    3. Developed a basic understanding of HTML and CSS to be able to design and edit the wiki moving forward.
    4. Wiki edits
      1. header, footer template
      2. notebook page and project description page skeletons
    5. Social media: started project reveal
  3. Dry Lab Team
    1. Developing a comprehensive range of project approaches
    2. Collecting protocols: culturing Microcystis a. in optimized conditions for toxin production
    3. Met with CA Department of Water Resources for project development and possible future approval/viability for deployment
    4. Guidelines for the genetic engineering of Microcystis communities based on phage and conjugative plasmids
      1. methods for the evaluation of success metrics (toxin concentrations, cell viability, propagation rates)
    5. Drafted various protocols related to molecular cloning, phage assembly, various assays including HPLC protocol for studying degradation products
    6. Coordinated meeting with Dr. Glenn Milhauser to discuss HPLC methods, usage, protocols, etc.
  4. Wet Lab Team
    1. Made chemicompetent e.coli cells to be used the rest of the summer
    2. Transformed chemicompetent e.coli to check competency
    3. Organized the lab space
    4. Replenish commonly used reagents
    5. Prepped, cleaned, and performed maintenance on the autoclave we will be using
    6. Took inventory of everything in the lab

July

  1. Wiki Team
    1. Edited existing notebook branch to isolate notebook updates from main
    2. Started team branch to keep updates to team page and associated styles isolated
    3. Gathered team information and bios
    4. Setup team page skeleton with placeholder information and pictures
      1. Created template and added usage for future reference
  2. Social Media:
    1. Project teasing on instagram
      1. Target location
      2. Target Species
      3. Microcystin vs microcystis nomenclature
    2. Thanked IMPACT grant sponsor
    3. First educational critter o'clock post- turtle
  3. Dry Lab Team
    1. Refined a conjugative transposon approach for knocking-out microcystin production.
    2. Looked for shuttle vectors that have a selectable marker, replicate in E. Coli and M. aeruginosa, and conjugate in M. aeruginosa.
    3. Drafted protocols for transforming M. aeruginosa based on the 2016 SSH iGEM team that succeeded in transforming M aeruginosa with a dead Cas-9 that silenced the McyB gene essential for microcystin production.
    4. Made protocol for Bold 3N growth media.
    5. Met with Glenn Millhauser and Kevin Singewald about using the HPLC to measure microcystin levels very accurately
  4. Wet Lab Team
    1. Completed Inoue protocol for chemicompetent cells and evaluated transformation efficiency of PET28-GFP plasmid
      1. Very low, restarted protocol from scratch
    2. Received and started an M.aeruginosa CPCC 300 strain (UTEX LB2385) culture
      1. Transferred from original vial to mammalian cell culture flask for increased surface area
    3. Started a second M.aeruginosa culture using provided Bold 3N (B3N) media inoculated with 0.5ul of original M.aeruginosa culture

  1. Fundraising
    1. Editing the grant applications
    2. Reaching out to potential sponsors
  2. Social Media:
    1. Created threads account
    2. Stories on pinto lake visit, national slushee day
    3. Updated bio
    4. Second critter o'clock update- double crested cormorant
  3. Dry Lab Team
    1. Coordinated additional meeting with Kevin Singewald to learn how to use the HPLC.
    2. Started drafting additional general protocols and HPLC based on personal protocols and literature
    3. Introduction to Stealth bioinformatics tool to identify potential restriction motifs
    4. Fully annotated pSPIN-R plasmid to determine all of the INTEGRATE components necessary for toxin disruption.
    5. Fully annotated pSHDY plasmid to determine all of the necessary components for broad host range conjugation.
  4. Wet Lab Team
    1. Competent cells with high transformation efficiency
      1. Top10 with pET28a
    2. 1st time DNA extraction / troubleshooting
      1. Low DNA yield, contamination
    3. Received pSHDY plasmid from Addgene (Addgene plasmid # 137661)
    4. Created 1% M.aeruginosa inoculation from initial culture
      1. Done to keep planktonic cell culture
  5. Thesis Team
    1. Creating the outline
    2. Drafting the abstract, acknowledgements, introduction
  6. Human Practice
    1. Presented project to Girls in Engineering, included learning activities
    2. Got in contact with Pinto Lake lab to research the long-term effects of our projec

  1. Social Media
    1. IDT shoutout
    2. Critter o'clock
    3. Instagram story updates
    4. Promega shoutout
  2. Dry Lab Team
    1. Met with Kevin Singewald for an HPLC tutorial.
    2. Drafted a co-culturing protocol for E. coli and M. aeruginosa.
    3. Coordinated with the wet lab team to test parameters for co-culturing.
    4. Designed pINTO: a conjugative plasmid capable of toxin disruption derived from pSHDY and pSPIN-R components.
  3. Thesis Team
    1. Editing the outline
    2. Writing the thesis
    3. Adding and editing figures
  4. Wet Lab Team
    1. Tried culturing M.aeruginosa and E.coli together
    2. DNA extraction of microcystis for sequencing

  1. Dry Lab Team:
    1. Focused primarily on drafting a thesis of our project.
    2. Tailored pINTO for expression in M. aeruginosa
    3. Considered plasmid delivery mechanisms alternative to conjugation to bypass a helper plasmid requirement.
    4. Assessed Stealth, a bioinformatics tool, for integration with our approach based on the horizontal propagation of our genetic construct.
      1. Wrote several small scripts to help with Stealth output processing
  2. Thesis Team
    1. Reading, editing, and adding citations to the thesis
    2. Writing personal chapters 2 and 3 for end of Summer session 1
  3. Wet Lab Team
    1. DNA extraction of microcystis for sequencing

  1. Team Progress
    1. Pivoted project approach and began brainstorming alternative directions
    2. Considered many alternative approaches for toxin disruption, including: plasmid-mediated cell lysis, siRNA silencing, enzyme degradation, and insertional mutagenesis.
    3. All team members met and discussed the pros and cons of each proposed project path
      1. Ultimately, the team decided to pursue insertional mutagenesis after we met with Diego Gelsinger from Colombia University.
  2. Human Practices
    1. Met with Diego Gelsinger from Columbia University for advice on expression of CAST (CRISPR-associated transposons) systems and conjugation in prokaryotes.
    2. Prepared for iGEM meetup
  3. Wet Lab
    1. DNA extraction of Microcystis
    2. Prepared for creation of a DNA library for nanopore sequencing
    3. Received materials from Diego Gelsinger, including: pSPIN, pSPIN-R, and EcGT2 (conjugative, auxotrophic E. coli).
  4. Interlab Study
    1. Transformed DH5a cells with 8 provided interlab devices
    2. Interlab Calibration - measuring fluorescence
    3. Used materials in measurement kit to complete serial dilution on 96 well plate; read @ different wavelengths
      1. Results:
        1. Didn't use a opaque plate, need to redo experiment
        2. Accidental exposure of light sensitive reagents during transfer between two labs

August

  1. Social Media
    1. Promega Shoutout
    2. iGEM UCSC Meet-Up promotion / advertising
  2. Wet Lab
    1. Transformation of pSPIN and pSPIN-R into TOP10 cells
    2. pSHDY transformation into M. aeruginosa
      1. Results:
        1. Transformation was successful, hypothesize that DNA might be getting methylated
  3. Sequencing
    1. Created DNA library
    2. Loaded minION and ran it, stopped running within 24 hrs due to weekend power outage
  4. Interlab Study
    1. Repeated interlab experiment 3
      1. DH5a transformation
      2. Inoculation & Serial Dilutions
      3. Talked to Rebecca DuBois' lab for initial plate (wrong plate)
      4. Plate measurements
  5. Dry Lab
    1. Drafted protocols for end-point conjugation between EcGT2 E. coli and M. aeruginosa based on Diego Gelsinger's Nature protocols preprint.
    2. Redesigned a smaller version of pINTO with hopes of improving conjugation efficiency.
    3. Considered more foundational contributions to the iGEM community, like a software pipeline (Chameleon) that tailors plasmids to the RM profile of a specific bacteria.
  6. Software
    1. Started development of a software pipeline centered around the Stealth program titled Chameleon
    2. Began converting previously written scripts into dedicated programs
  7. Outreach
    1. Prepared for and hosted iGEM team meetup with Washington Highschool

  1. Wet Lab
    1. Miniprep and serial decade dilutions of pSHDY, transforming into M. aeruginosa
  2. Dry Lab
    1. Designed a broad-host range, conjugative plasmid (pSPDY) that we would use to validate Chameleon.
      1. pSPDY was derived from pSPIN and pSHDY.
      2. Planned to order two versions of pSPDY: one that was unmodified and another that was optimized by Chameleon.
    2. Designed gene blocks and flagged primers for synthesizing unmodified pSPDY with golden gate.
    3. Order flagged primers and gene blocks from IDT for synthesizing the unmodified pSPDY.
    4. Designed pSPDY with a “miniT” so that it was still capable of targeted gene disruption with the addition of INTEGRATE.
    5. Designed experiments for validating Chameleon through natural transformation and conjugation of pSPDY.
  3. Sequencing
    1. Contacted Brady and had assistance on loading the minion
      1. Learned how to properly empty waste port
      2. Learned how to wash and reload flow cell mid run
    2. Ran the minION again
  4. Software
    1. Explored alternative approaches to pattern constraint module in order to enhance cohesion between codon optimization and pattern avoidance activities.
    2. Brainstormed method to identify “mutable” regions of a plasmid to prevent modifications of non-coding sequence
    3. Converted all Stealth output processing scripts to command-line programs
    4. Drafted outline of the total pipeline including inputs, outputs, and run options

  1. Wet Lab
    1. Serial dilutions of pSHDY and transformation into M. aeruginosa
    2. Conjugation with pSPIN delivery of E. coli EcGT2 and M. aeruginosa
  2. Interlab Study
    1. Repeat Interlab (Used wrong plate previous run):
      1. Switched to Experiment 2 due to lack of Experiment 3 devices
      2. Transformation, Inoculation, Plate Read
    2. Submitted data to iGEM HQ
  3. Dry Lab
    1. Worked closely with the software team to prepare Chameleon for modifying non-overlapping protein coding regions of pSPDY.
    2. Focused on revising our thesis.
  4. Sequencing
    1. Looked at the results of previous sequencing and tried to differentiate the DNA between species
  5. Software
    1. Decided on GenBank record and FastA compatibility
    2. Finished parsing mutable CDS regions from a plasmid
    3. Test ran modules on pSPDY
  6. Outreach
    1. Reaching out to local schools and Watsonville Wetlands Watch
  7. Thesis
    1. Divided the writing task portions to be written and updated

  1. Thesis
    1. Updated and completed thesis
  2. Wet Lab
    1. Selection of transconjugants
    2. Rehydrated primers for new plasmid
    3. Ran PCR on pSPDY
  3. Sequencing
    1. Created new DNA library
  4. Software
    1. Modular testing of software pipeline, continued development
    2. Brainstorm module-to-module integration to begin forming complete pipeline
    3. Drafted project plan towards final submission

September

  1. Wet Lab
    1. Performed natural transformation
    2. Sequencing:
      1. Fragments small -> Made clean DNA library and prepped
      2. Reloaded flow cell
    3. PCR and golden gate of pSPDY
  2. Dry Lab
    1. Worked closely with the software team to use Chameleon on pSPDY
    2. Designed gene blocks for synthesizing modified pSPDY with golden gate
    3. Order flagged primers and gene blocks from IDT for synthesizing modified pSPDY.
  3. Wiki
    1. Updating the notebook
      1. Caught wiki up with back-log entries
    2. Writing attributions and editing project descriptions
    3. Drafted team logo and wiki colorscheme
  4. Software
    1. Developed Stealth output parsing algorithm to screen global underrepresented motifs down to workable level.
    2. Applied pipeline modules to the pSHDY plasmid file, creating fragments for IDT synthesis.
    3. Code Reformatting:
      1. Each module can act as a standalone command line interface
      2. Separate pipeline that uses all modules to seamlessly generate single output

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