NOTEBOOK

Education graphic.

Notebook

June

  1. Fundraising/Planning Team
    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 M. Aeruginosa 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
    6. Took inventory of lab reagents and materials

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 biographies
    4. Setup team page skeleton with placeholder information and pictures
      1. Created template and added usage for future reference
  2. Social Media Team
    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
  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
  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 Team
    1. Edited the grant applications
    2. Reached out to potential sponsors
  2. Social Media Team
    1. IDT and Promega shoutout on social media
    2. Social media story updates
    3. Updated bio
    4. Critter o'clock story updates
  3. Dry Lab Team
    1. Coordinated additional meeting with Kevin Singewald to learn how to use the HPLC
    2. Drafted additional general protocols and HPLC based on personal protocols and literature
    3. Introduction to Stealth bioinformatics tool to identify potential restriction motifs
    4. Annotated pSPIN-R plasmid to determine all of the INTEGRATE components necessary for toxin disruption
    5. Annotated pSHDY plasmid to determine all of the necessary components for broad host range conjugation
    6. Started work on the Chameleon project
  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. Human Practices Team
    1. Presented to and completed learning activities with Girls in Engineering
    2. Began reaching out to Pinto Lake authorities to identify researchers able to speak to us regarding the long-term considerations of our project

  1. Social Media
    1. IDT shoutout
    2. Critter o'clock post
    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 and troubleshooting
    4. Designed early iteration of plasmid: a conjugative plasmid capable of toxin disruption
  3. Wet Lab Team
    1. Extracted M. aeruginosa DNA for sequencing

  1. Dry Lab Team
    1. Considered plasmid delivery mechanisms alternative to conjugation to bypass a helper plasmid requirement
    2. Assessed Stealth for integration with our approach based on the horizontal propagation of our genetic construct
    3. Wrote several small scripts to help with Stealth output processing
    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. Worked on streamlining use of Stealth via software pipeline
  2. Wet Lab Team
    1. Reattempted DNA extraction of M. aeruginosa DNA for sequencing

  1. Human Practices Team
    1. Met with Diego Gelsinger from Columbia University for advice on expression of CAST (CRISPR-associated transposons) systems and conjugation in prokaryotes.
  2. Dry Lab Team
    1. Considered alternative approaches for toxin disruption including:
      1. Plasmid-mediated cell lysis, siRNA silencing, enzyme degradation, and insertional mutagenesis
      2. Decided to pursue insertional mutagenesis after meeting with Diego Gelsinger from Columbia University
  3. Software Team
    1. Began development of a powerful software pipeline in order to enhance the potential and accessibility of the Stealth algorithm - Chameleon
    2. Began converting previously written scripts into dynamic programs
  4. Wet Lab Team
    1. Interlab Calibration - measuring fluorescence
      1. Did not use opaque plate, necessitating redoing experiment
    2. Reattempted DNA extraction of M. aeruginosa DNA for sequencing
    3. Prepared for nanopore sequencing and creation of a DNA library.
  5. Outreach Team
    1. Prepared for iGEM meetup
  6. Interlab Team
    1. Transformed DH5a cells with 8 provided interlab devices

August

  1. Social Media Team
    1. Promega Shoutout
    2. iGEM UCSC Meet-Up promotion / advertising
    3. Critter o’clock
  2. Wet Lab Team
    1. Transformation of pSPIN and pSPIN-R into TOP10 cells
    2. Successful pSHDY transformation into M. aeruginosa
  3. Sequencing Team
    1. Created DNA library
    2. Loaded minION and ran it, stopped running due to power outage
  4. Interlab Team
    1. Repeated interlab experiment 3
      1. DH5a transformation
      2. Inoculation & Serial Dilutions
      3. Plate measurements
  5. Dry Lab Team
    1. Drafted protocols for end-point conjugation between EcGT2 E. coli and M. aeruginosa based on Diego Gelsinger's Nature protocols preprint
    2. 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 Team
    1. Started development of a software pipeline centered around the Stealth program - Chameleon
  7. Outreach Team
    1. Presented at the iGEM meet up

  1. Wet Lab Team
    1. Miniprep and serial decade dilutions of pSHDY, transforming into M. aeruginosa
  2. Dry Lab Team
    1. Designed a broad-host range, conjugative plasmid (pSPDY) that we would use to validate Chameleon.
      1. 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 experiments for validating Chameleon through natural transformation and conjugation of pSPDY
  3. Sequencing Team
    1. Contacted Brandy McNulty (Miga Lab) 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 for nanopore sequencing
  4. Software Team
    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 Team
    1. Serial dilutions of pSHDY and transformation into M. aeruginosa
    2. Conjugation with pSPIN delivery of E. coli EcGT2 and M. aeruginosa
  2. Interlab Team
    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
  3. Dry Lab Team
    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 Team
    1. Looked at the results of previous sequencing and tried to differentiate the DNA between species
  5. Software Team
    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 Team
    1. Reaching out to local schools and Watsonville Wetlands Watch
    2. Met with the Santa Cruz County Water Quality Lab

  • Wet Lab Team
    1. Selection of transconjugants
    2. Rehydrated primers for new plasmid
    3. Ran PCR on pSPDY
  • Sequencing Team
    1. Created new DNA library
  • Software Team
    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 Team
      1. Performed natural transformation
      2. Sequencing:
        1. Fragments small -> Made clean DNA library and prepped
        2. Reloaded flow cell
      3. PCR and Golden Gate Assemble of pSPDY
    2. Dry Lab Team
      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
      4. Designed and edited promotional video
    3. Wiki Team
      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 Team
      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

    1. Software Team
      1. Command line interfaces reformatted to use new code structure
      2. Drafted plan to integrate Stealth software into iGEM submission
      3. Started work towards a Chameleon python package
        1. Simple pip installation and command line interface use
    2. Wiki Team
      1. Began writing and organization for wiki writing. Assigned members to different pages of the wiki
    3. Wet Lab Team
      1. Transformations of Golden Gate product pSPDY unmodified
      2. Golden Gate troubleshooting
      3. Transferred microcystis cultures into new BG11 for cultivation

    1. Software Team
      1. Adopted a more modular package layout for pipeline
      2. Integrated Stealth into package
      3. Started building final pipeline
    2. Wet Lab Team
      1. Troubleshooting Golden Gate Assembly by running Colony PCR on suspected GG product
      2. Drafting new ways to perform natural transformation in a liquid culture rather than a solid culture
    3. Outreach Team
      1. Gave a tour of our lab and a run down of our project to the Mayor of Santa Cruz, Fred Keely

    1. Software Team
      1. Renamed all submodules and reformatted code
      2. Created a Stealth sub-class to encapsulate Stealth output into an object
      3. Created a new codon analyzer
      4. Published a working version of the pipeline
    2. Wet Lab Team
      1. Gene amplification of the Stealthed fragments for pSPDY modified
      2. Golden Gate Assembly of pSPDY, still having some problems verifying unmodified pSPDY
      3. Ethanol precipitation of pSPDY

    October

    1. Software Team
      1. Added code documentation
    2. Wet Lab Team
      1. Colony PCR of unmodified pSPDY transformed in E. coli
      2. PCR amplification of gene fragments for modified pSPDY
      3. Miniprep and sequencing of unmodified pSPDY
    3. Outreach Team
      1. Presented info presentation on our project and performed experiments with Mission Hill Middle School

    1. Wiki Team
      1. Finalization of wiki
    2. Wet Lab Team
      1. Colony PCR was performed in order to verify the magnitude of success of our Golden Gate assembly
      2. Performed and troubleshooted Golden Gate Assembly of modified pSPDY including gel electrophoresis
      3. Electroporation of unmodified pSPDY into M. aeruginosa
      4. Gene fragment amplification of new iterations of fragments