Researching arrangements for travel to iGEM Jamboree
Filling out funding applications
Wiki Team
Initial writing of project description, attributions, notebooks, safety page, and human practices
Research on iGEM criteria
Developed a basic understanding of HTML and CSS to be able to design and edit the wiki moving forward.
Wiki edits
header, footer template
notebook page and project description page skeletons
Social media: started project reveal
Dry Lab Team
Developing a comprehensive range of project approaches
Collecting protocols: culturing Microcystis a. in optimized conditions for toxin production
Met with CA Department of Water Resources for project development and possible future approval/viability for deployment
Guidelines for the genetic engineering of Microcystis communities based on phage and conjugative plasmids
methods for the evaluation of success metrics (toxin concentrations, cell viability, propagation rates)
Drafted various protocols related to molecular cloning, phage assembly, various assays including HPLC protocol for studying degradation products
Coordinated meeting with Dr. Glenn Milhauser to discuss HPLC methods, usage, protocols, etc.
Wet Lab Team
Made chemicompetent e.coli cells to be used the rest of the summer
Transformed chemicompetent e.coli to check competency
Organized the lab space
Replenish commonly used reagents
Prepped, cleaned, and performed maintenance on the autoclave we will be using
Took inventory of everything in the lab
July
Wiki Team
Edited existing notebook branch to isolate notebook updates from main
Started team branch to keep updates to team page and associated styles isolated
Gathered team information and bios
Setup team page skeleton with placeholder information and pictures
Created template and added usage for future reference
Social Media:
Project teasing on instagram
Target location
Target Species
Microcystin vs microcystis nomenclature
Thanked IMPACT grant sponsor
First educational critter o'clock post- turtle
Dry Lab Team
Refined a conjugative transposon approach for knocking-out microcystin production.
Looked for shuttle vectors that have a selectable marker, replicate in E. Coli and M. aeruginosa, and conjugate in M. aeruginosa.
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.
Made protocol for Bold 3N growth media.
Met with Glenn Millhauser and Kevin Singewald about using the HPLC to measure microcystin levels very accurately
Wet Lab Team
Completed Inoue protocol for chemicompetent cells and evaluated transformation efficiency of PET28-GFP plasmid
Very low, restarted protocol from scratch
Received and started an M.aeruginosa CPCC 300 strain (UTEX LB2385) culture
Transferred from original vial to mammalian cell culture flask for increased surface area
Started a second M.aeruginosa culture using provided Bold 3N (B3N) media inoculated with 0.5ul of original M.aeruginosa culture
Fundraising
Editing the grant applications
Reaching out to potential sponsors
Social Media:
Created threads account
Stories on pinto lake visit, national slushee day
Updated bio
Second critter o'clock update- double crested cormorant
Dry Lab Team
Coordinated additional meeting with Kevin Singewald to learn how to use the HPLC.
Started drafting additional general protocols and HPLC based on personal protocols and literature
Introduction to Stealth bioinformatics tool to identify potential restriction motifs
Fully annotated pSPIN-R plasmid to determine all of the INTEGRATE components necessary for toxin disruption.
Fully annotated pSHDY plasmid to determine all of the necessary components for broad host range conjugation.
Wet Lab Team
Competent cells with high transformation efficiency
Top10 with pET28a
1st time DNA extraction / troubleshooting
Low DNA yield, contamination
Received pSHDY plasmid from Addgene (Addgene plasmid # 137661)
Created 1% M.aeruginosa inoculation from initial culture
Done to keep planktonic cell culture
Thesis Team
Creating the outline
Drafting the abstract, acknowledgements, introduction
Human Practice
Presented project to Girls in Engineering, included learning activities
Got in contact with Pinto Lake lab to research the long-term effects of our projec
Social Media
IDT shoutout
Critter o'clock
Instagram story updates
Promega shoutout
Dry Lab Team
Met with Kevin Singewald for an HPLC tutorial.
Drafted a co-culturing protocol for E. coli and M. aeruginosa.
Coordinated with the wet lab team to test parameters for co-culturing.
Designed pINTO: a conjugative plasmid capable of toxin disruption derived from pSHDY and pSPIN-R components.
Thesis Team
Editing the outline
Writing the thesis
Adding and editing figures
Wet Lab Team
Tried culturing M.aeruginosa and E.coli together
DNA extraction of microcystis for sequencing
Dry Lab Team:
Focused primarily on drafting a thesis of our project.
Tailored pINTO for expression in M. aeruginosa
Considered plasmid delivery mechanisms alternative to conjugation to bypass a helper plasmid requirement.
Assessed Stealth, a bioinformatics tool, for integration with our approach based on the horizontal propagation of our genetic construct.
Wrote several small scripts to help with Stealth output processing
Thesis Team
Reading, editing, and adding citations to the thesis
Writing personal chapters 2 and 3 for end of Summer session 1
Wet Lab Team
DNA extraction of microcystis for sequencing
Team Progress
Pivoted project approach and began brainstorming alternative directions
Considered many alternative approaches for toxin disruption, including: plasmid-mediated cell lysis, siRNA silencing, enzyme degradation, and insertional mutagenesis.
All team members met and discussed the pros and cons of each proposed project path
Ultimately, the team decided to pursue insertional mutagenesis after we met with Diego Gelsinger from Colombia University.
Human Practices
Met with Diego Gelsinger from Columbia University for advice on expression of CAST (CRISPR-associated transposons) systems and conjugation in prokaryotes.
Prepared for iGEM meetup
Wet Lab
DNA extraction of Microcystis
Prepared for creation of a DNA library for nanopore sequencing
Received materials from Diego Gelsinger, including: pSPIN, pSPIN-R, and EcGT2 (conjugative, auxotrophic E. coli).
Interlab Study
Transformed DH5a cells with 8 provided interlab devices
Interlab Calibration - measuring fluorescence
Used materials in measurement kit to complete serial dilution on 96 well plate; read @ different wavelengths
Results:
Didn't use a opaque plate, need to redo experiment
Accidental exposure of light sensitive reagents during transfer between two labs
August
Social Media
Promega Shoutout
iGEM UCSC Meet-Up promotion / advertising
Wet Lab
Transformation of pSPIN and pSPIN-R into TOP10 cells
pSHDY transformation into M. aeruginosa
Results:
Transformation was successful, hypothesize that DNA might be getting methylated
Sequencing
Created DNA library
Loaded minION and ran it, stopped running within 24 hrs due to weekend power outage
Interlab Study
Repeated interlab experiment 3
DH5a transformation
Inoculation & Serial Dilutions
Talked to Rebecca DuBois' lab for initial plate (wrong plate)
Plate measurements
Dry Lab
Drafted protocols for end-point conjugation between EcGT2 E. coli and M. aeruginosa based on Diego Gelsinger's Nature protocols preprint.
Redesigned a smaller version of pINTO with hopes of improving conjugation efficiency.
Considered more foundational contributions to the iGEM community, like a software pipeline (Chameleon) that tailors plasmids to the RM profile of a specific bacteria.
Software
Started development of a software pipeline centered around the Stealth program titled Chameleon
Began converting previously written scripts into dedicated programs
Outreach
Prepared for and hosted iGEM team meetup with Washington Highschool
Wet Lab
Miniprep and serial decade dilutions of pSHDY, transforming into M. aeruginosa
Dry Lab
Designed a broad-host range, conjugative plasmid (pSPDY) that we would use to validate Chameleon.
pSPDY was derived from pSPIN and pSHDY.
Planned to order two versions of pSPDY: one that was unmodified and another that was optimized by Chameleon.
Designed gene blocks and flagged primers for synthesizing unmodified pSPDY with golden gate.
Order flagged primers and gene blocks from IDT for synthesizing the unmodified pSPDY.
Designed pSPDY with a “miniT” so that it was still capable of targeted gene disruption with the addition of INTEGRATE.
Designed experiments for validating Chameleon through natural transformation and conjugation of pSPDY.
Sequencing
Contacted Brady and had assistance on loading the minion
Learned how to properly empty waste port
Learned how to wash and reload flow cell mid run
Ran the minION again
Software
Explored alternative approaches to pattern constraint module in order to enhance cohesion between codon optimization and pattern avoidance activities.
Brainstormed method to identify “mutable” regions of a plasmid to prevent modifications of non-coding sequence
Converted all Stealth output processing scripts to command-line programs
Drafted outline of the total pipeline including inputs, outputs, and run options
Wet Lab
Serial dilutions of pSHDY and transformation into M. aeruginosa
Conjugation with pSPIN delivery of E. coli EcGT2 and M. aeruginosa
Interlab Study
Repeat Interlab (Used wrong plate previous run):
Switched to Experiment 2 due to lack of Experiment 3 devices
Transformation, Inoculation, Plate Read
Submitted data to iGEM HQ
Dry Lab
Worked closely with the software team to prepare Chameleon for modifying non-overlapping protein coding regions of pSPDY.
Focused on revising our thesis.
Sequencing
Looked at the results of previous sequencing and tried to differentiate the DNA between species
Software
Decided on GenBank record and FastA compatibility
Finished parsing mutable CDS regions from a plasmid
Test ran modules on pSPDY
Outreach
Reaching out to local schools and Watsonville Wetlands Watch
Thesis
Divided the writing task portions to be written and updated
Thesis
Updated and completed thesis
Wet Lab
Selection of transconjugants
Rehydrated primers for new plasmid
Ran PCR on pSPDY
Sequencing
Created new DNA library
Software
Modular testing of software pipeline, continued development
Brainstorm module-to-module integration to begin forming complete pipeline
Drafted project plan towards final submission
September
Wet Lab
Performed natural transformation
Sequencing:
Fragments small -> Made clean DNA library and prepped
Reloaded flow cell
PCR and golden gate of pSPDY
Dry Lab
Worked closely with the software team to use Chameleon on pSPDY
Designed gene blocks for synthesizing modified pSPDY with golden gate
Order flagged primers and gene blocks from IDT for synthesizing modified pSPDY.
Wiki
Updating the notebook
Caught wiki up with back-log entries
Writing attributions and editing project descriptions
Drafted team logo and wiki colorscheme
Software
Developed Stealth output parsing algorithm to screen global underrepresented motifs down to workable level.
Applied pipeline modules to the pSHDY plasmid file, creating fragments for IDT synthesis.
Code Reformatting:
Each module can act as a standalone command line interface
Separate pipeline that uses all modules to seamlessly generate single output