June

  1. Wiki
    2. - Brainstormed organization and illustrated design of wiki layout on whiteboard in dry lab
    - Learned and practiced languages for wiki design using W3schools and other resources as needed
  2. Human Outreach
    3. To date: have met with endocrinologists, nephrologists, medical doctors, directors of health centers, and diabetes support program coordinators.
    Dr. Rayhan Lal (5/14/22), Doctors Without Borders (5/17/22), Dr. Patricia Wu (6/22/22), Dr. John Demko (6/23/22), Dr. Sunder Mudaliar (6/24/22), Dr. Lawrence DeGhetaldi (6/29/22), Dr. Matt Sparke (6/30/22) provided thorough insight for current Type II Diabetes treatment that shaped project development preparation
  3. Host Organisms
    4. Yeast:
    - Researched yeast and microbial strains, bioencapsulation methods, half-life extenders, Ex-4 functionality, plasmids for select strains
    - Looked into strains and backbones for our experiment
    - Met with Prof. Rohinton Kamakaka at UCSC for yeast protein synthesis and received yeast strain and plasmid backbone from his lab
    Microalgae:
    - Completed a microalgae protocol, timeline, list of materials, and line itemized cost list
  4. Plasmid Design
    5. - Constructed Ex-4 insert in pET28:GFP and yeast backone plasmids
    - Ordered plasmid parts from IDT

July

  1. Wiki
    2. - Coded in IDE for wiki pages
    - Brainstormed for theme and aesthetics
  2. Human Outreach
    3. - Admin team: sent thank you emails for giving month
    - Prepared investment portfolio; meeting with Paul L. Koch, PBSci Dean at UCSC
    - connected with iGEM teams for collaboration (Chihuahua, Mexico and Stoneybrook, New York)
    - Met with Dr. Somen Nandi (7/5/22), from UC Davis, for project development input and Ted Malpass (7/6/22), from UCSC, who has Type 1 Diabetes and works at a nonprofit for accesible medical treatment
  3. Host Organisms
    4. Yeast:
    - Finalized new plasmid design for available materials and ordered insert gene block
    - Checked lab stock, developed protocols, timelined for experimental work, and conceptualized bioencapsulation methods
    Microalgae:
    - Determined feasibility of microalgae strains from research: comparison of strains (chose Chlamydomonas reinhardtii), methodology of transformation (chose AMT), and target location (chose nuclear genome)
    - Prepared protocols for various selections of the above criteria
  4. Plasmid Design
    5. - Gave Golden Gate Assembly (GGA) lectures to establish a collective understanding within the team
    - Finished GGA design and ordered primers
    - Drafted general protocols for chemicompetent cell transformation, plasmid miniprep, colony touchdown PCR, protein purification using a HisPur™ Cobalt Purification Kit (ThermoFisher), and HisPur Cobalt column sterilization for reuse
    - Discussed GLP-1 R design attributes, size, cost, human vs. rat, post translational modifications, understanding and verifying GLP-1 R interaction to Ex-4

  1. Human Outreach
    2. - Finalized iGEM project track selection (7/18/22)
    - Met with Alexander Wolf, Baskin Engineering Dean at UCSC, for story approval
  2. Host Organisms
    3. Yeast:
    - Prepared LB and SOC media
    - Lab preparation for E. coli and S. cerevisiae transformations, including taking inventory
  3. Plasmid Design
    4. - Nicholas Lorig-Roach, member of DuBois Lab at UCSC, provided cells with His-TEV protease and cells for producing His-TEV protease
    - Cells were cultured, given IPTG, and pelleted for cell lysing
    - Lysis performed using glass beads and yield extracted through Histidine protein purification protocol by Gravity
    - Given His-TEV protease cells were used for protein purification practice
    - Results were inconclusive

  1. Wiki
    2. - Implemented 'sticky' navbar and fancy hyperlink underlines
  2. Human Outreach
    3. - Completed first set of website commits of various elements: sticky navbar, fancy hyperlinks mk.1
    - Used bootstrap scrollspy with a benzene ring design
    - Formatted footer on wiki page
    - Chose team name
  3. Computational Modeling
    4. - Start of computational modeling
    - Met with SBU for mathematical protein modeling and explained basics of MATLAB
  4. Host Organisms
    5. Yeast:
    - Practiced E. coli transformation and plasmid miniprep to test plasmid
    - Prepared Gibson Assembly protocol
    - Performed E. coli transformation for plasmid part cloning (materials provided by Rohinton Kamakaka lab: plasmid gal1 promoter, terminator, LEU- non integrative, G418 LEU- integrative, Hyg TRP1 integrative)

  1. Human Outreach
    2. - Created google form for weekly subgroup updates
    - Researched 3D modeling display, but shifted focus to more crucial elements for the website design
    - Reformatted google form for weekly submissions
  2. Computational Modeling
    3. - Introduction to using SimBiology
    - Researched how ligand-receptor binding functions in depth and how Ex-4 maintains homeostasis in the body
  3. Host Organisms
    4. Yeast:
    - Prepared LB and SOC media
    - Lab preparation for E. coli and S. cerevisiae transformations, including taking inventory
  4. Interlab
    5. - Completed Calibration Experiment
    - Completed Experiment 1

August


  1. Wiki
    2. - Worked on creating and implementing SVGs into the website: included a loading screen and worked on resizing SVG elements for compatibility with end user's screen space
  2. Computational Modeling
    3. - Researched and worked on schematics for ligand-receptor binding, GLP-1 R binding downstream effects, and recombinant protein production rates in both hosts
    - Created a schematic for downstream insulin production in pancreatic beta cells, a mathematical model for ligand-receptor binding, and a graph for pharmacological effect vs. dosage
    - Members of the modeling team began familiarizing themselves with the software and conducted background research.
  3. Human Outreach
    4. - Correspondence with Karianne Terry (8/5/22) for safety guidelines
    - IRB research to ensure project safety
    - Emailed various affected communities
    - Met with Dr. Tércio Rocha (8/5/22) who provided perspective on the health care system in Brazil
  4. Host Organisms
    5. Yeast:
    - Innoculated LEU+ yeast strain (ROY6783) and LEU- yeast strain (JRY4012) in 10ml YPD media, incubated overnight at 30 Celsius on shaker, lid loose
    - Prepared selection plates for S. cerevisiae transformation
    - Performed cleavage using manufacture protocol for Thermofisher FastDigest NotI Restriction Enzyme
    - Yielded unsuccessful DNA precipitation
    - Performed yeast transformation with miniprep plasmid product using the ZYMO Yeast Transformation Kit
    - Transformed non-integrative plasmids (1-1, 1-3) in LEU- yeast strain (JRY4012)
    - Transformed G418 LEU2 integrative plasmids (2-1, 2-2) in LEU+ yeast strain (ROY6783), plated on G418 antibiotic plates
    - Repeated transformation for successful enzyme digest
    - Transformed Hyg-TRP1 integrative plasmids (3-2, 3-4) in LEU+ yeast strain (ROY6783), plated on Hygromycin B antibiotic plates
    - Replica plating
  5. Interlab
    6. - Started Experiment 2

  1. Wiki
    2. - Learned microformatting for SEO for viewers to readily find our website
  2. Host Organisms
    3. Yeast:
    - Obtained LEU2 Cas9 plasmid from Prof. Rohinton Kamakaka
    - Innoculated S. cerevisiae strains (LEU+ ROY6783 and LEU- JRY4012) in 10ml culture 25℃ overnight
    - Transformed S. cerevisiae strains
    - Made protein expression media
    - Replica plating
    - Ran gel of colony PCR product from S. cerevisiae transformation
    E. coli:
    - Transformed pET28:GFP into TOP10
    - Inverse PCR, gel, clear band showing linearized backbone
    - Dpn1 + heat treatment on some of the iPCR rxns, Golden Gate Assembly, pET28:Ex-4 transformed into DH10B (8/9/22)
    - Transformed more pET28:Ex-4 into DH10B
    - Inoculated DH10B, performed colony PCR, index plates, and gels: had successful yield of Ex-4 bands
    - Miniprep on innoculated pET28:Ex-4, sent for Sanger Sequencing
    - Received successful sequencing results
  3. Interlab
    4. - Completed Experiment 2

  1. Wiki
    2. - Reorganized website layout and delegated pages with iGEM criteria to subgroups
    - Wiki development: touched up jump to top button, fixed banner carousel at top of page, started implementing the loading screen
  2. Host Organisms
    3. Yeast:
    - Innoculated S. cerevisiae strains (LEU+ ROY6783 and LEU- JRY4012) in 5mL culture for protein purification
    - Plasmid miniprep
    - PCR
    - Genomic DNA isolation
    - Performed gel electrophoresis on Genomic DNA to check for integration of Ex-4 at homology sites, protein purification, and sent for Sanger Sequencing of Genomic DNA to check for integration of Ex-4 at homology sites
    - Lab preparation for E. coli and S. cerevisiae transformations, including taking inventory
    E. coli:
    - Transformed pET28:Ex-4 into BL21, innoculated transformants, index plate, and colony PCR/gel
    - Ran an agarose gel on PCR samples of transformed pET28:Ex-4 in BL21 and successful yield of Ex-4 bands
    - Miniprep on innoculated pET28:Ex-4 and sent for Sanger Sequencing
    - Received confirmation from sequencing that pET28:Ex-4 was successfully transfected into the E. coli

  1. Wiki
    2. - Notebook page development and testing; developed an SVG design for the page

  1. Wiki
    2. - Continued coding and testing various elements for notebook page (buttons and collapsibles)
    - Various redesigns of notebook page format
  2. Human Outreach
    3. - Met with NGO founder Annette Oshin (9/1/22)
    - promo video submitted (9/2/22)
  3. Computational Modeling
    4. - Devised a detailed schedule for modeling work due by wiki freeze
    - Identified literature values for protein production rate constants, derived formulas used in the competitive binding model, coded both models
  4. Host Organisms
    5. Yeast:
    - S. cerevisiae culture innoculation for second round of protein purification

September


  1. Wiki
    2. - A hexagonal grid using flexboxes was achieved for the Team/Attributions page
  2. Computational Modeling
    3. - Development of competitive binding model: researched parameter values, derived fractional occupancy equation, and coded the model in Python
    - Eric Malekos helped troubleshoot our code
  3. Human Outreach
    4. - Follow up with Dr. Rayhan Lal (9/7/22)
  4. Host Organisms
    5. Yeast:
    - Performed protein purification
    - Successful SDS-PAGE gel showing expression of His-tag Golden Gate Assembly protein from genomic DNA
    E. coli:
    - Transformed Ex-4 again into C41 E. coli since it has been in freezer for a long time
    - Used freshly purified Ex-4 for our pancreatic beta cell test, binding affinity test, and Western Blot
    - Transformation was successful and colonies were innoculated
    - Colony PCR gel was performed
    - Expected bands were shown proving that Ex-4 was successfully transfected into our protein producing C41 cells

  1. Wiki
    2. - Worked on the color pallet, structure of the nav bar, and reorganized layout of the wiki
    - The flask jinja template documentation was utilized to achieve some reorganization
    - Began organization of content for wiki pages
  2. Computational Model
    3. - Worked on the dose-effect model: Searched for parameter values, derived Emax equation, and coded the model in Python
    - Eric Malekos viewed our graphs and provided feedback for optimization
    - Slight changes were made to the previous competitive binding model (developed week of 9/5/2022) to better format information
  3. Human Outreach
    4. - Safety form submitted (9/16/22)
  4. Host Organisms
    5. E. coli:
    - Performed protein purification using an FPLC machine, but to our dismay, the FPLC needs maintenance
    - Performed SDS-PAGE to verify if GLP-1R was purified, despite our faulty equipment
    - Our SDS-PAGE test to confirm the FPLC purification displayed that Ex-4 was not purified
    - Performed IMAC protein purification by gravity
    - Ex-4 was grown in LB/Kan and innoculated with IPTG to prepare for protein purification

  1. Wiki
    2. - Made miscellaneous shifts in webpage architecture to make implementation for page content more intuitive
  2. Computational Model
    3. - Worked on the protein production model: Searched for parameter values, drew up model in SimBiology, edited reaction equations for result concentration diagram
    - Drafted a plan for modeling work to complete during Fall quarter
  3. Human Outreach
    4. - Parts page submitted (9/22/22)
  4. Host Organisms
    5. Yeast:
    - Innoculated Int-TRP and Int-LEU in 5mL of YPD each
    - Incubated innoculated solutions at 30℃ for overnight growth
    - Innoculated each of the 5mL cultures into 500 mL of YPG respectively to activate gal1 promoter
    - Incubated innoculated solutions at 30℃ for overnight growth or until 2 OD
    E. coli:
    - Pelleted cells and stored in 4℃ until following day
    - Resuspended and lysed cells (for both C41 and BL21)
    - Performed IMAC protein purification of protein samples
    - SDS-PAGE gel of purified protein

  1. Wiki
    2. - Finalized website color scheme and most wiki page content
  2. Computational Model
    3. - Continued to look for appropriate parameters for the protein production model; difficult to find ribosome binding kinetics
    - Shifted to computational modeling wiki page development
  3. Human Outreach
    4. - Title and Abstract submitted (9/30/22)
  4. Host Organisms
    5. Yeast:
    - Pelleted cells and stored in 4℃ until following day
    - Resuspended and lysed cells (for both LEU and TRP)
    - Performed IMAC protein purification of protein samples
    - SDS-PAGE gel of purified protein

October


  1. Wiki
    2. - Integration of content
    - Created classes for consistency in page structure
    - Style redesign; added column artwork for content pages
  2. Computational Model
    3. - Finished the protein production model by circumventing ribosome binding, assuming any ribosome that can bind to mRNA will, at a rate of 1 per second
    - Sent protein production model to Stony Brook University to show how their recombinant protein would act in yeast
    - Continued to develop computational model wiki page

  1. Wiki
    2. - Full team meeting to finalize wiki content
  2. Computational Model
    3. - Finished uploading computational modeling figures
  4. Host Organisms
    5. E. coli:
    - Diafiltration (on previously purified E. coli and S. cerevisiae Ex-4) to swap elution buffer with Tris-HCL
    - Add enterokinase to those samples and incubate overnight to cut off His-tag on Ex-4
    - Packed new IMAC columns
    - Protein purification for TRP1, LEU2, BL21, and C41
    - Batch purification on enterokinase samples was unsuccessful
    - SDS-PAGE of new protein purification samples from S. cerevisiae and E. coli
    - Nothing showed up on SDS-PAGE so we ran a new SDS-PAGE with other samples from protein purification on Wednesday
    - Octet assay on previously purified E. coli Ex-4 and purchased GLP-1 R