Notebook

This serves as a chronological record of our team's progress throughout the season. It contains weekly summaries of our activities, experiments, discussions, and decisions.

Week 25

June 9 - June 13

We honed the project plan for this year's iGEM project and contacted researchers/professors (Petroutsos lab) to get feedback and help with materials for our project.

Strains such as starch-filled and WT with and without cell walls and promoters such as salt-induced and heat shock were also chosen after feedback. Practical/Industrial applications for our project were discussed.

We also had some lectures on BioBricks, biofuels and biosensors (Forster, Danielson). There was also a course on waste management where we were taught the correct disposal of chemical, biological and general lab waste.

We started planning the Swedish iGEM Conference which we are hosting this year. We will have teams from Stockholm, Lund and Gothenburg coming to Uppsala to discuss ideas and connect.

The team was divided into subgroups responsible for the Wiki, Swedish Meet, Team Building, Co-operations, PR, Sponsorship, Documentation, Graphical Design, Video, and Human Practice. Lab groups were made for Induction, Flocculation Genes, Pair Flocculation, Modelling, and Assay Prep which will work in parallel during the summer.

Week 26

June 16 - June 19

A shorter week due to Midsommar

This week we designed the Z17/18 cassettes for transformation into Chlamydomonas including codon optimization and intron insertion. After which we designed primers for Gibson assembly into the pLM005 plasmid, and validated them in Benching.

We were given an in-person tour of the Petroutsos lab to familiarize the team with available equipment and space and claimed Course Lab 5 for future cultivation work. After seeing the resources available for us we developed written protocols to familiarise ourselves with working with Chlamydomonas (sedimentation, flocculation, growth curves).

Work was also conducted in E. coli, as we began experimenting with transforming Z17 and Z18 into E. coli, including low-volume DNA transformations in DH5α strains. We purified our pLM005 plasmid from the E. coli colonies we received from the Petroutsos Lab.

The Human Practices group continued their research and documentation tasks and performed further outreach to companies. The Team Building group finalized the date and scope for the Swedish Meet and the Social Media group drafted a production plan and script for the promotional video, designed multiple logo versions and researched vendors for team apparel.

Week 27

June 23 - June 27

This week we focused on preparing key genetic parts, running transformations, and advancing our algae experiments. We completed multiple E. coli transformations with Z17/Z18, tested transformation efficiency, and made more glycerol stock. We also ran digests and gels (including pLM005), performed minipreps, and prepared plasmids for electroporation into Chlamydomonas. We finalised the design of the FLO1 plasmid, which needed to be split into three parts for ordering; thereby requiring us to design it for reassembly using Gibson assembly, designed and ordered primers from IDT, and had a meeting to refine the assembly strategy.

On the algae side, we started to collect data to plot growth curves for our Chlamydomonas strains, started additional cultures for nitrate and cell-wall-less strains, and ran experiments to attempt to quantify the sedimentation rate.

Additional work included planning Human Practices, writing a project abstract for the Maastricht journal, designing PCR primers for the GP1 gene, and continuing our team's outreach materials (photos, Instagram presentation template, logo work).

Week 28

June 30 - July 4

What we did this week:
  • Refreshed algal cultures and prepared them for electroporation
  • Identified contamination in TAP media and planned improved storage practices
  • Took final measurements for the growth curve experiment and plotted results in RStudio
  • Prepared and autoclaved fresh TAP-agar, poured plates for Chlamydomonas
  • Performed multiple electroporations of CC-125, refining the protocol with 1 mm cuvettes in EBC buffer
  • Plated transformed Chlamydomonas cultures onto agar plates
  • Maintained overnight cultures of pLM005 for plasmid work
  • Completed digestions of pLM005 with XbaI, BamHI, and HpaI, with successful agarose gel analysis and fragment purification
  • Purified pLM005 and mRFP1 plasmids
  • Ran SDS-PAGE gels to assess Z17 purification
  • Conducted colony PCR (cPCR) and analysed products via gel electrophoresis
  • Researched the Fus1-Mar1 receptor pair as an alternative flocculation strategy
  • Held gene design and Human Practices meetings to align subteam goals
  • Sent our abstract to Maastricht for submission
  • Shared lab progress by posting presentations on Instagram to engage the public and document our journey

Reflections
This week was a true exercise in problem-solving, persistence, and teamwork. We faced contamination setbacks but adapted quickly, used repetition to master electroporation, and strengthened our molecular workflows with careful documentation and analysis. Our meetings helped keep everyone aligned, and our social media updates reminded us of the importance of sharing science openly. Overall, we kept momentum despite challenges, demonstrating the resilience and collaboration at the heart of our iGEM project.

Week 29

July 7 - July 11
Weekly Progress
  • Z17-GFP Protein Expression:
    • Performed IPTG-induced expression in BL21 cells.
    • Several SDS-PAGE gels were run, and no strong Z17 band was detected initially.
    • Explored urea-based extraction for improved solubility.
    • Conducted mini-preps and digestions of Z17-GFP and Z18 plasmids in various E. coli strains (DH5α, JM109).
  • Sedimentation & Flocculation Assays:
    • Carried out tests on wild-type and starch-producing strains.
    • Began data processing and analysis.
    • Investigated the effectiveness of cell counting for quantification.
  • PCR & Cloning:
    • Designed and ordered primers for GP1 and the expression cassette.
    • Troubleshot PCR with longer extension times; positive controls worked.
    • Ran agarose gels for PCR analysis.
    • Prepared for colony PCR for GP1.
  • Strain Work & Transformation:
    • Initiated new BL21 cultures for protein comparison.
    • Electroporation plans began for membrane-deficient Chlamydomonas strains.
    • Wrote draft electroporation protocol and started corresponding salt stress experiments.
  • Human Practices & Documentation
    • Planned Human Practices activities.
    • Began finalising some protocols for addition to the wiki
    • Published presentations and social media stories, including a new Facebook profile picture and poster draft.
  • Media & Protocols
    • Prepared fresh TAP media and agar for Chlamydomonas cultures.
    • Continued adjusting lab protocols and started searching for improved membrane-strand protocols.
  • Research & Investigations
    • Investigated the salt promoter (optimal concentration for stress experiments).
    • Researched the unknown anchor gene and reviewed FLO1 assembly protocols.
  • Team & Organisational Updates
    • Held an association meeting and elected new board members.
    • Learned to use the microscope at EBC, capturing images for documentation

Week 30

July 14 - July 18
Weekly Progress
  • Z17/Z18 Protein Expression & Analysis:
    • Performed IPTG induction of Z17-GFP in E. coli BL21.
    • Conducted multiple SDS-PAGE gels on induced and uninduced Z17/Z18 samples, including urea-based extractions to improve solubility.
    • Began troubleshooting protease digestion and TEV cleavage of Z17, including plans for size-exclusion chromatography.
    • Prepared glycerol stocks for Z17 and Z18 strains and performed urea extractions under various conditions.
  • PCR, Cloning & Assembly:
    • Ran multiple PCRs, including FLO1, Z17 Gibson cassette, and Chlamydomonas lysate templates optimized for high GC content.
    • Successfully visualized PCR bands and continued troubleshooting where necessary.
    • Performed digestion and ligation of Z17-GFP/Z18 fragments (XbaI, BamHI), with ongoing troubleshooting of pLM005 ligation using different T4 ligase kits.
    • Ran agarose gels for verification and troubleshooting of PCR, digestion, and Gibson assembly results.
  • Transformation & Strain Work:
    • Performed multiple electroporations of wild-type and membrane-deficient Chlamydomonas reinhardtii at different voltages, followed by plating and recovery.
    • Refreshed Chlamydomonas cultures in preparation for electroporation and subsequent experiments.
    • Transformed Z18 plasmids into E. coli and monitored overnight cultures and restreaks.
    • Prepared cultures for protein overexpression and performed mini-preps on transformed plasmids.
  • Sedimentation & Salt Tolerance Assays:
    • Continued salt tolerance experiments, including day 5 measurements.
    • Analyzed flocculation and sedimentation results, preparing statistical summaries for wiki documentation.
  • Human Practices & Documentation
    • Drafted plans for future Human Practices activities.
    • Continued extensive wiki work, including uploading protocols, building protocol pages, and updating team member contributions.
    • Merged updates into the main branch and prepared summary documents.
    • Created social media content, including Instagram posts, and prepared poster drafts.
  • Media & Protocols
    • Prepared and refreshed TAP media and agar plates for Chlamydomonas work.
    • Documented and updated electroporation and transformation protocols.
    • Started developing and optimizing new membrane-deficient strain electroporation protocols.
    • Began planning for TEV cleavage workflows and protocol integration into downstream analysis pipelines.
  • Research & Investigations
    • Investigated unknown components related to the FLO1 assembly.
    • Continued exploring optimal salt promoter concentrations for stress response experiments.
    • Reviewed and optimized PCR strategies, including high GC content templates and primer design considerations.
  • Team & Organisational Updates
    • Worked on team coordination, teambuilding, and planning for future outreach activities.
    • Captured microscopy images of Chlamydomonas for documentation and analysis.
    • Focused on troubleshooting key steps across cloning, protein expression, and transformation workflows to improve reproducibility and yield.

Week 31

July 21 - July 25
Weekly Progress
  • Z17/Z18 Protein Expression & Analysis:
    • Performed IPTG induction of Z17-GFP and Z18 from overnight cultures and recent transformations.
    • Prepared buffers and carried out purification of Z17 protein using Ni2+ affinity chromatography with two different protocols.
    • Analyzed protein purification results via SDS-PAGE, with further gels planned for follow-up validation.
    • Continued exploring protein refolding from urea extractions to improve yield and solubility.
  • PCR, Cloning & Assembly:
    • Ran PCR for Z18 cassette and GP1 constructs, followed by gel electrophoresis for verification.
    • Digested pLM005 and Z17-GFP plasmids using multiple restriction enzymes (HpaI, BamHI, XbaI) and performed ligation reactions with Z18 inserts.
    • Conducted Gibson assembly for FLO1 constructs with the salt promoter and the “China” sequence, followed by transformation.
    • Troubleshot ligation issues involving Z18 and pET plasmids, including control reactions and alternative strategies.
  • Transformation & Strain Work:
    • Completed transformations with ligation and Gibson products.
    • Restreaked all four Chlamydomonas strains for continued use in downstream experiments.
    • Maintained growth curves across multiple strains and performed cell counts to support biomass and flocculation analyses.
  • Sedimentation & Salt Tolerance Assays:
    • Planned and prepared for large-scale sedimentation and flocculation experiments with Chlamydomonas.
    • Conducted first-round assays and repeated them with improved timelapse imaging to track sedimentation dynamics.
    • Continued refining experimental conditions for quantitative comparisons between strains.
  • Human Practices & Documentation
    • Continued filming and documentation of experiments for communication and Human Practices outputs.
    • Took updated team and lab photos for wiki and outreach purposes.
    • Maintained detailed records of cloning, transformation, and purification protocols for future reproducibility.
  • Media & Protocols
    • Prepared necessary buffers and reagents for protein purification and column chromatography.
    • Optimized purification workflows and planned improvements based on early results.
    • Gel-purified digested plasmids to improve downstream assembly and ligation efficiency.
  • Research & Investigations
    • Investigated protein solubility challenges by testing alternative purification strategies and refolding approaches.
    • Evaluated performance of Gibson assembly and ligation approaches, troubleshooting based on gel and transformation outcomes.
    • Collected and analyzed growth data for different Chlamydomonas strains to better understand biomass dynamics in flocculation assays.
  • Team & Organisational Updates
    • Coordinated planning for upcoming large-scale experiments and data collection phases.
    • Captured new laboratory media (photos and videos) for future presentations, wiki content, and outreach.
    • Continued collaborative troubleshooting sessions to resolve ongoing cloning and purification challenges.