Notebook
Notebook
May/June
MAY 1-7
- End of recruitment period
- Finalized initial project idea and approach
- Lab safety
training with Dr. Bartelle
- Established preliminary project deadline
- Compiled
pre-existing basic lab protocols from 2021 (Western Blot, C. reinhardtii transformation)
- Researched filter prototyping basics
MAY 8-14
- First official team meeting
- Researched genes of interest and began uploading sequences to
Benchling
- Troubleshot in-silico virtual assemblies of genes of interest
- “Work Sessions”
to make progress on codon-optimization of genes of interest and running Golden Gate assemblies
in-silico
- Determined goals for human practices team
MAY 15-21
- Began brainstorming water filter ideas related to hydrocyclone separation
- Purchased lab
supplies
- Troubleshot construct design
- Researched 2A peptide sequences for use in C.
reinhardtii
MAY 22-28
- Delegated human practices tasks based on previously discussed goals
- Finalized and ordered
constructs
MAY 29 - JUNE 4
- Researched MoClo kit for C. reinhardtii
- Re-ordered constructs after further
troubleshooting
- Purchased more reagents and lab supplies
- Met with team to discuss
final construct design
- Round 1 of sketching out filter designs
- Wrote out basic lab
protocols
- Collaboration meeting with QGEM
- Updated project timeline
JUNE 5-11
- Researched FRET protocol
- Started discussing collaboration with UBCO iGEM
- Initial CAD
attempts for filter design
- Wrote industrial stakeholder profiles to prepare for human practices
interviews
- Received Chlamydomonas Reinhardtii MoClo Toolkit and prepared cultures for
glycerol stocks as well as minipreps of parts in the kit needed for our project
JUNE 12-18
- Received vector to be used in our project and prepared a culture for a glycerol stock as well as a
miniprep of it First attempt at Golden Gate Assembly of plasmid containing genes of interest was
unsuccessful
- Second attempt at Golden Gate Assembly of plasmid yielded red colonies resulting
from an mScarlet reporter, which were picked and subjected to restriction digestion analysis
-
Designed variants of potential filter designs
- Safety forms
- HP stakeholder meeting with
Dr. Bruce Rittmann
- Collaboration meeting with SBU iGEM
- Wrote scientific stakeholder
profiles to prepare for human practices interviews
- Began planning Phototroph Community with
UBCO
- Collaboration meeting with iGEM Concordia
JUNE 19-25
Constructs from the second attempt at Golden Gate assembly which were confirmed by restriction
digestion analysis were subjected to an additional one-fragment Golden Gate assembly in which the
mScarlet reporter was replaced with a TaV 2A peptide, yielding the final construct
- The final
construct, named A2, underwent restriction digestion analysis in order to confirm correct assembly
- Phototrophs outreach and recruitment
- Finished safety forms
- QGEM podcast outreach
and interview requests
- Initial wiki planning and drafts
- Filter prototyping and
conceptual design sessions
JUNE 26 - JULY 2
- Our final A2 construct was additionally confirmed as correct by whole-plasmid DNA sequencing
- Tested glass bead transformation with unsequenced plasmid containing antibiotic resistance gene
- Researched assays to confirm successful transformation for final plasmid
- Wrote article for
SBU journal collaboration
- CAD designs for filter, continued
October
OCTOBER 2-8
- The FLAG blot from the last Western Blot trial was reprobed, confirming that the bands apparent on
it were background and did not result from the production of the tagged protein of interest
-
Completed 4-day testing cycle to determine arsenic uptake abilities of mutant strains
- Edited
phototroph handbook and video protocol submissions
OCTOBER 9-12
- Wiki writing
- Data analysis
- Model validation
- Podcast editing
July
JULY 3-9
- First attempt at glass bead transformation with sequenced plasmid proved unsuccessful
-
Podcast interview #1 - Dr. Stephen Brown
- Began planning modeling strategy
Defining and
finding samples of parts needed for water filter
- Met with UBCO to plan Phototroph Community
meetings for the summer
- More podcast outreach
JULY 10-16
- First attempt at transformation via electroporation with sequenced plasmid proved unsuccessful
- Researched specific parts needed to maintain algal growth in water filtration system
-
Podcast Interview #2 - Dr. Arul Varman
- Submitted IRB application for approval
JULY 17-23
- Resolved contamination issue in C. reinhardtii agar plates
- Podcast interview #3 -
Dr. Rosy
- Received IRB approval
- Modeling strategy and meeting with Dr. Andrew Marcus
- Sequencing plasmids
- Phototroph community outreach and planning
- Visited Dr.
Weiss at ASU’s AzCATI facility
- Filter prototyping: researched sedimentation strategy after
rethinking approach
JULY 24-30
- Continued attempts at transformation of C. reinhardtii with A2 plasmid via the glass bead
protocol
- Preparation for first Phototroph Community meeting
- Transcribed podcasts for
later editing
- Promotional video preparation
- Refined modeling approach and researched
existing models of similar systems
JULY 31 - AUGUST 6
- Planned bioremediation conference with iGEM Patras and iGEM Tec-CEM
- Ordered new strains of
C. reinhardtii for further testing and troubleshooting
- Moved base of operations to Dr.
Redding’s lab
- Tested transformation and material preparation
- Continued glass bead
transformation trials of C. reinhardtii and established transformation log to track
protocol changes and time required for transformant colonies to appear
- iGEM online
meetup hosted by iGEM Worldshaper-HZ
September
SEPTEMBER 4-10
- Chlamy maintenance while preparing for assays
- First two attempts of colony PCR as described
by Nouemssi et al. 2020 on successful CC-400 transformants containing A2 were unsuccessful
-
Prepared for transformations of 2A peptide test plasmids
SEPTEMBER 11-17
- First attempt at colony PCR using Promega Wizard Genomic DNA Preparation Kit followed by PCR was
successful and indicated three positive transformants
- Finished handbook and video protocol
contributions to phototroph handbook
- Miniprepped plasmids needed for 2A peptide testing
-
Began coordinating with Dr. Westerhoff’s lab for running ICP-MS on our samples
SEPTEMBER 18-24
- Completed 15 transformations of 2A peptide test plasmids for FRET testing
- Prepared cultures
of positive cPCR hits for western blotting
- A protein lysate of one of the positive cPCR hits
has prepared and a BCA assay was performed
- A Western Blot using the protein lysate from the
positive cPCR hit was performed yet bands were only apparent for the control, not the tagged proteins
of interest
SEPTEMBER 25 - OCTOBER 1
- Protein lysates were prepared from cultures of all three positive cPCR hits and a BCA assay was
performed
- A Western Blot using the protein lysate from the positive cPCR hit was performed, and
bands were apparent on the FLAG blot as well as the control blot but not the HA blot
- Another
Western Blot was performed using the same protein lysate samples with a skipped lane between the wild
type and transformant C. reinhardtii protein lysate samples to prevent contamination of the
untransformed lane, and additionally, a control was included to confirm the efficacy of the
antibodies used
- Transformants of “FRET plasmids” appeared on plates
August
AUGUST 7-13
- Recorded promotional video
- Podcast outreach for final interviews
- Phototroph
community meetup 2 preparation
- Continued glass bead transformation trials of C. reinhardtii
with alterations including testing the Kindle protocol (1990) with SGII media as well as the Kao
and Ng protocol (2017) with polyethylene glycol
AUGUST 14-20
- Continued glass bead transformation trials of C. reinhardtii using the Kindle protocol
(1990) with SGII media as well as the Kao and Ng protocol (2017) with polyethylene glycol
-
Phototroph community meetup 2 preparation
- Phototroph community meetup 2 (theme:
troubleshooting)
- Outreach for HP stakeholder meetings
AUGUST 21-27
- Obtained transformants of CC-400 strain of C. reinhardtii and A2 plasmid
- HP
stakeholder meeting 1 & 2
- Phototroph community virtual conference preparation
-
Started cultures of transformant strains for further testing
- Prepared for colony PCR
AUGUST 28 - SEPTEMBER 3
- Podcast interview #4 - Dr. Sundaram
- Began working on handbook and video protocol
contributions to phototroph handbook