Notebook

Spring 2024

January

  • Dry Lab
    • Reviewed past iGEM Projects and began to read primary synthetic biology literature to introduce the team to iGEM, synthetic biology and to get ideas for potential iGEM Projects.

February 1 - February 4

  • Dry Lab
    • Continued to review past iGEM Projects and primary literature for potential iGEM projects; focused on primary literature to identify/develop projects. Discussed numerous ideas with the entire team contributing multitude of ideas. 

February 5 - February 11

  • Dry Lab
    • Began to settle on a few project ideas, particularly the underutilized potential of satellite phages. Reviewed past iGEM Projects and literature related to using satellite phage systems for control for narrowing down potential iGEM Projects. 
    • Looked for available satellite systems to work with.

February 12 - February 18

  • Dry Lab
    • Looked for available satellite systems to work with and phage metagenomics software. 
    • Assigned individual project responsibilities and did our respective literature reviews. 

February 19 - February 25

  • Dry Lab
    • Using Phaster to screen for partial prophage (satellites) in strains of Mycobacteria.
    • Beginning to use Satellite Finder to search for satellites. Searched for other software; documented issues with existing software.

February 26 - March 3

  • Dry Lab
    • Wet lab team has meetings about different satellite systems we could work with and applications that could make a good iGEM project. Discussed wide range of applications. 
    • Discussing what our model should focus on.

March 4 - March 10

  • Dry Lab
    • Writing literature reviews on all relevant satellite phage literature.
    • Wet lab team meets to discuss satellite systems to potentially work with.

March 11 - March 17

  • Dry Lab
    • Continued looking into more relevant applications of satellites including biofilm destruction and creating novel satellites from pieces of DNA.

March 18 - March 24

  • Wet Lab
    • Began lab training and preparations for the meeting with Noblis and the Hackathon.
  • Outreach and IHP
    • Presented at and met with Noblis.
    • Hosted outreach event with Jamestown High School.

March 25 - March 31

  • Dry Lab
    • Reviewed literature on satellite phage isolation and satellites available for purchase. Intensive search for what was available. 

April 1 - April 7

  • Dry Lab
    • Utilizing DePhT to identify partial prophage, potential satellites, in strains of Mycobacteria.
    • Started working with CarveMe, a software for formulating genome scale metabolic models.
  • Outreach and IHP
    • Prepared our presentation for the 2024 Future of Biology Conference.

April 8 - April 14

  • Wet Lab
    • Reviewed protocols ahead of summer lab session; continued training.
  • Outreach and IHP
    • Prepared workshop for the Hackathon.

April 15 - April 21

  • Wet Lab
    • Reviewed protocols ahead of the summer lab session; continued training on basic techniques.
  • Outreach and IHP
    • Presented on “Introduction to Genome Scale Metabolic Modeling” at the Future of Biology 2024 Conference. 
    • Hosted a presentation and workshop on machine learning and AI applications to biology for the TribeHacks William & Mary Hackathon.

April 22 - April 28

  • Wet Lab
    • Found the P2/P4 system designed by Dr. Fa-arun to purchase.
    • Initial discussions of model colon design for hardware - began initial plans.
  • Dry Lab
    • Began discussions for an alternative software tool to Satellite Finder (to become SaPhARI).
  • Outreach and IHP
    • Presented at a William & Mary Biomath seminar.

April 29 - May 5

  • Wet Lab
    • More discussions of P2/P4 system applications and Mycobacterium satellites.
  • Dry Lab
    • Identified expectations for modeling and software projects.
    • Decided to move away from a GEM as part of the modeling.
    • Began coding web scraper for satellites in the NCBI database.
    • Completed safety training modules.
    • Officially divided up tasks for the project and established end goals.

May 6 - May 12

  • Wet Lab
    • Met with Jonathan Frey to begin selecting materials for the model colon.
  • Dry Lab
    • Began compiling tail fiber interaction database for E. coli and Mycobacteria.
    • Made improvements to the NCBI satellite web scraper.
    • Working on literature reviews on project sub-topics.

May Break 2024

May 13 - May 19

  • Wet Lab
    • Drafted protocols to prepare for summer session.
    • Drafted designs for model colon at different cost levels; determined volumes and retention times for each chamber.

May 20 - May 26

  • Wet Lab
    • Completed literature reviews on project sub-topics.
    • Developed engineering plan for the P2/P4 system.
    • Finalized materials list and began to discuss choice of inoculum for model colon.
    • Began designing soil microcosms.
  • Dry Lab
    • Began to discuss using Prodigal for the initial ORF identification in SaPhARI.
    • Deciding on metagenomic softwares to use for analysis.

Summer 2024

May 27 - June 2

  • Wet Lab
    • Continued lab training.
    • Met with Jonathan Frey to discuss mixing mechanism and cover design for model colon.
    • Began design of P4 Cosmid with RFP insert (P4-RFP) and detailed steps for assembly.
  • Dry Lab
    • Utilizing Prodigal and Pyrodigal for annotation of BLAST+ outputs for SaPhARI.
    • Running Kraken2/Bracken and Metalign on metagenomic nucleotide database.
    • Working with BBMerge for metagenomic read merging.
    • Writing preliminary safety forms.

June 3 - June 9

  • Wet Lab
    • Created and quantified transducing units.
    • Met about experimental design for screening our engineered systems in vitro and in the colon and soil models.
    • Finalized flow rates and feeding schedule for the model colon. 
    • Decided to use a kanamycin resistance gene as the gene target proxy to evaluate P4 cosmid system.
  • Dry Lab
    • Curated database to contain more information and altered NCBI web scraper to better target certain satellite families.
    • Attempting to use VirFinder for satellite identification in metagenomic data, but realized it is not suitable for large amounts of data.
    • Submitted preliminary safety forms.

June 10 - June 16

  • Wet Lab
    • Found that the transducing units had degraded within a week exponentially.
    • Beginning to make M. smegmatis lysogens.
    • Switched from Raspberry Pi to Arduino to control the model colon and began pump calibration.
    • Began working with Mycobacterium satellites
  • Dry Lab
    • Continued developing annotation pipeline for SaPhARI.
    • Finished compiling tail fiber interaction database for E. coli.
    • Using AlphaFold3 to find shared proteins between P1 and P2 tail fibers.
    • Began developing predictive and generative software for phage-host interaction.
    • Running Metalign with different cutoff scores.
    • Wrote project description.

June 17 - June 23

  • Wet Lab
    • Created a new batch of transducing units.
    • Quantified the titer of the transducing units, which did not deteriorate.
    • Enrichment plating of soil samples.
    • First cloning of kanamycin targeting sequence into P4 cosmid, first transformation failed, second succeeded.
    • Continued making M. smegmatis lysogens.
    • Worked with isolation/characterization of Mycobacterium satellites.
    • Attempted assembly and transformation of P4-RFP cosmid.
  • Dry Lab
    • Assembled a pipeline for SaPhARI that analyzes identified open reading frames in bacterial genomes with known satellites and performs DIAMOND BLAST.
    • Began using InSilicoSeq for satellite metagenomics.
  • Outreach and IHP
    • Preparing for the BLAST program.
    • Began mentoring a local high school senior.

June 24 - June 30

  • Wet Lab
    • Created a replicative system with the P4 transducing units.
    • Phage purification of potential satellites.
    • Completed final pump calibration for model colon.
    • Analytical work including colony PCR and digest to check for successful clones of kanamycin targeting P4 cosmid.
    • Continued making M. smegmatis lysogens.
  • Dry Lab
    • Conducting literature review on satellite structure to refine SaPhARI pipeline.
    • Analyzing phagelet genomes for potential phage tail proteins to use for host expansion.
    • Running predictive model for phage-host interaction.
    • Finished compiling tail fiber interaction database for Mycobacteria.
  • Outreach and IHP
    • Hosted the BLAST Camp program for two full days. 

July 1 - July 7

  • Wet Lab
    • Began attempting to resurrect the phagelets.
    • Phage purification of potential satellites.
    • Designed soil microcosms.
    • Began in vitro measurements of kanamycin targeting P4 cosmid.
    • Finished in vitro testing of M. smegmatis lysogens.
    • Began physical screening for satellites in M. aichiense.
  • Dry Lab
    • Debugging generative model for phage-host interaction.
    • Strengthening training data for predictive and generative phage-host interaction models.
    • Began running MegaHIT and MetaSPAdes.
  • Outreach and IHP
    • Had an IHP meeting with Dr. Fa-arun.

July 8 - July 14

  • Wet Lab
    • Phage purification of potential satellites.
    • Began design of automated pH control system for the model colon.
    • Continued physical screening for satellites in M. aichiense.
    • Due to lack of fluorescence, redesigned P4-RFP.
  • Dry Lab
    • Conducting literature review on PICMI satellites to compile parameters for SaPhARI.
    • Conducted tests of the SaPhARI annotation pipeline.
    • Began drafting organizational code for SaPhARI.
    • Determining which gene in phage HerbertWM to target for successful tail fiber engineering with phagelets.
    • Gained access to phage-host interaction predictive model source code and modifying model.
    • Analyzing MegaHIT outputs.
  • Outreach and IHP
    • Had an IHP meeting with Dr. Otchy.
    • Had an IHP meeting with Dr. Sheila Van Cuyk and Dr. Anand Kumar.

July 15 - July 21

  • Wet Lab
    • Continued plating the phagelets.
    • Screening for satellites in soil samples collected on campus.
    • Repeated enrichment plating with E. coli.
    • Planned experimental designs for model colon and soil microcosms.
    • Designed a single-chamber ‘mini’ model colon.
    • Had to restart measurement work because indicator E. coli strain showed weak chloramphenicol resistance independent of any plasmid.
    • Continued physical screening for satellites in M. aichiense.
    • Began physical screening for satellites in M. smegmatis lysogens.
  • Dry Lab
    • Began analysis of the satellite database.
    • Finalized training data for phage-host interaction predictive model.
    • Decided to use the A2 phage, L5, for tail fiber engineering with HerbertWM.
    • Improving metagenomic pipeline code for efficiency and finalized threading.
    • We presented our project to Vice Provost of Research Dennis Manos.

July 22 - July 28

  • Wet Lab
    • Ran experiments on the phagelets to determine their sizes and properties.
    • PCI extraction of potential satellites.
    • Began building the soil microcosms and designed rainfall mechanism.
    • Altered model colon from one five-chamber model to two separate three-chamber models.
    • Began preliminary work with pdCas9; began assembly of dCas9 Cosmid.
    • Assembled and transformed P4-RFP.
  • Dry Lab
    • Began initial coding for the wiki.
    • Analyzing initial SaPhARI output.
    • Filtering contigs from soil metagenomes.

July 29 - August 4

  • Wet Lab
    • Made transducing units with P1 tail fibers.
    • Began first run of the model colon.
    • Finished cloning of dCas9 Cosmid.
    • Evaluated growth of HL 713 (KanR E. coli strain for colon and soil measurement) in colon and soil-like conditions.
    • Verified P4-RFP assembly via colony PCR.
    • Reattempted E. coli potential satellite DNA extractions for higher purities.
  • Dry Lab
    • Debugging SaPhARI code.
    • Designing chimeric tail fibers with L5 and HerbertWM.
    • Automating assembly of SRA metagenomes.

August Break 2024

August 5 - August 11

  • Wet Lab
    • Sent E. coli potential satellites for sequencing.
    • Attempted transformation of Mycobacterium aichiense by electroporation. 
    • Finished constructing soil microcosms and rainfall mechanism.
    • Maintenance of the model colon.
    • Assembling chimeric tail fibers.
  • Dry Lab
    • Automating assembly of SRA metagenomes.
    • Worked on wiki and write-ups.
    • Writing promotional video script.

August 12 - August 18

  • Wet Lab
    • Maintenance and measurement of the model colon.
    • Assembling chimeric tail fibers.
  • Outreach and IHP
    • Had an IHP meeting with Shanthi Parkar from Seed Health.
  • Dry Lab
    • Worked on wiki and write-ups.
    • Continued running metagenomes and improving SaPhARI

Fall 2024

August 26 - September 1

  • Wet Lab
    • Maintenance and measurement of model colon.
    • Making graphics and animations for promotional video.
    • Began assembly of dCas9-w cosmid & inserting kanamycin targeting spacer insert into dCas9 cosmid.
  • Dry Lab
    • Debugging SaPhARI code.
    • Merged and assembled sequencing returns for phagelets and putative E. coli satellite phage.
    • Creating graphics and editing project promotional video.
  • Outreach and IHP
    • Had an IHP meeting with Dr. Katherine Jennings from Noblis.
    • Had an IHP meeting with Dr. Negron from Noblis.

September 2 - September 8

  • Wet Lab
    • Created transducing units and grew out phagelets for gut and soil inoculation.
    • Maintenance and measurement of model colon.
    • Taking soil samples and bacterial plating from soil microcosms.
    • Preliminary work for CI dependent terminator characterization.
    • Made P4-RFP transducing units.
  • Dry Lab
    • Running SaPhARI on metagenomic samples.
    • Continuing assembly of phagelet backbone with chimeric tail fiber inserts.
    • Finalizing metagenomic assemblies for phagelet and putative E. coli satellite sequencing data.
    • Submitted final safety forms.
    • Submitted project promotional video.

September 9 - September 15

  • Wet Lab
    • Taking samples from soil microcosms and bacterial plating.
    • Inoculated soil microcosms 9-12 with phagelets and 13-16 with phage. 
    • Began phage plating of samples from soil microcosms 9-16 with M. aichiense
    • Maintenance and measurement of model colon.
    • Designing engineering plan for inserting the chimeric tail fibers into the pKW08 plasmid.
  • Dry Lab
    • Running SaPhARI on metagenomic samples.
    • Reassembling the putative E. coli satellite phage reads.
  • Outreach and IHP
    • Had a booth at the William & Mary SCI-FRI community event. 
    • Had an IHP meeting with Dr. Birgit Scharf.
    • Presented at a Willaim & Mary Biomath seminar.
    • Finalized Gut Busters board game.

September 16 - September 22

  • Wet Lab
    • Taking samples from soil microcosms and bacterial plating.
    • Continuing to phage plate of samples from soil microcosms 9-16 with M. aichiense 
    • Maintenance and measurement of model colon.
    • Inoculated soil microcosms with P4-RFP transducing units.
    • Attempting to insert chimeric tail fibers into pKW08.
  • Dry Lab
    • Running SaPhARI on metagenomic samples.
    • Writing up wiki pages.
  • Outreach and IHP
    • Had an IHP meeting with Dr. Fa-arun.
    • Had an IHP meeting with Dr. Bradley Abrahamson and Dr. Daniel Negrón from Noblis.

September 23 - September 29

  • Wet Lab
    • Used TEM to attempt to image the two systems of phage satellites.
    • Maintenance and measurement of model colon.
    • Taking samples from soil microcosms and bacterial plating.
    • Continuing to phage plate samples from soil microcosms 9-16 with M. aichiense
    • Attempting to insert chimeric tail fibers into pKW08.
  • Dry Lab
    • Running SaPhARI on metagenomic samples.
    • Writing up wiki pages.

September 30 - October 2

  • Wet Lab
    • Taking samples from soil microcosms and bacterial plating.
    • Continuing to phage plate samples from soil microcosms 9-16 with M. aichiense
    • Maintenance and measurement of model colon.
  • Dry Lab
    • Running SaPhARI on metagenomic samples.
    • Completing wiki.