Weekly Progress
Click on each week for more details.
Before Lab work (Dec 23-July 24)
Recruitment (December-January)
- Applications to join the team were due by 1st December
- Interviews with members of the CU Synthetic Biology Society committee were carried out during December
- The team was finalised in mid-January
Brainstorming and training (January-March)
- Weekly brainstorming sessions to discuss and develop project ideas
- Information sessions on synthetic biology, iGEM, human practices, etc.,
- Meetings with academics and industry experts for advice on idea feasibility
Detailed project design (Early July)
- Literature review on previous methods in magnetogenetics and possible pathways that could be manipulated e.g. EGFR, EPG, Hog1, HSF1, MscS, Piezo, Ste2, TcI, TlpA, Wsc1
- Literature review on magnetic nanoparticles and their purchase and synthesis
- Meetings with academics for advice on project feasibility and suggestions
Getting ready for lab work
- Risk assessment for iGEM and the Department of Engineering lab space
- Created template for wet lab notebook
- Discussed logistics of ordering reagents and plasticware with advisors
- Parts mining for plasmid design through iGEM registry and scientific papers
- Workshop on primer design
- Induction and training in lab
- Researching and compiling protocols
- Inventory of items left by previous iGEM teams
- Design and ordering of primers and gene fragments for the construction of plasmids needed to test EPG, TlpA, and TcI
- Design of AFM circuit and fluorometer
Outreach
- Designing materials for Smallpeice outreach event
Week 1 (15/07-21/07)
Wet lab
- Receiving, suspension, and storage of primers
- Receiving and preservation of required E. coli and S. cerevisiae strains for TlpA/TcI, Wsc1, HSF, and EPG pathways
- Test of antibiotics that are required for transformation
- Preparation of competent cells for Golden gate
- PCR of parts required for Golden gate
Hardware
- Box no.1, for imaging plates on the large electromagnet, constructed
- Raspberry Pi 3 obtained
Modelling
- Modelling workshop with instructor
- Construction of ODE model for TlpA, TcI, and Wsc1 started
- Literature review on model parameters
Human Practices
- Start reading on neuroscience, organoid, and policies
- Start communication with academics
Week 2 (22/07-28/07)
Wet lab
- Antibiotics testing
- Test transformation of pUC19 and luciferase from the kit to test the competency of competent cells and efficiency of chemical transformation protocol
- Start transformation of kit parts required for all pathways and get ready for the golden gate
- Yeast HSF pathway construction starts
- 250 nm magnetic nanoparticles visualisation with a light microscope
Hardware
- ZVS heating of ferrofluids obtained from Cavendish lab
- ZFS coil parameter analysis
- Design of box with moving magnets and imaging system
Modelling
- Construction of model calculating forces generated by 250 nm magnetic nanoparticles started
Human Practices
- Detailed feedback from researchers in organoid development and neuroscience
- Plan for structures of human practices deliverable
Week 3 (29/07-04/08)
Wetlab
- Attempting minipreps on previously transformed cells
- Validation of miniprep parts on gel
- Troubleshooting chemical transformation
- Testing and trials of electroporation
- Continuous working on TlpA/TcI pathway construction
- More MNP observation under light microscope, observing random movement change to biassed movement upon application of a magnetic field
Hardware
- Coil design iterations
Modelling
- Modelling of magnetic field shape and strength, used to inform coil design iterations
- Modelling of nanoparticle heating and dissipation of heat across plate
- Modelling of the HSF and Wsc1 pathways
Human Practices/Outreach
- Making the table of stakeholder areas and people we have communicated with
Making a comparison table of sonogenetics, optogenetics, magnetogenetics, and chemical methods
- Contacting more stakeholders in a more diverse range
- Biomedical engineering summer course outreach event
Week 4 (05/08-11/08)
Wet lab
- Chemical transformation has relatively low efficiency. Electroporation works better. We’re switching the transformation method to electroporation
- Previous miniprep is not working and we need to re-transform all parts required.
- Construction of EPG pathway starts
- First attempt at golden gate assembly parts available for TlpA/TcI
Hardware
- Testing of relays, servo, camera, thermal camera, motors, and display
- Initial development of GUI for control and measurement of magnet
Modelling
- Further iterations on modelling of TlpA/TcI, HSF, and Wsc1 pathways
- Calculation of magnetic field gradient
- Calculation of force acting on magnetic nanoparticles in water
- Modelling of further EPG constructs, with LacI and TEV
Human Practices
- Meeting with Stuart Ibsen about sonogenetics - costs of technology, how the technology works, use cases.
- Meeting with Mightex about optogenetics - found out rough costs of equipment and cost ranges. Found out more about who is using optogenetics (mostly universities).
Week 5 (12/08-18/08)
Wet lab
- Golden gate of EPG started
- MNPs attachment test to see if MNPs bind to his-tagged Wsc1
- Preparation and testing for yeast Leu- Ura- His- selection plate
- Transformation of Y1 for Wsc1 pathway
- Miniprep is not working, figuring out what's the problem and changing the kit for miniprep
- Troubleshooting of previous TlpA / TcI golden gate by gel and digest validation
Hardware
- Development of imaging system
- Further design of the “magenta” box with the moving magnet
Modelling
- Further modelling on HSF and discussion with the instructor about the temperature function
- EPG-TEV model constructed
Human Practices
- Meeting set up with neuro-oncologist at Addenbrooke’s next Tuesday 10am
- Responses from CAR-T cells researcher
- Girls in Math educational event at St Johns’ college Cambridge
Week 6 (19/08-25/08)
Wet lab
- Transformation of Y1 with pHPS100H for wsc1 construct
- Testing of X-gal and sorbitol on HAS100L
- Golden gated plasmids are not working. We need to check from the parts, miniprep, transformation, golden gate with gel validation to see what went wrong
Hardware
- Construction of the “magenta” box with the moving magnet and imaging system
Modelling
- Initial model of HSF finished and had a discussion with the instructor
- Integrating HSF model with thermal magnetic modelling
Human Practices
- Meeting with Dr Rajesh Jena (neuro-oncologist) who had a lot of interesting thoughts about glioblastoma recurrence prevention and CAR T-cell therapy application ideas
- Draft of Education page
Week 7 (26/08-01/09)
Wet lab
- Successful transformation of Y1 with pHPS100H. Conducting sorbitol and X-gal test on Y100H to validate the construct is working
- MNPs binding test to Wsc1
- Golden gate troubleshooting
- Toxicity test of 20 nm MNPs on E. coli DH5alpha and BL21
- Re-transforming, miniprep, digest, and golden gate the parts
Hardware
- Testing on a large coil to validate if a magnetic field with a low frequency, high field strength could be produced
- 20 nm MNPs heated up when a magnetic field with high frequency and high field strength was applied
Modelling
- Improvement of TcI and Wsc1 modelling
Human Practices
- Preparation of posters and slides that will be used for UK 2024 iGEM meetup at Sheffield
Week 8 (02/09-08/09)
Wet lab
- The transformation and direct miniprep did not work well, and we decided to colony PCR the parts we need and validate it
- Figuring out a working protocol to conduct X-gal testing on yeast
- Testing of sonicator on liquid X-gal testing with yeast
- Wsc1 MNPs testing with magnetic field in a liquid based culture
- Toxicity testing of yeast with 250 nm MNPs
- Continuous attempts of golden gate of EPG parts
Hardware
- Set up the algorithm for the camera fluorescence measuring
- Movement of magnets, and calibration with limit switches
- Testing on the motor.
Modelling
- Further improvement of modelling of TlpA/TcI and EPG
Human Practices
- Writeup in progress
- UK iGEM Meetup 2024 this weekend
Week 9 (09/09-15/09)
Wet lab
- Colony PCR of EPG, validation with gel, transformation with BL21 and test with nanoluc
- MNPs-resuspended in LB agar and testing the bacterial growth and heating of the plate
- Arrival of synthesised TlpA and TcI constructs
- Miniprep of TlpA and TcI constructs from Dh5alpha
- Wsc1 MNPs testing with magnetic field in both solid and liquid method
Hardware
- Software set-up and testing interactions of the “magenta” box
- Camera test and calibration
Modelling
- Summarising TlpA and TcI modelling
Human Practices
- Writeup approaching the end
Week 10 (16/09-22/09)
Wet lab
- Colony PCR, miniprep, and gel validation of EPG and TcI construct
- Troubleshooting of the TcI construct
- Heat-testing of TcI and testing of EPG luminescence with magnet
Hardware
- New coil design and ferrofluid heating test
- Testing of three constructed boxes with instructors and getting user’s feedback
Modelling
- Summary of all models (TlpA/TcI, wsc1, HSF, magnetic force, magnetic heating) and final writeup planned
Human Practices
- Write-Up content done. Planning the final format to be displayed on wiki
Week 11 (23/09-29/09)
Wet lab
- Colony PCR, miniprep, and gel validation of TcI construct
- Repeats of TcI heat-activation to test new golden gate 1pV3
- Miniprep of some constructs for sequencing
- Summarising all testing results and uploading new basic parts/constructs to iGEM registry
Hardware
- Construction of the final box finished
- Hardware writeup in progress
Modelling
- Summarising all models. The final write up is uploaded to wiki
Human Practices
- Write-Up content uploaded to wiki
Detailed wet lab notebook
Click on the contents link to be taken directly to the page or scroll down for the second part of the notebook.