USMA-West_Point
Notebook
Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.
What should this page have?
- Chronological notes of what your team is doing.
- Brief descriptions of daily important events.
- Pictures of your progress.
- Mention who participated in what task.
Inspiration
You can see what others teams have done to organize their notes:
Week of 11 September 2017
- Attempted transfection of iGEM plasmid, and altered protocol as needed.
- Prepared bacteria culture plates for vector amplification.
- Purified DNA from cultured bacteria.
Week of 18 September 2017
- Continued transformation of iGEM plasmid.
- Prepared chloramphenicol resistant plates.
- Cultured psb1c3 cells and made stock.
- Created fluorescein calibration curve.
- Vectors amplified and purified.
Week of 25 September 2017
- Ran iGEM interlab protocol.
- Made and ran gel of plasmids.
- Created new glycerol stocks, using resistant bacteria for the process.
Abbreviations
Perfusion System with Bioreactor Design
Programming and Electronics (Bioreactor focus)
WO RG AM KP MM
WO RG AM KP MM PB
WO RG AM KP MM PB
JG
WO RG MM PB
WO RG MM PB
JG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
JG
WO RG
WO JG
WO RG
WO
WO
WO
WO RG AM KP
WO RM AM KP
WO RG AM KP MM
WO RG AM KP MM
WO RG AM KP MM
WO RG AM KP MM
WO RG AM KP MM
WO RG AM KP MM
WO RG AM KP MM PB
WO RG AM KP MM PB
WO RG AM KP MM PB
WO RG AM KP MM PB
WO RG AM KP MM PB
WO RG MM PB
WO RG MM PB
WO RG MM PB
WO RG MM PB
WO RG MM PB
WO RG MM PB
WO RG MM PB
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO RG
WO
WO
WO
WO
WO
WO RG
WO RG
WO RG
WO RG
- AM - Angela Marsh
- JG - Dr. Joel Gaston
- KP - Kanak Patel
- MM - Matthew McDonough
- PB - Payton Boylston
- RG - Rayonna Gordon
- WO - Winston Ou
Perfusion System with Bioreactor Design
Programming and Electronics (Bioreactor focus)
Neurons
9 Jun 2017WO RG AM KP MM
- Received HT-22 Immortalized Mouse Hippocampal Cell Line and related materials from West Point
WO RG AM KP MM PB
- Unfroze neurons
- Plated neurons for amplification
WO RG AM KP MM PB
- Change media
JG
- Changed media
WO RG MM PB
- Split cells - Passage 1 from receipt of neurons
WO RG MM PB
- Changed media
- Froze excess cells
JG
- Changed media
WO RG
- Made and changed media
WO RG
- Thawed and plated cells - Passage 2
WO RG
- Split cells - Passage 3
WO RG
- Changed media
WO RG
- Split cells - Passage 4
WO RG
- Changed media
JG
- Cells were killed
WO RG
- Plated Passage 2 HT22 cells that were previously frozen into a 6 well plate
WO JG
- Coated MEA with polylysine
- Seeded 20,000 HT22 cells onto coated MEA
- Incubated said cells
WO RG
- Transfected cells on MEA with 3200ng of OR2W1
- Incubated transfected cells on MEA in the bioreactor
Perfusion System with Bioreactor Design
23 May 2017WO
- Began bioreactor design based on former models to be used in a perfusion system
WO
- Used AutoDesk Inventor to design a general bioreactor
- Incorporated shape and general items such as holes for screws and cavity for neuronal culturing
- Determined location of a multielectrode array (MEA) in bioreactor design, where the neurons would be transfected and tested
WO
- Adjusted design of bioreactor design
- Began a custom design for a MEA
WO RG AM KP
- Continued MEA designing: location of electrodes, leads, contact pad spacing, materials
WO RM AM KP
- MEA
- Altered the electrode arrangement and size for a single well of a 96 well plate
- Predetermined specifications: Gold or TiN wires, embedded in glass
WO RG AM KP MM
- Determined custom MEA design specs, particularly spacing
WO RG AM KP MM
- Revised MEA spacing
WO RG AM KP MM
- Discussed the location of holes for media flow and a bubble trap
WO RG AM KP MM
- Fleshed out design for bubble trap
- Began design for clamp to keep pins on the multielectrode array (extension of bioreactor) - KP
WO RG AM KP MM
- Began discussion on removal of olfactant contaminated media while maintaining a perfusion system via syringes
- Continued clamp design-KP
- Requested a quote for custom MEA
WO RG AM KP MM
- Confirmed the use of a peristaltic pump for media flow
- Finalized MEA design
WO RG AM KP MM PB
- Contacted other MEA fabrication companies for quotes
WO RG AM KP MM PB
- Continued search of MEA vendors
- Worked on specifics regarding flow into and out of the bioreactor/media reservoirs - decided to use luer locks
WO RG AM KP MM PB
- Continued contacting MEA vendors
WO RG AM KP MM PB
- Determined best luer lock connectors for tubing
- Finished printing of clamp design
- Explored photomasking vendors
WO RG AM KP MM PB
- Determined design of media reservoirs to be conical flasks
- Explored pre-made MEA vendors
WO RG MM PB
- Determined design of media reservoirs to be conical flasks
- Explored pre-made MEA vendors
WO RG MM PB
- Ordered MEAs from Multichannel Systems
- Researched on systems to control the peristaltic pump from the computer
WO RG MM PB
- Find tubing for bioreactor and pump
WO RG MM PB
- Designed media reservoirs in Inventor, obtaining a cubic appearance, as opposed to the original conical tube appearance
WO RG MM PB
- Ordered tubing
WO RG MM PB
- Redesigned clamp system for pins using a platform and screws system
- Revised bioreactor design to have internal channels in one side of the bioreactor
WO RG MM PB
- Inserted a conical steeple for the bubble trap
- Made minor modifications to bioreactor design
WO RG
- Inserted threading into media reservoirs
- Made measurements of ordered o-rings to be fitted
WO RG
- Printed out models to help test fittings for the O-ring
WO RG
- Printed array to narrow down sizing for O-Ring
- Modified media reservoir due to space constraints
- Printed model MEAs to test pin setup and general fittings
WO RG
- Modified media reservoir for better fluid flow
WO RG
- Printed first part of bioreactor
WO RG
- Adjusted dimensions and reprinted first part of bioreactor
WO RG
- Adjusted dimensions and printed a third version of the first part of bioreactor
WO RG
- Printed array to determine necessary dimensions to fit luer locks
WO RG
- Applied dimension changes to bioreactor design and reprinted the first part of bioreactor
WO RG
- Determined screw size and lengths needed to apply appropriate pressure on pins
WO RG
- Printed out top and bottom of bioreactor and clamp
- Perforated MEA Bioreactor modified
- Verified operation of the pump
- Tested calibration of pump
WO RG
- Printed bioreactor top in Dental SG and media reservoir in clear
- Tested perfusion system with bioreactor and MEA to test for leakage and overall flow
WO RG
- Printed bioreactor bottom in Dental SG and waste reservoir in clear
- Revised bioreactor design for better hole sizing and placement
- Tested perfusion system with bioreactor, media reservoir, and MEA to test for leakage and overall flow
- Redesigned reservoir for more efficient drainage
WO RG
- Printed bioreactor top in Dental SG
- Tested perfusion system with all components to test for leakage and overall flow
WO RG
- Printed both reservoirs in Dental SG
- Tested perfusion system with Dental SG bioreactor top/bottom and MEA to test for leakage and overall flow
- Cured bioreactor pieces in UV
WO RG
- Performed dry run with cured pieces
- Autoclaved epoxy-containing piece
WO RG
- Autoclaved entire perfusion/bioreactor system
WO RG
- Enclosed MEA with transfected cells in between top and bottom piece of bioreactor
- Placed in incubator
WO RG
- Ran first trial: recorded impedance, started recording voltage at 1,000Hz and injected 0.5mL of 2mM 1-hexanol olfactant, waited 7 minutes and inject 0.5mL more of olfactant, waited 7 minutes, stopped recording, pumped in 1mL of fresh media
- Bioreactor and reservoirs were placed back in incubator
WO RG
- Ran two trials:recorded impedance, started recording voltage at 20,000Hz and injected 0.5mL of 2mM 1-hexanol olfactant, after a total of 1 minute stopped recording, pumped out olfactant with 1mL of fresh media
- ran an additional trial without recording voltage reading data, analyzed data for a total of 10 minutes
- ran an additional trial without recording voltage reading data injecting 20mM of olfactant, analyzed data for a total of 10 minutes
Programming and Electronics (Bioreactor focus)
23 May 2017WO
- Introduced to labview - use to take voltage readings from electrodes
- Determined to set peak threshold to +/- 2 SD of base activity
WO
- Built time-averaging method to determine frequency and amplitude of peaks
- Generated preliminary statistics for data
- Built code to write to a file
WO
- Inserted method to adjust thresholds before data collection
WO
- Adjusted labview program to read in data from electrodes using Intan codes (as opposed to theoretical inputs)
- Finished labview program - can select filename/type
WO
- Implemented impedance program from Intan to determine the presence of cells on electrodes
WO RG
- Soldered pins to electrode adapter boards
WO RG
- Tested program that ran impedance tests and voltage readings
- Soldered more electrode adapters
WO RG
- Soldered pins to electrode adapter board ensuring to have pins on the ground and reference channels on adapter board
WO RG
- Set voltage reading program to read at 20,000Hz