Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

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

• AM - Angela Marsh
• JG - Dr. Joel Gaston
• KP - Kanak Patel
• MM - Matthew McDonough
• PB - Payton Boylston
• RG - Rayonna Gordon
• WO - Winston Ou

Neurons
Perfusion System with Bioreactor Design
Programming and Electronics (Bioreactor focus)

Neurons

9 Jun 2017
WO RG AM KP MM

• Received HT-22 Immortalized Mouse Hippocampal Cell Line and related materials from West Point

13 Jun 2017
WO RG AM KP MM PB

• Unfroze neurons
• Plated neurons for amplification

15 Jun 2017
WO RG AM KP MM PB

• Change media

17 Jun 2017
JG

• Changed media

19 Jun 2017
WO RG MM PB

• Split cells - Passage 1 from receipt of neurons

21 Jun 2017
WO RG MM PB

• Changed media
• Froze excess cells

23 Jun 2017
JG

• Changed media

27 Jun 2017
WO RG

• Made and changed media

30 Jun 2017
WO RG

• Thawed and plated cells - Passage 2

5 Jul 2017
WO RG

• Split cells - Passage 3

7 Jul 2017
WO RG

• Changed media

10 Jul 2017
WO RG

• Split cells - Passage 4

13 Jul 2017
WO RG

• Changed media

17 Jul 2017
JG

• Cells were killed

25 Jul 2017
WO RG

• Plated Passage 2 HT22 cells that were previously frozen into a 6 well plate

31 Jul 2017
WO JG

• Coated MEA with polylysine
• Seeded 20,000 HT22 cells onto coated MEA
• Incubated said cells

2 August 2017
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 2017
WO

• Began bioreactor design based on former models to be used in a perfusion system

25 May 2017
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

30 May 2017
WO

• Adjusted design of bioreactor design
• Began a custom design for a MEA

31 May 2017
WO RG AM KP

• Continued MEA designing: location of electrodes, leads, contact pad spacing, materials

1 Jun 2017
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

2 Jun 2017
WO RG AM KP MM

• Determined custom MEA design specs, particularly spacing

5 Jun 2017
WO RG AM KP MM

• Revised MEA spacing

6 Jun 2017
WO RG AM KP MM

• Discussed the location of holes for media flow and a bubble trap

7 Jun 2017
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

8 Jun 2017
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

9 Jun 2017
WO RG AM KP MM

• Confirmed the use of a peristaltic pump for media flow
• Finalized MEA design

12 Jun 2017
WO RG AM KP MM PB

• Contacted other MEA fabrication companies for quotes

13 Jun 2017
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

14 Jun 2017
WO RG AM KP MM PB

• Continued contacting MEA vendors

15 Jun 2017
WO RG AM KP MM PB

• Determined best luer lock connectors for tubing
• Finished printing of clamp design
• Explored photomasking vendors

16 Jun 2017
WO RG AM KP MM PB

• Determined design of media reservoirs to be conical flasks
• Explored pre-made MEA vendors

16 Jun 2017
WO RG MM PB

• Determined design of media reservoirs to be conical flasks
• Explored pre-made MEA vendors

19 Jun 2017
WO RG MM PB

• Ordered MEAs from Multichannel Systems
• Researched on systems to control the peristaltic pump from the computer

20 Jun 2017
WO RG MM PB

• Find tubing for bioreactor and pump

21 Jun 2017
WO RG MM PB

• Designed media reservoirs in Inventor, obtaining a cubic appearance, as opposed to the original conical tube appearance

22 Jun 2017
WO RG MM PB

• Ordered tubing

26 Jun 2017
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

27 Jun 2017
WO RG MM PB

• Inserted a conical steeple for the bubble trap
• Made minor modifications to bioreactor design

30 Jun 2017
WO RG

• Inserted threading into media reservoirs
• Made measurements of ordered o-rings to be fitted

3 Jul 2017
WO RG

• Printed out models to help test fittings for the O-ring

5 Jul 2017
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

7 Jul 2017
WO RG

• Modified media reservoir for better fluid flow

10 Jul 2017
WO RG

• Printed first part of bioreactor

12 Jul 2017
WO RG

• Adjusted dimensions and reprinted first part of bioreactor

13 Jul 2017
WO RG

• Adjusted dimensions and printed a third version of the first part of bioreactor

14 Jul 2017
WO RG

• Printed array to determine necessary dimensions to fit luer locks

17 Jul 2017
WO RG

• Applied dimension changes to bioreactor design and reprinted the first part of bioreactor

18 Jul 2017
WO RG

• Determined screw size and lengths needed to apply appropriate pressure on pins

19 Jul 2017
WO RG

• Printed out top and bottom of bioreactor and clamp
• Perforated MEA Bioreactor modified
• Verified operation of the pump
• Tested calibration of pump

20 Jul 2017
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

21 Jul 2017
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

24 Jul 2017
WO RG

• Printed bioreactor top in Dental SG
• Tested perfusion system with all components to test for leakage and overall flow

25 Jul 2017
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

26 Jul 2017
WO RG

• Performed dry run with cured pieces
• Autoclaved epoxy-containing piece

27 Jul 2017
WO RG

• Autoclaved entire perfusion/bioreactor system

2 August 2017
WO RG

• Enclosed MEA with transfected cells in between top and bottom piece of bioreactor
• Placed in incubator

3 August 2017
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

4 August 2017
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 2017
WO

• Introduced to labview - use to take voltage readings from electrodes
• Determined to set peak threshold to +/- 2 SD of base activity

25 May 2017
WO

• Built time-averaging method to determine frequency and amplitude of peaks
• Generated preliminary statistics for data
• Built code to write to a file

30 May 2017
WO

• Inserted method to adjust thresholds before data collection

29 Jun 2017
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

12 Jun 2017
WO

• Implemented impedance program from Intan to determine the presence of cells on electrodes

13 Jul 2017
WO RG

• Soldered pins to electrode adapter boards

19 Jul 2017
WO RG

• Tested program that ran impedance tests and voltage readings
• Soldered more electrode adapters

3 August 2017
WO RG

• Soldered pins to electrode adapter board ensuring to have pins on the ground and reference channels on adapter board

4 August 2017
WO RG

• Set voltage reading program to read at 20,000Hz