Safety

In an effort to ensure safety, we detail our safe protocols below

The APUS team is committed to safety and security in the lab, and have taken multiple steps to ensure the safety of the team and APUS users

We have taken a set of trainings before working in the lab and beginning our iGEM project, including:

  • Lab access and rules
  • Responsible individuals
  • Differences between biosafety levels
  • Biosafety equipment
  • Good microbial technique
  • Disinfection
  • Emergency procedures
  • Rules for transporting samples between labs or shipping between institutions
  • Chemical, fire, and electrical safety

APUS works on an open lab bench, wearing all necessary PPE. Members consulted professionals to learn how to safely handle all equipment including 3D printers, laser cutters, syringe pumps, plasma surface treatment machine.

Before conducting transformations to create the sender and receiver strains, the team trained with the DAMP lab to safely perform the experiment. All microbes were handled and stored in accordance with Boston University’s safety procedures and all hardware that came in contact with bacteria were also treated as biohazard.

In solving the PDMS chip leakage problem, we have also reduced the risk of coming in contact with bacteria. Before our solution sealed up the PDMS chip, media and bacteria leaked out of the chip via its ports. Now, the system is fully closed and liquid remains in the system from media to waste.

Below are protocols to safely set up and run experiments or download as PDF:

Cell preparation

  • Grow cells to OD of 0.2-0.5 (exponential phase)

PDMS chip preparation

  • Look under 10x microscope to confirm chip was well-constructed
  • Choose a new chip if there is dust, hair, or collapsed monolayer chambers
  • To construct and seal PDMS chips, see “PDMS Chip Construction” protocol

Loading cells into PDMS chip

  • Use 1/16 tubing and 3mL syringe pump, connecting them via Luer Lock needle connections
  • Syringe pump cells into PDMS chip
    • Diameter: 4.6 mm
    • Infuse rate: 500uL/min
    • Refill rate: 500uL/min

PDMS chip for centrifugation

  • Syringe pump cells into one side of PDMS chip
    • Place chip in 3-printed chip holder, glass flush with the barrier
      • Tape lengthwise opposite of barrier, taping glass to holder, not PDMS
    • Place chip in centrifuge cylinder M, sender side inclined and on the inner side of the centrifuge
      • H, M, L is in terms of bacterial abundance
    • Centrifuge at 1328Gs for 2 minutes
    • Confirm under microscope that cells are trapped in monolayer chambers
  • Syringe pump cells into left side of PDMS chip
    • Centrifuge at 664 Gs for 2 minutes 
    • Lower force so the cells on the other side stay trapped
    • Confirm under microscope that cells are trapped in monolayer chambers on left side and right side

Place PDMS chip into APUS holder and begin experiments

Software notes if measuring fluorescence on a Nikon Eclipse Ti2 microscope

  • Stop collecting data at 24 hours, make a new ND file
    • So data stays uncorrupted
  • 2 episcopic channel (YFP and CFP), 1 diascopic channel (phase contrast)
    • Image phase contrast every 5 minutes
    • But fluorescence every 15 (or 10) minutes
      • To prevent photobleaching/phototoxicity
    • Take 10-15 XY (monolayer chamber) locations for images
    • Software will analyze images and produce graph