Team:MichiganState/InterLab

InterLab Notebook Documentation, 7/16-8/3

Monday, 7/16

  • Planned on doing the transformation of the 8 sample plasmids into NEB DH 5-alpha competent E. coli, but couldn't find the cells
  • Instead, pipetted the 8 sample plasmids out of iGEM 2018 Distribution Kit Plate 7 and suspended in 10 uL of npH2O, then stored in -20 C freezer as follows:
Table of InterLab Sample Numbers and Names

Tuesday, 7/17

  • Met with Dr. Velasquez to learn about using the He Lab's plate reader on the 4th floor
  • Transformed the 8 sample plasmids into NEB 5-alpha competent E. coli according to NEB high-efficiency transformation protocol
  • Plated original transformation as well as 1:10 dilution for each sample plasmid
  • Plated on LB + chloramphenicol plates
  • Incubated at 37 C overnight

Wednesday, 7/18

  • Observed growth on every original transformation plate, but no dilution plates
  • Discarded dilution plates and stored plates with growth in the cold room

Thursday, 7/19

  • Completed three calibration protocols and entered data into the Interlab MichiganState Excel sheet
  • Prepared two overnight cultures of each of the 8 devices (16 cultures total) as follows:
    • 5 mL LB
    • 5 uL chloramphenicol
    • 1 colony from a plate
    • Incubated cultures at 37 C overnight

Saturday, 7/21

  • Made 16 new cultures (in LB + chloramphenicol) to use on Monday

Monday, 7/23

  • Performed Day 3 protocol for InterLab:
  • Measured OD600of 1:10 dilutions of the 16 overnight cultures on the spectrophotometer (blanked with LB + chloramphenicol)
  • Diluted further to get each culture to target OD600of 0.02
  • Kept 500 mL of 0 hr cultures in Eppendorf tubes on ice
  • Put 0.02 OD600cultures back in 37 C incubator for 6 hours
  • At 6 hours, remove cultures from incubator and take 500 mL samples into Eppendorf tubes on ice
  • Measured OD600and fluorescence of 4 replicates of each culture at each time point
  • Data seemed to be off; will re-try later this week

Thursday, 7/26

  • Made overnight cultures for Day 3 re-run tomorrow
    • 5 mL LB + chloramphenicol
    • 1 colony from plate

Friday, 7/27

  • Performed Day 3 protocol for InterLab:
  • Measured OD600of 1:10 dilutions of the 16 overnight cultures on the spectrophotometer (blanked with LB + chloramphenicol)
  • Diluted further to get each culture to target OD600of 0.02
  • Kept 500 mL of 0 hr cultures in Eppendorf tubes on ice
  • Put 0.02 OD600cultures back in 37 C incubator for 6 hours
  • At 6 hours, remove cultures from incubator and take 500 mL samples into Eppendorf tubes on ice
  • Measured OD600and fluorescence of 4 replicates of each culture at each time point
  • Turned in all data to iGEM HQ before the midnight deadline

Note: CFU Protocol was not turned in on 7/27, so it was done after the deadline with iGEM HQ's permission.

Monday, 7/30

  • Made four 3 mL overnight cultures to prepare for CFU protocol tomorrow
    • 2 cultures of the negative control device
    • 2 cultures of the positive control device
  • Incubated in 37 C shaker overnight

Tuesday, 7/31

  • Performed InterLab CFU Protocol:
  • Diluted overnight cultures in triplicate to OD600of 0.1 in LB + chloramphenicol media (1 mL total volume)
  • Performed serial dilutions for each of the 12 cultures and plated dilutions 3, 4, and 5 on LB + chloramphenicol plates
  • Incubated at 37 C overnight (or so we thought)

Wednesday, 8/1

  • Checked plates and saw no sign of growth
    • The plates were accidentally incubated at 30 C instead of 37 C, which is why they didn't grow
  • Incubated 4 more overnight cultures and prepared supplies for tomorrow's repeat of the CFU protocol
  • New LB+chloramphenicol agar plates were made from heated LB agar + antibiotic
    • Plates from yesterday were LB plates with CAM spread on top

Thursday, 8/2

  • Performed InterLab CFU Protocol
  • Diluted overnight cultures in triplicate to OD600of 0.1 in LB + chloramphenicol media (1 mL total volume)
  • Performed serial dilutions for each of the 12 cultures and plated dilutions 3, 4, and 5 on LB + chloramphenicol plates
  • Incubated plates at 37 C overnight

Friday, 8/3

  • Observed growth on all 36 plates after 18-20 hrs and photographed for evidence and analysis
  • Colonies were counted on each plate, and results were submitted to and accepted by iGEM HQ
  • Thus concludes Michigan State iGEM 2018's InterLab experience! 🎉🎉
  • Seen below: second (A1-Y) and third (A1-Z) replicates of the negative control
    • 8x10^6 dilutions in the left column
    • 8x10^5 dilutions in the center column
    • 8x10^4 dilutions in the right column

Table of InterLab Sample Numbers and Names