Purdue/23 June 2008

From 2008.igem.org

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Looked up QC information for the SOS and LacZ parts (J22106 and I732005). SOS is OK and consistent, but I732005 is Inconsistent and BAD for all analyses.

Found new part that is more consistent and OK:

  • I732017: LacZ (full-length) + RBS in front
  • Location: Plate 1009, Well 11H
  • Plasmid: pSB1A2 (AmpR)
  • RBS efficiency = 1.0 (based on Elowitz 1999 Repressilator)

Edited by Janie Stine

Transformation of LacZ and SOS parts

  • Used OneShot MAX Efficiency DH5alpha-T1 cells (Invitrogen) and used a combo of their protocol and the iGEM protocol
  • Thawed on ice
    • We were not ready with the DNA/filter paper, so these cells were left thawing on ice for at most 30 minutes
  • Cut out parts from filter paper, warmed in 5 uL of TE 20 min.
    • LacZ/RBS (I732015), plate 1009, well 11H, plasmid pSB1A2
    • SOS promoter (J22106), plate 1003, well 12F, plasmid pSB1A2
    • QC for both parts read "OK"
  • Added all TE/DNA (~3uL...the filter paper absorbs a lot of the TE) to 25uL of cells for each part
  • Let sit on ice 30 min. in 2 mL Eppendorfs
  • Put cells in 42C water bath EXACTLY 30s (no mixing or shaking)
  • Place on ice for a couple minutes
  • Add 250uL room temp SOC to each vial
  • Shake EXACTLY 1 hour at 300rpm and 37C
  • Spread 200uL from ea. vial onto LB plates (store remainder at 4C)
  • Invert plates and let incubate at 37C until we leave (to get a kick-start to their growth)
  • Let incubate at room temperature overnight (We can't get to the cells for more than 14 hours, so we're forcing them to grow more slowly)

Edited by Janie Stine

Sol-Gel Vapor Spray

Fluorescence checked with confocal microscope with Jennie.

One plate that was visibly confounded has fluorescence that ranged over all red/green/blue ranges. Most likely silica that has clumped and refracted light.

One sprayed plate showed few bacteria that fluoresced green, confirming our suspicion that the bacteria are encapsulated. Great result for future work.

The sol-gel native (sol-gel pipetted into the plate) showed approximately the same as the spray. Good for a control of sol-gel.

Janie and Jennie both looked at the fluorescence. (Craig is color blind and could not distinguish.) Both concurred that what was seen in the two positive plates were what was expected of GFP bacteria.

Edited by Craig Barcus