4 - Lac Operon

Qualitative lac operon experiment

Grow GFP E. coli in different sugar media, then photograph in a 12 well plate on a blue light box through the orange filter.

• Streak GFP E. coli on an LB-glucose-kan plate, grow overnight

• Pick a colony, grow in overnight in liquid LB-glucose-kan (to prevent it turning green)

• Spin down the ON culture (take a picture of the pellet for other use!)

• Rinse several times (to get rid of the glucose)

• Fill 12 well plate as indicated below. 2 mL per well:

additive	none	IPTG 0.01mM	lac 0.25M	gal 0.25M
none
glu 0.25M
mal 0.25M

• Photograph
  • in white light
  • in blue light

• Read in plate reader for blanks (OD and FL)

  • 12-well plate is too tall for reader!!!
  • Sample 100 uL of each to read blanks (OD & FL in a 96 well plate)

• Inoculate each well with a small amount of culture.

• Incubate on the shaker.

• Photograph

  • in white light
  • in blue light
• Read in plate reader (OD and FL)
  • Take 100 uL (?) samples to read in 96 well plate

Media

Need enough for the qualitative experiment ~5 mL each

Need enough for the quantitative experiment: 96 well plate @ 250 uL/well = ~25 mL total

Make 25 mL of each high concentration stock; mix the combinations from that

Add 50 µg/mL Kanamycin as indicated here.

Lac Operon PowerPoint from 2020 remote class