Su24 FPOP prep and setup
Contents
Exercise #1: Identifying CRISPRi targets
No setup required; however, FPOP team should be familiar with the reading in case participants have questions.
Exercise #2: Preparing CRISPRi experiment
The following steps are needed to ready the E. coli cultures:
- 48 hours prior to laboratory session, MG1655 and MG1655 + CRISPRi freezer stocks should be streak plated and incubated ON at 37 °C.
- MG1655 strain streaked onto LB agar plates.
- MG1655 + CRISPRi transformants streaked onto LB + amp + cam agar plates.
- 24 hours prior to laboratory session, MG1655 and MG1655 + CRISPRi colony from each streak plates should be inoculated into 5 mL of broth and incubated ON at 37 °C.
- MG1655 strain inoculated into LB broth.
- MG1655 + CRISPRi transformants inoculated into LB + amp + cam broth.
The following aliquots should be prepared prior to the laboratory session (one aliquot of each per team):
- ~3mL of blue dye
- ~10 mL of water
- 30 mL of LB broth
- 100 μL of MG1655 culture
- 100 μL of MG1655 psgRNA_ldhA culture
- 100 μL of MG1655 psgRNA_pta culture
- 100 μL of MG1655 psgRNA_ack culture
- 100 μL of MG1655 psgRNA_adhE culture
The following reagents should be available at the front laboratory bench (not aliquoted, instead participants will retrieve needed volume when at the front laboratory bench):
- ~1 mL of ampicillin
- ~1 mL of chloramphenicol
- ~100 μL of aTc (anhydrotetracyline)
The following supplies should be available at the front laboratory bench (numbers indicate what each team needs):
- 10 cuvettes
- 5 screw cap tubes
The following should be checked after the laboratory session:
- Ensure that all screw-cap tubes are hand-tightened and evenly distributed in the rotating wheel in the 37 °C incubator.
Exercise #3: Measuring ethanol yield
The following aliquots should be prepared prior to the laboratory session (one aliquot of each per team):
- ~10 mL of LB broth
The following reagents should be available at the front laboratory bench (not aliquoted, instead participants will retrieve needed volume when at the front laboratory bench):
- All reagents from ethanol assay kit (this is to reduce loss from aliquots):
- Assay Buffer
- Enzyme A
- Enzyme B
- NAD Solution
- MTT Solution
- Stop Reagent
The following supplies should be available at the front laboratory bench (numbers indicate what each team needs):
- 6 cuvettes
- 1 96-well plate (only one needed as all teams will share)
- 1 print-out of 96-well plate map from wiki (only one needed as all teams will share)
The following steps are needed to ready the ethanol assay standards:
- Label four microcentrifuge tubes with #1, #2, #3, #4.
- Prepare a 0.1% ethanol standard by mixing 25 μL of the 1% ethanol stock with 225 μL of water.
- Prepare the ethanol standards according to the chart:
| tube | final % ethanol | volume of 0.1% ethanol standard | volume of water |
| #1 | 0 | 0 μL | 100 μL |
| #2 | 0.03 | 30 μL | 70 μL |
| #3 | 0.06 | 60 μL | 40 μL |
| #4 | 0.10 | 100 μL | 0 μL |
- Transfer 10 μL into the appropriate wells of a clear 96-well plate according to the map below.
- Prepare the Working Reagent in a microcentrifuge tube by mixing the following:
- 640 μL of Assay Buffer
- 8 μL of Enzyme A
- 8 μL of Enzyme B
- 20 μL of NAD Solution
- 112 μL of MTT Solution
- Use the vortexer to completely mix the solution.
- Add 90 μL of Working Reagent into each of the wells.
- Mix completely by pipetting the solution up and down.
- Be sure to change pipet tips between wells!
- Cover the plate and incubate for 30 minutes at room temperature.
- After the incubation, add 100 μL of Stop Reagent to each of the wells.
- Mix completely by pipetting the solution up and down.
- Be sure to change pipet tips between wells!
- Acquire the A565 readings using the plate spectrophotometer and distribute to participants.
The following should be checked after the laboratory session:
- Ensure that all ethanol assay reagents are returned to -20 °C freezer.
