BE FPOP(Su24):Preparing CRISPRi experiment

Introduction

Now that you are familiar with the strategy you will use for this project, you will prepare cultures to test the effect of different gene targets on ethanol yield. The targets you will test are the genes that encode the enzymes responsible for the upstream and downstream reactions in the ethanol pathway of the E. coli chassis.

In total you will test five conditions:

1. MG1655 is the E. coli chassis without the CRISPRi system.
2. MG1655 psgRNA_ldhA is the E. coli chassis with the CRIPSRi system programmed to target the ldhA gene (Cas9 protein + sgRNA complementary to ldhA sequence).
3. MG1655 psgRNA_pta is the E. coli chassis with the CRIPSRi system programmed to target the pta gene (Cas9 protein + sgRNA complementary to pta sequence).
4. MG1655 psgRNA_ack is the E. coli chassis with the CRIPSRi system programmed to target the ack gene (Cas9 protein + sgRNA complementary to ack sequence).
5. MG1655 psgRNA_adhE is the E. coli chassis with the CRIPSRi system programmed to target the adhE gene (Cas9 protein + sgRNA complementary to adhE sequence).

Protocols

Practice pipetting skills

To ensure you are familiar with the best practices demonstrated by the FPOP team for pipetting liquids you will practice preparing dilutions. For this, complete the following steps to dilute a blue dye (0.1% food coloring).

1. Obtain an aliquot of blue dye, an aliquot of water, and ten cuvettes from the front laboratory bench.
2. Using your P20, measure 10, 15 and 20 μL of the 0.1% food coloring stock solution into the bottom of three cuvettes.
   • With your P1000, add water to bring the final volume to 1 mL (=1000 μL) and pipet up and down to mix.
3. Using your P200, measure 20, 50 and 100 μL of the 0.1% food coloring stock solution into the bottom of three more cuvettes.
   • With your P1000, add water to bring the final volume to 1 mL.
4. Using your P1000, measure 100, 200, and 400 μL of 0.1% food coloring solution into the bottom of three more cuvettes.
   • Add water to bring the final volume to 1 mL.
5. Add 1 mL of water to an empty cuvette.
   • This will be used to blank the spectrophotometer.
6. Carefully take all of your cuvettes to the spectrophotometer at the back of the teaching laboratory.
7. Using the touchscreen select "Fixed Wavelength" and ensure that the value is set to 630 nm.
8. Insert your blank into the cuvette holder.
   • Be sure the flat window of the cuvette (and not the indented sides) are in the light beam that travels from front to back!
9. Replace the blank with your first sample, close the door to the spectrophotometer and then select "Read."
   • Record this value in your laboratory notebook.
10. Measure the remaining samples and record the values in your laboratory notebook.
11. Use the template (provided here) to graph your data.
12. In your laboratory notebook, complete the following:
    • How closely do your data points fit the trendline?
    • What do these data suggest about your pipetting precision?

Prepare cultures for ethanol yield experiment

To test the effect of sgRNA_target on increasing the production of ethanol, the co-transformed E. coli MG1655 cells will be incubated for at least 24 hours. The cultures will be grown anaerobically using screw-top tubes. Remember from lecture that cells grown anaerobically should produce more ethanol; however, the goal of using the CRISPRi system is to further enhance the production of ethanol by manipulating gene expression of an enzyme in the anaerobic fermentative pathway.

Rather than using anaerobic chambers or gas replacement, a much simpler technique was employed to omit oxygen from the cultures for this experiment. Because screw-cap tubes maintain a tight seal, cultures can be maintained in a low-O₂ environment. For this technique cultures are grown at least 24 hrs to ensure the following: 1. that the oxygen present at the time of inoculation is depleted by the growing cells and 2. that the cells grow in the O₂-depleted environment long enough to undergo anaerobic fermentative metabolism.

1. Obtain 5 screw-top tubes from the front laboratory bench and label as shown below:
   • MG1655
   • MG1655 psgRNA_ldhA
   • MG1655 psgRNA_pta
   • MG1655 psgRNA_ack
   • MG1655 psgRNA_adhE
2. Attach one of the colored dot stickers from your drawer to each tube.
   • This will ensure we know which tubes belong to which teams!
3. Obtain an aliquot of LB broth from the front laboratory bench.
4. Transfer 5 mL of the LB broth into the MG1655 screw-top tube.
5. To the remaining LB broth add 25 μL of chloramphenicol and 25 μL of ampicillin antibiotic stocks.
   • The antibiotics are used to ensure the plasmids for CRISPRi system are maintained by the cells.
6. Also to the remaining LB broth add 0.25 μL of aTc (anhydrotetracyline) stock.
   • The aTc is used to turn-on expression of the gene that encodes the dCas9 protein.
7. Tightly close the tube that contains the LB broth + antibiotics + aTc and mix using the vortexer.
8. Transfer 5 mL of the LB broth + antibiotics + aTc to the four remaining screw-cap tubes.
9. To inoculate each of the screw-cap tubes, use the starter cultures at the front laboratory bench.
   • From each starter culture, transfer 5 μL into the appropriately labeled tube that you prepared.
   • Tightly close each screw-cap tube.
10. Place all of your cultures in the rotating wheel in the 37 °C incubator.
11. In your laboratory notebook, complete the following:
    • For each of the conditions that will be tested, do the cells contain the CRISPRi system?
    • Which culture conditions provide controls? What does each control indicate / validate?
    • Rank order the culture conditions from expected highest ethanol yield to expected lowest ethanol yield.
    • In which culture condition do you expect to see the lowest yield of ethanol? Why?
    • In which culture condition do you expect to see the highest yield of ethanol? Why?