Optimal caffeine concentration on plates
Overview
Before strains with the tetracycline-theophylline fitness module can be grown in broth, we need to know how much caffeine the cells need, to produce an amount of theophylline that grants survival. Previous experimentation shows that colonies on plates are able to grow in a ring around caffeine-soaked disks, leading to my hypothesis that there is an optimal range of caffeine in which cells can survive. Too much caffeine or too little caffeine prevents cells from growing. However, we do not know whether cells growing in broth require the same concentration of caffeine as cells growing in plates. This experiment was designed to determine the concentration required by cells growing on plates, specifically pertaining to JM109 strains #12 and #23, which have been demonstrated to be the most competent clones on agar plates.
Protocol
Prepare 80-mL batches of LB agar with incremental dilutions of caffeine from 0 mM to 10 mM. (This range was guessed based on a model of caffeine diffusion created by Morgan Spencer.) The agar-caffeine dilutions should have 20 μg/mL tetracycline and 0.5 mg/mL L-arabinose. The negative control should contain no caffeine. The first positive control should have 4 mM theophylline and the second positive control should have no tetracycline. See the below table for details. Add anhydrous caffeine and anhydrous theophylline to the respective agar batches before autoclaving. Add tetracycline and L-arabinose from liquid stocks after autoclaving and cooling to below 60°.
| # | Tetracycline (μg/mL) | Tet (uL) | Theophylline (mM) | Theo (g) | Caffeine (mM) | Caff (g) | L-arabinose (uL) |
|---|---|---|---|---|---|---|---|
| 1 | 20 | 320 | 0 | 0 | 0 | 0.000 | 160 |
| 2 | 20 | 320 | 0 | 0 | 1 | 0.016 | 160 |
| 3 | 20 | 320 | 0 | 0 | 2 | 0.031 | 160 |
| 4 | 20 | 320 | 0 | 0 | 3 | 0.047 | 160 |
| 5 | 20 | 320 | 0 | 0 | 4 | 0.062 | 160 |
| 6 | 20 | 320 | 0 | 0 | 5 | 0.078 | 160 |
| 7 | 20 | 320 | 0 | 0 | 6 | 0.093 | 160 |
| 8 | 20 | 320 | 0 | 0 | 7 | 0.109 | 160 |
| 9 | 20 | 320 | 0 | 0 | 8 | 0.124 | 160 |
| 10 | 20 | 320 | 0 | 0 | 9 | 0.140 | 160 |
| 11 | 20 | 320 | 0 | 0 | 10 | 0.155 | 160 |
| 12 | 20 | 320 | 4 | 0.058 | 0 | 0.000 | 160 |
| 13 | 20 | 320 | 4 | 0.058 | 10 | 0.155 | 160 |
Pour the agar dilutions into 60-mm plates. Flip plates after 3 hours and let dry for two days.
Incubate LB broth cultures of clones #12 and 23 overnight at 37°, with 0.5 mG/mL L-arabinose and either amp or amp+chlor. Clones with a chaperone require chloramphenicol to prevent curing.
Measure A590. Combine the clones and dilute with LB broth so that each is at 0.025 OD. For each caffeine dilution, put 5-8 plating beads onto the plate and pipette 20 uL cells on top. Swirl beads carefully without the lid, discard beads, and replace lid. Store plates inverted.
Incubate all plates at 37° overnight. Let grow for 2 days at RT. Inspect daily for growth.
