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 20-mL batches of LB agar with incremental dilutions of caffeine from 20 mM to 40 mM. (This range was guessed based on observation of the distance of colonies from the disk in MWSU photos.) The agar-caffeine dilutions should have 5 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 agar 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) | Comment |
|---|---|---|---|---|---|---|---|---|
| 1 | 5 | 20 | 0 | 0 | 0 | 0.000 | 40 | Negative control |
| 2 | 5 | 20 | 0 | 0 | 20 | 0.078 | 40 | |
| 3 | 5 | 20 | 0 | 0 | 21 | 0.082 | 40 | |
| 4 | 5 | 20 | 0 | 0 | 22 | 0.085 | 40 | |
| 5 | 5 | 20 | 0 | 0 | 23 | 0.089 | 40 | |
| 6 | 5 | 20 | 0 | 0 | 24 | 0.093 | 40 | |
| 7 | 5 | 20 | 0 | 0 | 25 | 0.097 | 40 | |
| 8 | 5 | 20 | 0 | 0 | 26 | 0.101 | 40 | |
| 9 | 5 | 20 | 0 | 0 | 27 | 0.105 | 40 | |
| 10 | 5 | 20 | 0 | 0 | 28 | 0.109 | 40 | |
| 11 | 5 | 20 | 0 | 0 | 29 | 0.113 | 40 | |
| 12 | 5 | 20 | 0 | 0 | 30 | 0.117 | 40 | |
| 13 | 5 | 20 | 0 | 0 | 31 | 0.120 | 40 | |
| 14 | 5 | 20 | 0 | 0 | 32 | 0.124 | 40 | |
| 15 | 5 | 20 | 0 | 0 | 33 | 0.128 | 40 | |
| 16 | 5 | 20 | 0 | 0 | 34 | 0.132 | 40 | |
| 17 | 5 | 20 | 0 | 0 | 35 | 0.136 | 40 | |
| 18 | 5 | 20 | 0 | 0 | 36 | 0.140 | 40 | |
| 19 | 5 | 20 | 0 | 0 | 37 | 0.144 | 40 | |
| 20 | 5 | 20 | 0 | 0 | 38 | 0.148 | 40 | |
| 21 | 5 | 20 | 0 | 0 | 39 | 0.151 | 40 | |
| 22 | 5 | 20 | 0 | 0 | 40 | 0.155 | 40 | |
| 23 | 5 | 20 | 4 | 0.014 | 0 | 0.000 | 40 | Positive control |
| 24 | 5 | 20 | 4 | 0.014 | 40 | 0.155 | 40 | Positive control |
Pour each agar dilution into two 35-mm plates, labeled A and B. Flip all plates after 3 hours and let dry for two days.
Incubate LB broth cultures of each clone 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 and dilute concentration to 0.05 OD with LB+antibiotic.
For the “A” series, place 2-μL spot of each clone onto the plates.
For the “B” series, spread 10 uL of each clone with 3-5 beads onto the plates. Swirl beads carefully without the lid, discard beads, and replace lid. Store plates inverted.
Incubate all plates at 37° overnight. Let grow for 4 days at RT. Inspect daily for growth. Count colonies per plate or colonies per area to determine the plate with the optimal caffeine concentration.
