Updated on Monday 29 Nov. 2021
This is a snapshot of the protocol we used on 27-29 Nov 2021 Saturday-Monday: Trying an approach where we first start the cells growing in LB, then add BLOSMM v0.1 to the culture to stress them osmotically. Today's focus is on seeing if transformed E. coli cells like our GFP Agar Art strain can be made to grow on BLOSMM v0.1.
The most up-to-date version of the protocol is here.
¶ (Draft) Protocol
¶ Materials
- at least 20 mL of LB media
- seven 14 mL culture tubes
- agar plate with DH5α E. coli colonies (in BosLab 4°C fridge)
- agar plate with GFP-expressing E. coli colonies (in BosLab 4°C fridge)
- 1000X chloramphenicol stock solution (in BosLab −20°C freezer)
- 5 mL serological pipettes and compatible pipettor
- P10 or P20 micropipettor with compatible tips
- two sterile inoculation loops
- at least 100 mL sterile deionized or distilled H2O
- at least 50 mL BLOSMM v0.1
- at least 50 mL 1 M trehalose
¶ Equipment
- OD600 (optical density) reader or spectrophotometer
- Dry culture carrier comprising either:
- sterile petri dishes and pieces of filter paper cut to fit in them, or
- new unused zip lock bags and pieces of filter paper cut to fit in them
- 37°C plate incubator to use for drying cells (consider drying at 30°C if cells don't revive)
- 37°C shaker incubator for liquid cultures
- tube vortexer
- PCR workstation, or laminar flow workbench, or clean work surface with bunsen burner to create a sterile airspace in which to work
¶ Procedure used
- Work in a sterile airspace such as the BosLab PCR workstation (a.k.a. “PCR hood”).
- I labeled 4 culture tubes with my initials, the date, and as follows:
- LB blank
- LB DH5α
- LB+chloro blank
- LB+chloro GFP
- With a 5 mL serological pipette, I transferred 3 mL LB media to each tube.
- With a P20 micropipette (P10 also OK), I transferred 4 µL of 1000X chloramphenicol stock solution to each of the “LB+chloro” tubes.
- I vortexed each tube for 10 seconds, making sure that the 4 µL of chloramphenicol gets thoroughly mixed into the LB.
- I used a sterile inoculation loop to transfer 1 DH5α colony from the agar plate to the LB DH5α tube.
- I used another sterile inoculation loop to transfer 1 GFP Agar Art E. coli colony from the agar plate to the LB+chloro GFP tube.
- I started incubating all four tubes in the orbital shaker at 37°C and 250 RPM.
- I noted the time I started incubation as 5:43 PM Saturday 27 Nov 2021.
- The next day, Sunday 28 Nov:
- I let it grow to saturation over the next 22 hours because I didn't have time to come in to look for beginning of exponential phase.
- FL 5 Apr 2022: In retrospect, leaving the culture incubating for 22 hours maybe wasn't the smartest thing to do. According to this growth curve we did at a later Novice Night in March 2022, saturation happens within 4 hours of inoculation-- 22 hours is really excessive and probably killed off most of the bacteria.
- Note: Next time consider using 4.2mL LB so we can take two OD600 measurements (each measurement consumes 0.6mL of the culture).
- Add 3 mL BLOSMM v0.1 to each tube.
- Add the phrase “+BLOSMM v0.1” to the label of each tube.
- Label three new sterile culture tubes as follows:
- 50% BLOSMM v0.1 blank
- 50% BLOSMM v0.1 DH5α
- 50% BLOSMM v0.1 + chloro GFP
- Pipette 3.5 mL sterile deionized H2O into each of these four new tubes.
- Then pipette 3.5 mL BLOSMM v0.1 into each of these tubes.
- Using a P20 or P10 micropipette, add 7 µL 1000X chloramphenicol stock solution to the “50% BLOSMM v0.1 + chloro GFP” tube.
- Using a P200 micropipette, add 100 µL of the “LB + chloro GFP” culture to the “50% BLOSMM v0.1 + chloro GFP” tube.
- Similarly, add 100 µL of the “LB + DH5α” culture to the “50% BLOSMM v0.1 + DH5α” tube.
- Measure OD600 of the contents of each inoculated tube, being sure to first zero the optical density reader using the corresponding blank. Note:
- Our OD reading machine uses non-sterile cuvettes with a minimum volume requirement of 0.6 mL. Hence we will need to throw away 0.6 mL of cell culture every time we take a reading.
- This is why I chose a total culture volume of 6 mL: it allows us to make 5 readings yet still have half the culture remaining.
- Continue incubating all tubes, noting the time the culture tubes are removed from or placed into the incubator. The LB-based cultures will look pretty cloudy at this point because we're only doing a 1:2 dilution with BLOSMM v0.1.
- Measure OD600 every hour or so, if possible.
- Plot the curve and show it here.
- I let it grow to saturation over the next 22 hours because I didn't have time to come in to look for beginning of exponential phase.
| Tube label | t0 | OD600(t0) | t1 | OD600(t1) | t2 | OD600(t2) | t3 | OD600(t3) | t4 | OD600(t4) | t5 | OD600(t5) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| LB DH5α |
8:59pm 28 Nov 2021 |
1.34 |
out of incubator at 9:52pm, back in incubator at 10:02pm |
1.36 |
out of incubator at 11:19pm, back in incubator at 11:31pm |
1.28 |
out of incubator at 12:21am 29 Nov 2021, back in incubator at 12:32am |
1.21 |
out of incubator at 9:53am, back in incubator at 10:03am | 0.93 | out of incubator at 8:37pm | 1.71 |
| LB+chloro GFP |
0.94 |
0.89 |
0.76 |
0.63 |
1.67 GFP fluorescence visble to eye with UV flashlight |
1.75 GFP fluorescence visible to eye with UV flashlight |
||||||
| 50% BLOSMM v0.1 DH5α |
0.02 |
0.02 |
0.03 |
0.04 |
0.33 |
0.27 slight GFP fluorescence to the eye with UV flashlight (surprisingly) |
||||||
| 50% BLOSMM v0.1+chloro GFP |
0.01 |
0.01 |
0.01 |
0.01 |
0.33 though no noticeable GFP fluorescence to the eye with UV flashlight |
0.27 slight GFP fluorescence to the eye with UV flashlight |
- It is interesting to note that the LB cultures seem to have decreasing OD600 the longer they incubate, while the 50% BLOSMM v0.1 group, the OD600 is either slowly increasing or staying steady. Curious if we may be seeing cell death in the LB cultures, and very slow growth in the 50% BLOSMM v0.1 tubes.
- 29 Nov 2021 morning: Looks like both 50% BLOSMM v0.1 cultures grew overnight! This suggests that BLOSMM has potential as a viable media, and that it may be worthwhile to spend effort on optimizing its recipe for best growth.
- 29 Nov 2021 night (9pm): Looks like there is contamination in the experiment. The LB+BLOSMM v0.1 Blank was cloudy after incubation. Also, it was curious that both 50% BLOSMM cultures showed slight but noticeable GFP fluorescence, which suggests that the GFP strain contaminated the DH5α culture.
¶ Procedure continued
As of Monday 29 Nov 2021, we've never gotten to the following steps:
Centrifuge to pellet cells. Discard supernatant.
Resuspend cells in 1 mL of 1 M trehalose.
Pipette 100 µL per storage spot on the filter paper. (Should the filter paper have storage spots marked then autoclaved ahead of time?)
Put paper in sterile petri dishes with lids closed but NOT parafilmed or sealed. Or put paper in new unused zip lock bags (leave bags open).
Place petri dishes or bags in plate incubator to dry. May take 1-3 days depending on ambient humidity.
Remove petri dishes and/or bags and store in a cool dry place.
TBD: rehydration procedure using LB. Consider rehydrating both in broth and in agar.
¶ (Draft) Protocol for making BLOSMM v0.1 and 1 M trehalose solution
¶ Materials
For BosLab Osmotic Stress Minimal Medium version 0.1 (BLOSMM v0.1)
| Desired composition | for 500 mL final volume of BLOSMM v0.1 |
|---|---|
| 0.01% Miracle-Gro Water Soluble All Purpose Plant Food (Miracle-Gro product 1001123) | 0.05 g Miracle-Gro powder: which is difficult to measure accurately, so I recommend making 10X stock solution by adding enough distilled H2O to 1 g Miracle-Gro to make 1 L, then aliquoting 50 mL for this recipe |
| 1% glucose | 5 g glucose (in BosLab the bottle is labeled “dextrose”) |
|
0.6 M NaCl intended but 1.2 M NaCl actual. Drat! Math error. I put 35.1 g NaCl into a 500 mL volume so I mistakenly made a 1.2 M NaCl concentration, twice the intended 0.6 M! |
17.55 g NaCl intended but 35.1 g NaCl actual |
| 0.1X LB (a.k.a. lysogeny broth, Luria-Bertani broth) | 50 mL LB |
| distilled H2O | enough to make 500 mL final volume |
For 1 mole/liter trehalose dessication solution (1 M trehalose)
| Desired composition | for 500 mL final volume of 1 M trehalose |
|---|---|
| 1 M trehalose | 189.1 g trehalose dihydrate |
| distilled H2O | enough to make 500 mL final volume |
Additional materials and equipment
- Two 1 L culture media bottles (make/autoclave BLOSMM v0.1 and 1 M trehalose)
- autoclave with space for the two 1 L bottles
¶ Procedure
TBD, but try something like:
Mix BLOSMM v0.1 ingredients in a clean 1 L culture bottle.
Mix 1 M trehalose ingredients in another clean 1 L culture bottle.
Autoclave both bottles.
Store at room temperature until needed.