Isolation of Stress-Adapted Bacteria through Single Colony Selection

Samenvatting

Source: Jeong, H., et. al. Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat. J. Vis. Exp. (2016).

This video demonstrates the selective isolation and growth of stress-adapted bacteria from a culture exposed to prolonged stress conditions. The culture is plated on stressor-containing agar, and a single colony is selected to inoculate a liquid medium with the stressor. Absorbance is measured at regular intervals to monitor the growth and confirm the successful isolation of the stress-adapted bacteria.

Protocol

1. Equipment Preparation

1. Obtain a chemostat jar (150-250 ml) or an Erlenmeyer flask (250 ml) containing an inlet port and an outlet port. Connect the ports with silicon tubing, allowing for flow rates of 10-100 ml/hr. Optionally, use an air vent, an air outlet port, and temperature-controlled water inlet and outlet ports.
2. Obtain a device suitable for the chemostat jar that provides for agitation and temperature control (or use a rotary shaking incubator).
3. Obtain two peristaltic pumps in order to deliver fresh medium and collect the culture.
4. Obtain a reservoir jar (10-20 L) containing a medium outlet port and an air inlet port.
5. Obtain silicon tubing suitable for the dilution rate (i.e., ID 0.8 mm, flow range 0.06-36 ml/min; L/S 13 tubing).

2. Medium Preparation and Sterilization

1. Initial Medium
   1. Dissolve 0.3 g glucose, 0.08 g ammonium chloride (NH₄Cl), 0.05 g sodium chloride (NaCl), 0.75 g disodium hydrogen phosphate dihydrate (Na₂HPO₄·2H₂O), and 0.3 g disodium hydrogen phosphate dihydrate (KH₂PO₄) in 90 mL distilled water (D.W.) in a chemostat jar.
   2. Seal the chemostat jar along with the tubing using clamps. Do not seal the air vent.
   3. Sterilize the chemostat jar in an autoclave at 121 °C for 15 min. After sterilization, store the chemostat jar at room temperature.
   4. Dissolve 0.02 g magnesium sulfate heptahydrate (MgSO₄·7H₂O), 0.01 g calcium chloride (CaCl₂), and 0.1 mg thiamine in 10 mL D.W. (solution A).
   5. Filter solution A using a syringe and a pre-sterilized syringe filter (a 0.45 µm pore filter).
   6. Add the solution A filtrate to the chemostat jar.
2. Stress Medium
   1. Dissolve 30 g glucose, 8 g NH₄Cl, 5 g NaCl, 75 g Na₂HPO₄·2H₂O, 30 g KH₂PO₄, and 300 g disodium succinate hexahydrate (Na₂·succinate·6H₂O; the stressor used in this experiment) in 9.9 L D.W. in a reservoir jar.
   2. Seal the reservoir jar along with the tubing using clamps. Do not seal the air vent.
   3. Sterilize the reservoir jar in an autoclave at 121 °C for 15 min. After sterilization, store the jar at room temperature.
   4. Dissolve 2 g MgSO₄·7H₂O, 1 g CaCl₂, and 10 mg thiamine in 100 ml D.W. (solution A).
   5. Filter solution A with a syringe and a pre-sterilized syringe filter (a 0.45 µm pore filter).
   6. Add the solution A filtrate to the reservoir jar.
   7. Aseptically connect the sterilized silicon tubing to the reservoir jar and attach the peristaltic pumps.
3. High-stress Medium
   1. Prepare the medium as in section 2.2, but with a greater concentration of stressor (i.e., 3-5 g/L higher in the succinate adaptation).
      Note: This protocol is for adaptation to a stress that can be delivered via the medium. In the case of physical stressors such as temperature, agitation, or illumination, the cultivation should be designed accordingly.

3. Initial Cultivation

1. Inoculate a single colony of wild-type Escherichia coli (E. coli) in a 15 ml test tube containing 4 ml of initial medium.
2. Incubate the test tube in a shaking incubator for 12 hr at 37 °C and 220 rpm.
3. Aseptically transfer 1 ml of preculture to the chemostat jar.
4. Incubate the chemostat jar, providing for aeration (air 50 ml/min) and agitation (200 rpm), at 37 °C for 6 hr.

4. Stress Adaptation

1. Aseptically connect the end of the silicon tubing from the pumps to the chemostat jar.
2. Start the outlet pump (10 ml/hr or higher) and collect culture.Note: The culture should be in the exponential phase, typically 4-8 hr after initial cultivation.
3. Check the optical density (600 nm) of the culture from the outlet tubing.
4. Start the inlet pump (10 ml/hr, corresponding to a rate of dilution of 0.1 hr-1).
5. Check the optical density of the culture at 600 nm from the outlet tubing every 24 hr.
6. Operate the chemostat for 96 hr (9.6-fold turnover) or more. If the optical density is stable, exchange the reservoir containing the high-stress medium. If the optical density is lower than 0.2, stop the feeding inlet pump for 6 hr. Restart the inlet pump and check that the optical density is over 0.2.
7. Gradually increase the concentration of the stressor by changing to a reservoir containing a higher stressor concentration.
8. Take samples of the adapted culture whenever it reaches a milestone (e.g., a strain adapted to 100 g/L succinate stress), and store them for further genomic analysis.
9. For sample storage, mix the culture sample (0.5 ml) with a sterilized 80% glycerol solution (0.5 ml) and store it at -80 °C.Note: If the microorganism acquires an ability to degrade the stressor during the ALE process, the stressor concentration in the fermentation jar is not the same as that in the fresh reservoir.

5. Single-colony Isolation of the Stress-adapted Strain

1. Prepare agar plate medium (1.6% agar) containing the same stressor and at the same concentration of medium.
2. Plate the outlet culture (0.1 ml) from the chemostat, and incubate at 37 °C for 16 hr.
3. Pick single colonies from the plate using a sterile toothpick and inoculate them in 15 ml test tubes containing the same stressor and at the same medium concentration as in the chemostat, and incubate for 6 hr.
4. Transfer 1 ml of culture broth into a 250 ml Erlenmeyer flask containing 50 ml of medium. Harvest 0.5 ml of the culture broth every 1 hr, and measure the O.D. at 600 nm. Compare the growth rate of the adapted strain to that of the wild-type strain given the stressor.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Mini-chemostat fermentor	Biotron Inc.	-	manufactured by special order
Silicon tubing	Cole-Parmer	Masterflex L/S 13	tubing size can be varied depending on the dilution rate and the size of fermentor jar.
Reservoir jar	Bellco	Media storage bottle	20 L
Chemicals	Sigma-Aldrich	-	reagent grade
Glucose	Sigma-Aldrich	G5767	ACS reagent
NH₄Cl	Sigma-Aldrich	A9434	for molecular biology, suitable for cell culture, ≥99.5%
NaCl	Sigma-Aldrich	746398	ACS reagent, ≥99%
Na₂HPO₄·2H₂O	Sigma-Aldrich	4272	98.5-101%
KH₂PO₄	Sigma-Aldrich	795488	ACS reagent, ≥99%
MgSO₄·7H₂O	Sigma-Aldrich	230391	ACS reagent, ≥98%
CaCl₂	Sigma-Aldrich	793639	ACS reagent, ≥96%
Thiamine·HCl	Sigma-Aldrich	T4625	reagent grade, ≥99%
Na₂succinate·6H₂O	Sigma-Aldrich	S2378	ReagentPlus, ≥99%