SOP for Shakeflask Experiments

Standard Operating Procedure (SOP) BaSysBio Project

Batch shakeflask cultures of Bacillus subtilis

Description

Standard cultivation protocol for batch shakeflask experiments with Bacillus subtilis. Experiments are performed in batch on minimal medium with glucose as carbon and energy source. In short, the tested strain is first plated on LB-agar plates, transferred to LB-medium for preculturing, transferred to M9 medium for a second preculturing step and finally cultured in 500 mL non-baffled shakeflasks on M9 medium. Unless explicitly stated, media compositions are as described by Harwood and Cutting (1). During the time course of the shakeflask experiment, the OD600 of the broth is determined for calculating the maximum growth rate of the tested strain. Optionally, samples can be collected for determining the extracellular concentration of glucose and several organic acids by HPLC analysis.

SUMMARY: Solution G (50 mM FeCl3) is supplemented with 100 mM citric acid. Citric acid acts as a chelating agent to keep the iron from precipitating in the medium, thereby aiding the uptake of iron by Bacillus subtilis. With respect to growth of B. subtilis, the addition of citric acid results in a shorter lag-phase and in a higher specific maximal growth-rate (see Appendix B).

REVISIONS ARE MARKED IN RED

Materials

Bacteria Stock:  Stock of Bacillus subtilis 168 trp+ frozen in 15% (v/v) glycerol at -80oC. Unlike the 168 parent strain, this strain is not auxotrophic for tryptophan due to the stable integration of the trpC gene into the genome. The procedure for making glycerol stocks is described in the SOP entitled: ‘Preparing glycerol stocks for Bacillus subtilis’. Important: Do not defrost the glycerol stock, freeze-thaw cycles influence the stability of the strain.

Required solutions (see Appendix A): A. Luria Bertani (LB) medium B. Luria Bertani (LB) medium with 1.5% (w/v) agar C. M9 minimal medium 5x stock D. Trace elements 100x stock E. 100 mM CaCl2 solution F. 1 M MgSO4 solution G. 50 mM FeCl3 in 100 mM citric acid solution H. 50% (w/v) glucose solution

Required equipment:  LB agar plates  500 mL non-baffled shakeflasks with sterile caps. For reproducibility reasons and to reduce foaming, non-baffled shakeflasks should be used.  10 mL Greiner culture tubes

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D:\Docs\2017-12-16\059268d3adc2b6ef26636708a429a4b2.doc  Incubator equipped with shaker. Temperature should be controlled at 37oC. Shaker should be able to reach speeds up to 300 rpm with a shaking diameter of 2.5 cm.  Spectrophotometer set to 600 nm for OD determination.

Method

Important: Make sure all steps are performed aseptically to prevent contamination of the shakeflask experiments.

Day -2: Creating LB plates

1. Heat up the LB-agar medium in the microwave (sol. B) 2. Add the appropriate amount of required antibiotics (dependent on grown strain). 3. Pour LB-agar plates (25 mL/plate). 4. Leave the plates standing until they solidify. 5. Add a few crystals from the glycerol stock on the LB-agar plates (1 plate/tested strain). 6. Streak the cells out over the agar plate. 7. Incubate overnight at 37°C.

The plating step is just to help the strain grow more easily when testing crippled mutants.

Day -1: Preculturing on LB and M9 medium

1. Check plates from previous day for growth. 2. Add 5 mL of LB medium (sol.A) to a 10 mL culture tubes (1 tube/tested strain). 3. Inoculate the culture tubes with 1 colony from the appropriate plate. 4. Incubate for 2-4 hours at 37°C, shaking at 200-300 rpm to an OD of 0.3-1.0. 5. Prepare the appropriate amount of M9 medium. For 1 liter of medium add in the following order:

 781 mL of nanopure H2O

 1 mL 100 mM CaCl2 (sol. E)  10 mL trace salts 100x stock (sol.D)

 1 mL 1 M MgSO4 (sol. F)

 1 mL 50 mM FeCl3/100 mM C6H8O7 (sol. G)  6 mL of glucose 50% (w/v) stock solution (sol. I)  200 mL M9 5x stock (sol. C)

6. Final glucose concentration in the M9 medium will thus be 3 g/L. 7. Add 35 mL of M9 medium to a 500 mL shakeflask or 18 mL of M9 medium to a 250 mL shakeflask. It is recommended to use 3 shakeflasks per tested strain. 8. Check the OD600 of the LB culture. Use appropriate dilutions to assure the measured OD600 remains below 0.250. The final OD600 of the LB culture should be between 0.3 and 1.0. 9. Prepare three dilutions of the LB preculture in M9 medium; 2000x, 4000x and 6000x. These M9 precultures should be made 12-16 hours before starting the shakeflask experiments. 10. Incubate the shakeflasks overnight in the shaker at 37°C, 300 rpm. 11. Store remaining M9 medium overnight in the fridge (4°C).

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D:\Docs\2017-12-16\059268d3adc2b6ef26636708a429a4b2.doc Day 0: Batch shakeflask experiments

1. Take the M9 medium out of the fridge. Shake the bottle, since precipitates may form during storage. 2. Add 35 mL of M9 medium to each shakeflask and preheat 15 minutes in the incubator at 37oC, 300 rpm. 3. Measure OD600 of the M9 precultures. Use appropriate dilutions to measure OD600 above 0.250. 4. Select the M9 preculture with an OD600 between 0.2 and 1.0 to inoculate the shakeflasks. Ideally, the selected preculture should still be in exponential phase, thereby minimizing the lag-phase of the shakeflask experiment. To verify this, one could determine the residual glucose concentration in the culture tubes using for example glucose strips or the GluCell. 5. Determine the amount of preculture that has to be added. Starting OD600 of the shakeflask experiment should be between 0.03-0.05. Pelletize the cells in the required amount of preculture by centrifugation at room temperature.(Recommended: 2 minutes at 5000- 10000g). Resuspend the pelletized cells in 100 µL of fresh medium. 6. Inoculate the shakeflasks with the resuspended cells. 7. Check the inoculation OD of the culture by sampling 1 mL and measuring the OD600. 8. Incubate the shakeflasks at 37°C in the shaker (300 rpm). 9. After measuring the OD600, transfer the undiluted sample into an eppendorf tube and spin down at 15000 g for 3 minutes. Decant the supernatant and store it in a separate eppendorf tube at -20°C for measuring extracellular concentrations of glucose and organic acids. Collecting the supernantant is optional, and is only needed when determining specific uptake rates. 10. Check the growth of the culture by every hour sampling 1 mL of broth and measuring the OD600. Use appropriate dilutions to assure the measured OD600 remains below 0.250. The time the shakeflasks are not shaken in the incubator should be minimized to prevent oxygen limitation, especially when taking samples at high OD values. Therefore, when sampling no more than two shakeflaks should be taken out of the incubator at the same. 11. Keep on taking samples until the OD600 reaches a plateau or starts to drop. At this point, the cells have reached the stationary phase and the glucose in the medium is depleted. With a glucose concentration of 3 g/L glucose the maximal OD600 will be approximately 2.0-2.5. Alternatively, the cultivation can be aborted at the required point in time or OD600. To prevent possible pre-culture effects, final samples (for e.g. transcriptomic, proteomic or metabolomic analysis) should always be harvested between an OD of 0.5 and 1.5. 12. Calculate the maximal specific growth-rate as described in the SOP entitled: ‘Calculating the maximum specific growth rate of Bacillus subtilis’. 13. Check if growth parameters match the provided reference values.

Things to consider:  Oxygen limitation is a potential problem when cultivating cells in shakeflasks. Under the following conditions oxygen limitation was not observed: i. Shaker with a 2.5 cm shaking diameter operated at 300 rpm ii. Initial glucose concentration of 3 g/L, resulting in a maximal OD600 of 2.0- 2.5 iii. Total working volume of 35 mL in 500 mL non-baffled shakeflasks When increasing the initial glucose concentration, increasing the working volume, or decreasing the shaker speed, one should again check whether the cells are not hampered in their growth by oxygen limitation.  Inoculating with an OD600 below 0.03 can sometimes result in very long lag phases, thereby prolonging the duration of the experiment.

References

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D:\Docs\2017-12-16\059268d3adc2b6ef26636708a429a4b2.doc 1. Harwood, C. R., and S. M. Cutting. 1990. Molecular Biological Methods for Bacillus. John Wiley and Sons Ltd., Chichester.

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D:\Docs\2017-12-16\059268d3adc2b6ef26636708a429a4b2.doc Appendix A: Contents, sterilization and storage of required solutions

A) Luria Bertani (LB) medium: 10 g Bacto-Tryptone (Becton Dickinson and Co) (per liter) 10 g NaCl (Merck) 5 g Yeast extract (Becton Dickinson and Co) Medium can be autoclaved at 121oC for 20 minutes and stored at RT.

B) Luria Bertani (LB) agar medium: 10 g Bacto-Tryptone (Becton Dickinson and Co) (per liter) 10 g NaCl (Merck) 5 g Yeast extract (Becton Dickinson and Co) 15 g Agar (Invitogen) Medium can be autoclaved at 121oC for 20 minutes and stored at RT.

. C) M9 minimal medium: 42.5 g Na2HPO4 2H2O(Merck) (5x stock solution, per liter) 15 g KH2PO4 (Merck) 5.0 g NH4Cl (Merck) 2.5 g NaCl (Merck) Adjust to pH 7.0 using 4M NaOH. Solution is autoclavable at 121oC for 20 minutes. Store at RT.

. D) Trace elements solutions: 100 mg MnCl2 4H2O (Merck) (100x stock solution, per liter) 170 mg ZnCl2 (Sigma) . 43 mg CuCl2 2H2O (Merck) . 60 mg CoCl2 6H2O (Merck) . 60 mg Na2MoO4 2H2O (Aldrich) Contrary to what is described by Harwood and Cutting (1) MgSO4, CaCl2 are added as separate solutions to prevent precipitation problems. FeCl3 is also added as a separate solution as this compound is unstable. Filter-sterilize the solution and store in the dark at RT.

. E) Calciumchloride solution: 1.47 g CaCl2 2H2O (Merck) (100mM, per 100 mL) Autoclavable and store at RT.

. F) Magnesiumsulfate solution: 24.6 g MgSO4 7H2O (Merck) (1M, per 100 mL) Autoclavable and store at RT.

. G) Ironchloride solution: 1.35 g FeCl3 6H2O (Sigma) . 2.10 g C6H8O7 H2O (Sigma) (50 mM, 100 mM citric acid, 100 mL) Filter sterilize and store in the dark at 4oC.

H) Glucose solution: 500 g C6H12O6 (Merck) (50% w/v, per liter) Dissolving the glucose takes time: use a warm waterbath to speed up the dissolving. Solution is autoclavable at 121oC for 20 minutes. Filter sterilization is also possible.

NOTE: For all solutions Nanopure H2O with a electric resistance of more than 17 MOhm/cm (= conductivity of less than 60 nS * cm) is used.

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D:\Docs\2017-12-16\059268d3adc2b6ef26636708a429a4b2.doc Appendix B: Effect of citric acid addition to Solution G

To illustrate the effect of citric acid addition to the FeCl3 solution G on the growth of Bacillus subtilis, M9 medium was prepared using: i. the original solution G (50 uM FeCl3) ii. the new solution G (50 mM FeCl3 and 100 mM citric acid) Pre-cultures were all grown on M9 medium containing the Fe-citrate solution (as specified in the above SOP).

The addition of citric acid results in a decreased lag-phase for Bacillus subtilis, enabling the cells to quicker enter the exponential growth phase. More importantly, the addition of citric acid significantly increased the maximal growth-rate. The previously reported maximal specific growth-rate in shakeflasks was 0.57±0.07. Based upon the first set of experiments the new maximal specific growth-rate was calculated to be 0.70±0.07. More experiments need to be performed to accurately determine the latter value and its corresponding standard deviation.

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