Culture Media

In our project we used different kinds of media for cultivation, transformation and chip preparation. While complex media such as LB offered an easy way of cultivation, minimal media such as M9 or HM provide a low autofluorescence for fluorescence measurements. All used media are listed below.

Complex media

Luria-Bertani Medium (LB)

1. weight components

   5 g/L NaCl

   10 g/L tryptone

   5 g/L yeast extract

   (15 g/L agar for plates)

2. fill up to 1 L with deionized water
3. mix well by shaking
4. autoclave
   1. autoclaving tape, caps slightly unscrewed
   2. base of the pot has to be covered with deionized water
   3. close lid
   4. heat level 3 until the pressure valve opens
   5. reduce heat level to 1.5
   6. set timer to 20 minutes
   7. turn heater off
   8. wait until the pressure valve retracts (30-45 minutes)
   9. open, close caps & shake
5. for plates, wait until you can touch the bottle (<60°C, clean bench!)
6. add antibiotics (1 µl/ml) and shake (gloves!)

Terrific-Broth-Medium (TB)

1. components for 1 :

   4 ml/L glycerol

   12 g/L tryptone

   24 g/L yeast extract

2. fill up to 900 ml with deionized water
3. mix well by shaking
4. autoclave
5. components 2:

   0.17 M KH₂PO₄

   0.72 M K₂HPO₄

6. dissolve in 100 ml deionized water and sterilize it by passing it through a filter
7. after autoclaving and cooling down, add sterile phosphate solutions

Nutrient Agar medium (NA)

1. Components for 1 L

   Enzymatic Digest of Gelatin 5 g

   Beef Extract 3 g

   Agar 15 g

2. Final pH: 6.8 ± 0.2 at 25°C
3. Suspend 23 g of the medium in one liter of purified water.
4. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
5. Autoclave at 121°C for 15 min.

Minimal Media

Hartmans Minimal Medium (HM)

This mineral salts medium is based on (Hartmans et al., 1989).

Three 100x stock solutions are prepared according to the following recipe and stored at 4°C:

• 100x Buffer,composed of 388 g dipotassium phosphate, 212 g monosodium phosphate dihydrate per Liter, adjusted to a pH of 7.0 and sterilized by autoclaving.
• 100x ammonium sulfate composed of 200 g/l ammonium sulfate and sterilized by autoclaving.
• 100x MM salts

1. Add 1 g EDTA to 25 ml water.
2. Add drops of 10 M sodium hydroxide until EDTA is completely dissolved.
3. Adjust pH back to 4.0 with concentrated hypochloric acid.
4. Fill up with water to 800 ml and dissolve following components:
   1. 10 g magnesium chloride sexahydrate
   2. 200 mg zinc sulfate heptahydrate
   3. 100 mg calcium chloride dihydrate
   4. 500 mg iron(II) sulfate heptahydrate
   5. 20 mg sodium molybdate dihydrate
   6. 20 mg copper(II) sulfate quintahydrate
   7. 40 mg cobalt(II) chloride sexahydrate
   8. 100 mg manganese(II) chloride sexahydrate

For 1x medium without carbon source, pool 10 ml of each 100x stock solution and fill up to 1000 ml with sterile, deionized water and store at 4°C.

M9 Minimal Medium (M9)

Transformation Medium

Super Optimal broth with Catabolite repression medium (SOC)

1. components

   0,5% yeast extract

   2% tryptone

   10 mM NaCl

   2.5 mM KCl

   20 mM MgSO₄

2. fill up with deionized water
3. adjust to pH 7.5 with NaOH
4. after autoclaving, add 20 mM sterile glucose solution (filter sterilization)

References

• Hartmans, S., Smiths J.P., Volkering F., and de Brondth, J.A. (1989). Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X. Applied and Environmental Microbiology, Nov. Available at: http://www.ncbi.nlm.nih.gov/pubmed/?term=PMC203180.