BG11_and_inducer_preparation

BG11 medium for interlab study (after van Alphen et al., 2018)

A	B	C
Macronutrients	mM	g/L
NaNO3	17.65	1.5
KH2PO4	0.294	0.04
MgSO4 * 7H2O	0.304	0.075
CaCl2 * 2H2O	0.244	0.036
FeCl3 * 6H2O	0.015	0.004
Na2EDTA * 2 H2O	0.05	0.0186
NaHCO3	9.44	1
HEPES	5	1.2
Micronutrients	µM	mg/L
H3BO3	46.25	2.86
MnCl2 * 4H2O	9.15	1.81
ZnSO4 * 7H2O	0.77	0.222
Na2MoO4 * 2H2O	1.62	0.391
CuSO4 * 5H2O	0.316	0.079
Co(NO3)2 6H2O	0.168	0.049

Molarity and mass concentration of all BG11 media components after van Alphen et al. 2018CuSO4 was omitted for the Interlab study to have a copper free media.

A	B
Component	mL/L
Buffer solution	5 mL
Stock 1	2.5 mL
Stock 2	2.5 mL
Stock 3	2.5 mL
Stock 4	10.5 mL
Fill with autoclaved water to 1L

Overview over the stock solutions used to create the final BG11 media

BG-11 is split into four stock solutions such that incompatible (i.e. because of low solubility) compounds at high concentration are avoided. Additionally, a minimal amount of HEPES buffer is added to ensure the same starting pH of the cultures.

The final medium is prepared by adding buffer and each stock solution to 500 ml autoclaved water measuring cylinder and filling it to a total of 1 liter.

A	B
HEPES buffer solution	g/L
HEPES, NaOH (pH 8)	240

Mass concentration of HEPES buffer solution

Dissolve 24g of HEPES in 40 ml of MilliQ water

Under stirring adjust pH to 8 using NaOH

Add MilliQ water to 100 ml

Filter sterilize the solution

A	B
Stock 1	g/L
NaNO3	600
CaCl2 * 2H2O	14.4

Mass concentration of stock solution 1

Dissolve 1.4g CaCl2*2H2O in 70 ml of MilliQ water

Add 60g of NaNO3 under vigorous stirring at 50°C until fully dissolved.

Cool solution to room temperature and add MilliQ water to 100 ml.

Filter sterilize the solution

A	B
Stock 2	g/L
MgSO4 * 7H2O	30
FeCl3 * 6H2O	1.62
Na2EDTA * 2 H2O	2.24
Micronutrients	40 mL in 100 mL

Mass concentration of stock solution 2

Dissolve 0.224g EDTA Na2*2H2O in 40 mL MilliQ water followed by 0.162g FeCl3·6H2O and leave the solution stirring over night

Dissolve 3g MgSo47H2O in 15 ml MilliQ water and add it to the EDTA Na22H2O + FeCl3·6H2O solution

Add 40 ml of Micronutrient solution (See Section Micronutrient stock solution preparation) to this mix and fill to 100 ml using MilliQ water

Filter sterilize the solution.

A	B
Stock 3	g/L
KH2PO4	16
Na2EDTA * 2 H2O	5.2

Mass concentration of stock solution 3

Dissolve 1.6 g K2HPO4 in 40 ml of MilliQ water

Dissolve 0.52 g EDTA Na2*2H2O in the same solution

Fill to 100 ml using MilliQ water

Filter sterilize the solution

A	B
Stock 4	g/L
NaHCO3	80

Mass concentration of stock solution 4

Dissolve 8 g NaHCO3 in 80 ml MilliQ water

Fill to 100 ml using MilliQ water

Filter sterilize the solution

A	B
Micronutrients	g/L
H3BO3	2.86
MnCl2 * 4H2O	1.81
ZnSO4 * 7H2O	0.222
Na2MoO4 * 2H2O	0.391
CuSO4 * 5H2O	0.079*
Co(NO3)2 6H2O	0.049

Mass concentration of Micronutrient stock solution

• Copper sulfate is not to be added for the interlab study as we need copper free media for PpetE induction

Add trace metal salts in the order they are listed to 100 ml of MilliQ water and wait for complete dissolution between each addition

Filter sterilize the solution

A	B	C
Rhamnose stock solution	g/L	mM
Rhamnose	164	1000

Mass concentration of Rhamnose inducer stock solution 1

Dissolve 8.2 g of rhamnose in 40 ml MilliQ water

Add MilliQ water to 50ml

Filter sterilize the solution

A	B	C
CuSO4 stock solution	g/L	mM
CuS04*5H20	0.0025	0.1

Mass concentration of copper sulfate inducer stock solution

Dissolve 25 mg CuS04*5H20 in 8 ml MiliQ water

Add to 10 ml using MilliQ water

Dilute 10 µl of this solution in 9.99 ml MilliQ water

Filter sterilize the solution