Fluorescence_activity_assay_Interlab_Study_PCC_6803

Precultures were started from cultures derived from cryoconserved cells after 48h of growth in copper free BG11-PC medium (hereafter referred to as BG11 medium).

4 Strains were used for the fluorescence activity assay in the Interlab study. All strains were Synechocystis PCC 6803 mutants carrying a fluorescence reporter gene.

Dilute all strains to an OD730 of 0.3 in 35ml of BG11 final volume in a 100ml Erlenmeyer flaks with a cotton plug

Grow all cultures in shaking incubators set at 100 rpm under 50 µmol photons · m-2 · s-1 constant white light illumination, ambient CO₂, and 30°C over night until OD730 0.5-0.6

Transfer all cultures into 50ml falcons

Adjust the OD730 of all cultures to 0.5 with BG11 containing 10 µg mL-1 chloramphenicol to a final volume of 50 ml

Measure the full 400-750 nm OD730 spectrum and OD730 nm of these cultures

Rinse the preculture flasks with 25 ml Copper-free BG11 twice

Fill 20 ml of each preculture adjusted to an OD730 of 0.5 back into the preculture flasks

Fill 20 ml of each preculture adjusted to an OD730 of 0.5 into new 100ml erlenmeyer flasks with cotton plugs

Add the respective inducer to the flasks prepared in Step 2.5

To each Synechocystis strain carrying the following plasmid add 200 µl of the following inducer:

A	B
strain	inducer
EVC	MilliQ water
prha_mVENUS	1M rhamnose
petE_mVENUS	100µM CuSo4
J23100_mVENUS	MilliQ water

List of strains and respective inducers used in the Interlab Study

Put all flasks in a shaking incubators set at 100 rpm under 50 µmol photons · m-2 · s-1 constant white light illumination, ambient CO₂, and 30°C

From each culture, take 1 ml sample at timepoint 0, 2, 4, 5, 6, 7 and 24h starting from the addition of the inducers and use this sample to perform the OD730 and fluorescence intensity measurements described in the following two section immediately after sampling

Dilute 500 µl of your sample with 500µl of BG11 in a spectrophometer cuvette

Use 1 ml of BG11 to blank your spectrophotometer

Measure the absorption at 730nm

For each strain, fill 3 wells of a black, flat-bottomed 96 well plate with 100 µl of your sample

Fill 4 wells with BG11 as blanks

Measure the absorbance of each well at 730nm

Measure fluorescence of each well with an excitation of 511 nm/ 12 nm bandwidth and an emission of 552 nm/ 20 nm bandwidth