TITLE: Fluorescence_activity_assay_Interlab_Study_PCC_6803
AUTHORS: maurice.mager1808

[DESCRIPTION]
Fluorescence activity assay for Synechococcus PCC 6803 strains during the interlaboratory study published by Mager et al. 2023.

[STEPS]
SECTION: Preculture conditions of the fluorescence activity assay
1. 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.

 

SECTION: Preparing main cultures
2. Transfer all cultures into 50ml falcons
 

SECTION: OD730 measurements in the spectrophotomer
3. Dilute 500 µl of your sample with 500µl of BG11 in a spectrophometer cuvette
 

SECTION: Fluorescence- and OD730 measurements in the plate reader
4. For each strain, fill 3 wells of a black, flat-bottomed 96 well plate with 100 µl of your sample
SECTION: Preculture conditions of the fluorescence activity assay
1.1. Dilute all strains to an OD730 of 0.3 in 35ml of BG11 final volume in a 100ml Erlenmeyer flaks with a cotton plug

 

SECTION: Preculture conditions of the fluorescence activity assay
1.2. 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
SECTION: Preparing main cultures
2.1. Adjust the OD730 of all cultures to 0.5 with BG11 containing 10 µg mL-1 chloramphenicol to a final volume of 50 ml
SECTION: Preparing main cultures
2.2. Measure the full 400-750 nm OD730 spectrum and OD730 nm of these cultures
 

SECTION: Preparing main cultures
2.3. Rinse the preculture flasks with 25 ml Copper-free BG11 twice

 

SECTION: Preparing main cultures
2.4. Fill 20 ml of each preculture adjusted to an OD730 of 0.5 back into the preculture flasks
 

SECTION: Preparing main cultures
2.5. Fill 20 ml of each preculture adjusted to an OD730 of 0.5 into new 100ml erlenmeyer flasks with cotton plugs
 

SECTION: Preparing main cultures
2.6. 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:

 


 

SECTION: Preparing main cultures
2.7. 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


SECTION: OD730 measurements in the spectrophotomer
3.1. Use 1 ml of BG11 to blank your spectrophotometer
SECTION: Preparing main cultures
2.8. 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
SECTION: OD730 measurements in the spectrophotomer
3.2. Measure the absorption at 730nm
 

SECTION: Fluorescence- and OD730 measurements in the plate reader
4.1. Fill 4 wells with BG11 as blanks
SECTION: Fluorescence- and OD730 measurements in the plate reader
4.2. Measure the absorbance of each well at 730nm
SECTION: Fluorescence- and OD730 measurements in the plate reader
4.3. Measure fluorescence of each well with an excitation of 511 nm/ 12 nm bandwidth and an emission of 552 nm/ 20 nm bandwidth