Apr 26, 2023
Fluorescence_activity_assay_Interlab_Study_PCC_6803
- 1HHU
Protocol Citation: maurice.mager1808 2023. Fluorescence_activity_assay_Interlab_Study_PCC_6803. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jdw5l1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 17, 2023
Last Modified: April 26, 2023
Protocol Integer ID: 77177
Abstract
Fluorescence activity assay for Synechococcus PCC 6803 strains during the interlaboratory study published by Mager et al. 2023.
Preculture conditions of the fluorescence activity assay
Preculture conditions of the fluorescence activity assay
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).
Note
Please refer to our protocol "Cryo_conservation_Synechocystis_PCC_6803" for more details on growing PCC 6803 from cryo conserved cells and our protocol "BG11_and_inducer_preparation" for the preparation of copper free BG11-PC 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.
Note
Please refer to our manuscript for more details on the plasmids. plasmid maps can be found in our figshare repository under ......
Dilute all strains to an OD730 of 0.3 in 35ml of BG11 final volume in a 100ml Erlenmeyer flaks with a cotton plug
Note
Supply 10 µg mL-1 chloramphenicol, which is added individually to each flask before inoculation.
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
Preparing main cultures
Preparing main cultures
Transfer all cultures into 50ml falcons
Note
Keep the flasks steril !
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
Note
This is your timepoint 0h value for the absorption spectrum. Refer to the last section of this manuscript for details on OD measurements.
Rinse the preculture flasks with 25 ml Copper-free BG11 twice
Note
This is used to remove any residual copper. These flasks will be used for the uninduced cultures
Fill 20 ml of each preculture adjusted to an OD730 of 0.5 back into the preculture flasks
Note
These will be your uninduced cultures !
Fill 20 ml of each preculture adjusted to an OD730 of 0.5 into new 100ml erlenmeyer flasks with cotton plugs
Note
These will be your induced cultures !
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
Note
For the preparation of the inducers please refer to our protocol "BG11_and_inducer_preparation".
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
OD730 measurements in the spectrophotomer
OD730 measurements in the spectrophotomer
Dilute 500 µl of your sample with 500µl of BG11 in a spectrophometer cuvette
Note
For the sample at timepoint 24h, instead dilute 200µl in 800µl of BG11.
All measured samples should only be measured in the assumed linear range of OD730 from 0.1-0.5.
Use 1 ml of BG11 to blank your spectrophotometer
Measure the absorption at 730nm
Note
For all samples at timepoint 0h and 7h, additionally measure the full 400-750 nm OD spectrum and of these cultures. This is your timepoint 0h and 7h value for the absorption spectrum.
Fluorescence- and OD730 measurements in the plate reader
Fluorescence- and OD730 measurements in the plate reader
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