TITLE: Dose response assay for inducible promoters in Synechocystis sp. PCC 6803
AUTHORS: Anna Behle, Pia Saake, Ilka Maria Axmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Inducible promoters are an important tool for synthetic biology. They enable temporal control of gene expression, as well as controlled expression of toxic genes.</div><div class = "text-block"><span>This protocol describes the methodology for fluorescence-based reporter assays in the unicellular cyanobacterium </span><span style = "font-style:italic;">Synechocystis sp</span><span>. PCC 6803 to quantify the dose-dependent response of an inducible promoter to its inducer. </span></div></div>

[STEPS]
?. [Inoculation of preculture]
Inoculate an appropriate amount of BG11 with your cyanobacterial strain of choice. Include appropriate controls, i.e. an empty vector control. The inoculation volume depends on the number of concentrations to be tested. For example: 6 concentrations x 3 replicates x 5 mL = 90 mL of preculture. Since the culture will be diluted ~1:3 before starting, 35 mL culture volume should be enough. Grow culture for 3-5 days, until an OD750 of ~1 is reached.
Include appropriate antibiotics, especially when using plasmid-based systems, since the plasmid will be lost in the absence of selection pressure.
?. [Dilution of preculture]
Dilute precultures to an OD750 of 0.2. Include the appropriate antibiotics.
This step is important for comparability. In order to compare cultures, precultures should be relatively fresh, i. e., no older than 1 week and no further than early stationary phase. Furthermore, their OD750 should be roughly the same.
?. [Dilution of preculture]
Grow preculture for 2-3 additional days, until OD750 has reached ~ 0.6.
?. [Dilution of main culture]
In a large sterile vessel, dilute the preculture to OD750 = 0.2, making sure to prepare a suffient amount of culture. Include appropriate antibiotics. Aliquot this main culture into clear 6-well culture plates, with 5 mL per well and at least three biological replicates per inducer concentration.
?. [Induction]
At the end, the total volume added should be equal for each replicate. This can be achieved either by preparing one stock solution for each concentration, or adding the difference in volume of the solvent used. The following three tables show the pipetting schemes for the three inducible promoters Prha, PL03 and PvanCC over a range of inducer concentrations.  ABCDEFGHIJK1L-rhamnose, final concentration [mM]00.010.050.10.512510202L-rhamnose stock solution-1010101001001001000100010003µL stock solution052550255010025501004µL MilliQ water, sterile1009575507550075500  ABCDEFG1aTc, final concentration [nM]0105010050010002aTc stock solution [µM]-105010050010003µL stock0555554µL EtOH, 100 %500000  ABCDEFG1vanillate, final concentration [µM]0100200500100020002vanillate stock solution [mM]-1001002502502503µL stock05101020404µL EtOH, 100 %40353030200
ABCDEFGHIJK1L-rhamnose, final concentration [mM]00.010.050.10.512510202L-rhamnose stock solution-1010101001001001000100010003µL stock solution052550255010025501004µL MilliQ water, sterile1009575507550075500
ABCDEFG1aTc, final concentration [nM]0105010050010002aTc stock solution [µM]-105010050010003µL stock0555554µL EtOH, 100 %500000
ABCDEFG1vanillate, final concentration [µM]0100200500100020002vanillate stock solution [mM]-1001002502502503µL stock05101020404µL EtOH, 100 %40353030200
?. [Fluorescence measurement]
To monitor fluorescence development, fluorescence measurements should be performed 6h, 24h and 48h post-induction. For each sample, pipette 150 µL into a black-walled, clear-bottom 96-well plate in technical triplicates.Include a well with an appropriate blank (BG11)Fluorescence measurement settings in the BMG Clariostar:Top opticExcitation λ: 511/12 nmExcitation λ: 552/20 nm# of flashes: 20To quantify cell density, measure the absorption at 750 nm.Shake before plate reading at 500 rpm for 30 seconds (double orbital)