TITLE: Toxicity assay for inducer compounds 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">However, prior to working with an inducible promoter system, it is important to assess whether the inducer compount might positively or negatively affect growth or viability of the cell.</div><div class = "text-block"><span>This protocol describes the methodology for toxicity assays in the unicellular cyanobacterium </span><span style = "font-style:italic;">Synechocystis sp</span><span>. PCC 6803 to quantify its growth in the presence of different concentrations of inducer. </span></div></div>

[STEPS]
?. [Inoculation of pre-culture]
Inoculate an appropriate amount of BG11 with your cyanobacterial strain of choice. The inoculation volume depends on the number of concentrations to be tested. For example: 3 concentrations x 3 replicates x 30 mL = 270 mL of preculture. Since the culture will be diluted ~1:3 before starting, 100 mL culture volume in a 250 mL Erlenmeyer flask should be enough. Grow culture for 3-5 days, until an OD750 of ~1 is reached.
If using a strain carrying a plasmid, make sure to include appropriate antibiotics, since the plasmid will be lost in the absence of selection pressure.
?. [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 four tables show the pipetting schemes for the four inducer compounds L-rhamnose, aTc, vanillic acid and CoCl2, over a range of concentrations.  ABCD1L-rhamnose, final concentration [mM]01102L-rhamnose stock solution [mM]-100010003µL stock solution0303004µL MilliQ water, sterile3002700  ABCD1aTc, final concentration [nM]010010002aTc stock solution [µM]-10010003µL stock030304µL EtOH, 100 %3000  ABCD1vanillate, final concentration [µM]0100020002vanillate stock solution [mM]-2502503µL stock01202404µL EtOH, 100 %2401200  ABCD1Cobalt (III) chloride, final concentration [µM]05102Cobalt (III) chloridestock solution [mM]-5103µL stock030304µL MilliQ water, sterile3000
ABCD1L-rhamnose, final concentration [mM]01102L-rhamnose stock solution [mM]-100010003µL stock solution0303004µL MilliQ water, sterile3002700
ABCD1aTc, final concentration [nM]010010002aTc stock solution [µM]-10010003µL stock030304µL EtOH, 100 %3000
ABCD1vanillate, final concentration [µM]0100020002vanillate stock solution [mM]-2502503µL stock01202404µL EtOH, 100 %2401200
ABCD1Cobalt (III) chloride, final concentration [µM]05102Cobalt (III) chloridestock solution [mM]-5103µL stock030304µL MilliQ water, sterile3000
?. [Dilution of pre-culture]
Dilute pre-cultures to an OD750 of 0.2. Include the appropriate antibiotics.
This step is critical for comparability. In order to compare cultures, pre-cultures 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 pre-culture]
Grow pre-culture 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 = 0.1, making sure to prepare a suffient amount of culture. Include appropriate antibiotics if necessary. Aliquot this main culture into smaller, 100 mL Erlenmeyer flasks, with 30 mL per well and at least three biological replicates per inducer concentration.
In order to compare cultures, it is important that all cultures are in the same growth phase and that you start the experiment at the same OD750!
?. [Growth monitoring]
Measure OD750 of each culture every day. If the OD is above 1.0, an additional dilution step is necessary for precise measurement. Monitor growth over at least 7 days, or until the culture becomes stationary.
If growing your cultures in an incubator with CO2-enriched air, it could be beneficial to measure the OD twice a day, since the growth rate is much higher under these conditions.