Cyanobacterial OD based Growth Assay - iGEM IISER Pune 2021

Start with a single colony of Synechococcus elongatus UTEX 2973 and inoculate it in 10mL of liquid BG-11 media containing 150millimolar (mM) of NaCl.

Allow the colony to grow to a dark green color for 1-2 days in a shaker incubator at 150 rpm, at the temperature for which you want a growth curve.

We are using the Thermo Scientific MaxQ 6000 shaking incubator.

Transfer the growing mixture into 90mL of liquid BG-11 media containing 150millimolar (mM) of NaCl.

Allow the 100mL the growing mixture to grow for 24 hours

Take 1mL of the culture and measure its optical density in a spectrophotometer at a wavelength of 730 nm, since it lies outside of the absorption spectrum of cyanobacterial pigments (Blankenship et al., 2011), after calibrating the spectrophotometer to the BG-11 media with 150 mM NaCl.

We are using the Eppendorf Biospectrometer Basic spectrophotometer.

If the OD of the culture is of a high-density (OD= 0.8-1) proceed with the next step. If not, allow the culture to grow for some more time before taking an OD measurement again.

If the OD of this culture is >1, subculture 10mL of the culture into 90mL of the BG-11 media with 150millimolar (mM) NaCl and allow it to grow again before taking an OD measurement.

After obtaining the OD from the previous step, use the formula O1.V1 = O2.V2 (where O is OD and V is the volume of the culture at that OD). O1 is the OD value at which you want to start measuring the growth and O2 is the OD value of the high-density culture (OD = 0.8 -1). V1 is the volume of culture that has the OD=O1. V2 is the volume of the high-density culture that needs to be diluted to V1 in order to obtain a culture with OD=O1.

For the purposes of this experiment, we want to start measuring growth from a 100mL culture of OD = 0.2. Let's say we measured the OD of the high-density culture to be 0.8. Thus, by the above formula; 0.2x100 = 0.8xV2

V2 = 25 ml. Thus we need to dilute 25 ml of the high-density culture to 100mL with the BG-11 media containing 150millimolar (mM) NaCl.

Once the 100mL culture of OD=0.2 is obtained, we can start making measurements. Take 1mL of this culture and record the absorbance value in the spectrophotometer.

Take 1mL of the culture and make absorbance measurements every 3 hours till an OD=1 is obtained.