Team:Stony Brook/Plant

Plant

Mobirise

Promoter Characterization

While investigating possible parts to apply to our project, we realized that the registry had few characterized cyanobacterial parts, more specifically promoters. Therefore, we set out to expand the selection by adding and characterizing several novel promoters specifically for our chassis organism Synechococcus elongatus PCC 7942. The promoters we chose to characterize include strong constitutive as well as light- and nutrient-dependent promoters.

The constitutive promoters we characterized are Pcpc from our chassis organism and Pcpc-560 from a close relative, Synechocystis sp. PCC 6803. The light-dependent promoter is PpsbA2, a high-light inducible promoter, and makes toggling genetic circuits on and off easier. The nutrient-dependent promoter is PidiA, a ferrous-ion repressible promoter. Since cyanobacteria consume ferrous ions as a nutrient, PidiA allows for automatic activation of genetic circuits once a certain culture density is reached. By adjusting the initial concentration of ferrous ions, the timing of activation is tuned.

These promoters should make working with cyanobacterial genetic circuits easier and open new possibilities for synthetic biology. For more information, check out our Basic Parts page!

Photobioreactor and Culture Conditions

We noticed that in most cyanobacteria and plant-related experiments, scientists try and recreate artificial sunlight to the best of their ability. Fluorescent lights are normally used; however, their wavelength distribution (Figure 1) does not match sunlight (Figure 2), which approximates a ~5600K blackbody:

Figure 1   This chart shows the spectral distributions of cold cathode fluorescent (CCFL), compact fluorescent (CFL), and light-emitting diode (LED) lights [1].

Figure 2  Solar spectrum and atmospheric absorbing gases [2].

To optimize growth conditions, we aimed to mimic natural sunlight with a custom-made LED panel. We calibrated the panel using a PAR meter.

In addition to controlling light, we controlled temperature, humidity, and carbon dioxide levels. To do so, we used a sterile carbon dioxide incubator set at 90% humidity, 5% carbon dioxide, ~120 uE/m^2/s, and 33°C. Although we did set the temperature to 33°C, fluctuations arose due to heat released from the light source shining on the metal inside. Fortunately we troubleshooted with some other teams, and we were able to fix the issue. We decided on these parameters based on our results from modeling and growth optimization experiments from Dr. David Kuan [3].