Light and oxygen response!

AppA/PpsR Regulation System

In this repressor/antirepressor system, under low oxygen tension, the GFP expression is possible because the repressor PpsR is forming a complex with the antirepresssor protein AppA. Moreover, when blue light fall upon the complex, a conformational change in AppA breaks the complex, and AppA avoid GFP expression. (See Rhodofactory section for a complete explanation).

We made two BioBricks (BBa_K776018 y BBa_K776020) to test the Light & Oxygen Control System, each one has GFP as a reporter gene and the functionality was related to the fluorescence detection.

Figure 1. This BioBrick will show if our dependent promoter is functional, using the constitutive (or natural) system from R. sphaeroides or the orthologue system from R.palustris.

Figure 2. This BioBrick will show if our complete system is functional because probably we need a synthetic system to promote GFP expression by binding its target sequence (dependent promoter) in R. palustris.

Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur Photosynthetic Bacteria, the plasmids were introduced in R. sphaeroides and R.palustris, by biparental and triparental conjugation.

The measurement approach was:

We used 3 environmental growing conditions:

For all data results, we considered a negative control: R. sphaeroides and R.palustris, conjugated bacteria with pRK415 vector without BioBrick.

Figure 3. Percentage of bacterial population expressing GFP..

Figure 4. Images obtained by fluorescence microscopy, where our systems were functional in the expected conditions.

Discussion

In R. sphaeroides, the best functionality of our system was in aerobic and darknesss condition, it was not the expected result, but probably the activation is because to the incomplete repression of the system because we were overexpressing the constitutive proteins. Altought, we obtained GFP expression with a lower level, in the expected condition, both BioBricks (BBa_K776018 y BBa_K776020) were functional.

In R.palustris, our best condition was in anaerobic and light condition, expected result. The low level of GFP in BioBrick BBa_K77608 it could be due to low affinity of orthologous proteins to our promoter sequence, but when we introduced the complete system BBa_K776020 GFP expression increases showing the funcionality of our system.

Conclusion

Both BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic bacteria R.palustris and R. sphaeroides. The best condicion was aerobic/darknes and anaerobic light respectively. This is a functional system for controlling genetic expression with Light and Oxygen signals.