Perspective

In this way of the global Project we have, design an On/OFF system which can be integrate into the global genetic network of the biodoseur bacteria. This system extract from Pseudomonas aeruginosa is translational regulation system. It consist of the expression of a small protein named RsmA which can bind on the target mRNA on a leader sequence. This sequence is localized upstream the coding sequence of the target gene and integrate the RBS. Concequntly, RsmA fixation avoids the ribosome binding for the translation. We choose fha and magA Leader Sequence because the first have 5 binding site versus on for the other. This translational repression can be avoid with rsmY RNA which have more binding site and better affinity for RsmA.

In this part of the project we performed the extraction and the BioBricks standardisation of compounds of this system: rsma, rsmY, fhaLS and rsmALS. Moreover we designed all construction which is necessary for the complete characterization of the system (see description part).

First step of the characterization have been done. Indeed, we succeed to obtain the characterization of the fhaLS and magALS by a comparison with the reference RBS part BBa_B0040 by a florescent measurement on flow cytometry. The first sequence has a relative strength of 10% while the second attain 0.4%.

Other experiment was performed has RsmA regulation rate by flow cytometry. First preliminary result led to thinks of a good inhibition rate of rsmA. But this experience has not be done in necessary conditions to confirm clearly this result. Moreover we plan to improve dynamic experiments with fusion. Indeed this experiment will gives us evolution of the repression during time by time fluorescence analysis. .. Consequently we have all necessary elements to completely characterize these systems.

The characterization of this system is very important. Today we know that the global genetic network we designed could be more accurate and faster. But we need experimental data in order to know if the RsmA regulation system is sufficiently powerful to repress the toggle switch. We can also imagine that this system could use as a global interrupter. Indeed this system has the advantage to regulate a complete network if all transcription unit possess one of fhaLS or magSL .