A post-transcriptional regulation system for our toggle switch

The toggle developed by the marmot’s team will switch to one or the other phenotype depending on the amount of both inductors inducer (mercury or tetracycline) and repressor (LacI) proteins. For instance, when no mercure mercury or tetracycline is added to the milieumedium, all bacteria should produce the CinR protein. Then, the more activator inducer is added, the more each cell is likely to change its phenotype. If the amount of LacI repressor is low, then this switch has more chances to occur, and will happen with a lower amount of the molecule to quantify. This prevision prediction has been demonstrated by the our modelling work. So iIn order to improve the accuracy of our device, we decided to develop a post-transcriptional translational control of the repressorss. This regulation mechanism has the specificity to very rapidly modulate the amount of a protein fabrication within the cell. It can of the most useful for numerous systems that need a quick induction.

We found out two mechanisms well documented that can be extracted from micro-organism and share to the foundation. The first one is from Pseudomonas aeruginosa. Called the RsmA regulation mechanism, it is composed of two RNA sequences and a protein. The second one, well characterised in coli, is called the rpoS regulation system. It only involves a hairpin leader sequence and an inducible regulatory small RNA.

We isolated and cloned the RsmA translational regulation system from Pseudomonas aeruginosa, and part of the rpoS system from Escherichia coli. We did not include those constructions into the rolling assembly of the toggle parts, and therefore worked on those as a sub-project. These new bricks are already documented and partly characterised. More tests will need to be made by the iGEM teams in the following years!

The RsmA translational regulation system

• How does it work?

The RsmA regulation system of Pseudomonas has homologs in many other bacteria, like CsrA of Escherichia coli1, for example. It is basically composed of:  A leader sequence in the 5’ end of the genes to be regulated. Many different sequences exist depending on the gene to regulate.  A regulatory protein named RsmA that binds to the GGA motif within the stem-loop structure of the transcribed leader sequences2. When bound to the mRNA, the latter cannot be translated and is degraded.  An inducible small RNA – the one we use is called rsmY – which sequesters the RsmA protein, having a greater affinity for it than the transcribed gene leader sequences.

So, at some state, the cell transcribes genes of which the translation is more or less repressed by RsmA, depending on their leader sequence (figure 2). The strength of the repression depends on the structure of the stem-loop as well as the number of GGA repeats.

Then, when an environmental signal activates the transcription of rsmY, rsmY RNA acts as an activator by sequestering the RsmA repressor (Figure 3).

• Fha1 and magA operon leader sequences

A microarray analysis revealed that RsmA regulates about 60 genes from two to more than one hundred fold3! Most of those genes are involved in secretion, or pili biogenesis. We decided to work on the leader sequences of magA and fha1. They are not strongly inhibited by RsmA, but are well documented, and the biobricks we made will be useful for our host lab.

• The rpoS regulation system

When nutrients become scarce, bacteria need to quickly shut down the expression of many genes an activate others. A major global regulator of this growth transition is RpoS, an alternative sigma factor (also called sigmaS). The RNA polymerase holoenzyme containing sigmaS recognizes a new set of promoters and thus changes the global transcriptional program in an appropriate manner1⁠.

Because of the central role of RpoS, its expression is tightly regulated. Much of this regulation is exerted at the level of translation. The mechanism has been intensely studied and we can therefore exploit the system to create a new biobrick that provides an on-off switch for the translation of a target genes

The 5'-untranslated RNA (5'-UTR) of the rpoS gene adopts a particular secondary structure that places the ribosome binding site into a double-stranded region and therefore prevents recognition by the ribosome2⁠. A small RNA, called dsrA, is produced when the cells enter starvation. This RNA interacts with the 5'-UTR and induces a change in its secondary structure that liberates the RBS and thus stimulates the translation of rpoS 3⁠. We use this mechanism to construct a new BioBrick that allows controlling the efficiency of translation of target gene cloned downstream.

The rpoS leader sequence has been exemplified by PCR and cloned into PSB1C3.

• 1. Ferenci, T., Galbiati, H.F., Betteridge, T., Phan, K. & Spira, B. The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in Escherichia coli. BMC microbiology 11, 62 (2011).
• 2. Resch, A. et al. Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5’-leader. RNA (New York, N.Y.) 14, 454-9 (2008).
• 3. Hopkins, J.F., Panja, S., McNeil, S. a N. & Woodson, S. a Effect of salt and RNA structure on annealing and strand displacement by Hfq. Nucleic acids research 37, 6205-13 (2009).

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 .