Justification of the need of a regulation system

In our project, we developed a translation regulation system in order to keep the system OFF until the pollutant is added. With modelling, we show that this system is really important and without it, the measure is really disturbed.

In all of our simulation, we considered the initial concentrations of both repressors equal to zero. This will be the case with the regulation system. Without this system, the initial concentrations of the repressors are higher. In the following figure, we performed a simulation for an aTc concentration of $1.10^{-6}$ with initial concentrations equal to zero and one with initial concentrations equal to $5\%$ of the concentrations in the steady state of the previous simulation.

Figure 1: Observation of the interface when the regulation system works and when it doesn't for an aTc concentration of $1x10^{-6}$.

When the regulation system doesn't work, the interface is not at the place it supposed to be. Because we can't havemeasures of the initial concentration of both repressors, to well predict where the interface will appear, we need to control these concentrations. That's why we developed the translation regulation system.

Can rsmA be transferred into E. coli?

The RsmA system from Pseudomonas has a homologue in Escherichia coli, named "CsrA". We know that these two systems are extremely similar. Consequently we ask ourselves whether the synthesis of RsmA in E. coli interferes with its survival. Figure 1 shows growth curves of E.coli cells transformed by a plasmid containing an IPTG-inducible rsmA sequence from Pseudomonas and control cells carrying the same plasmid without rsmA. The superimposed curves demonstrate that the synthesis of RsmA does not interfere with the growth of E. coli.

Figure 2 Influence of RsmA production on the growth of E coli DH5α. Bacteria transformed with pVLT31-rsmA or empty pVLT31 from overnight culture were re-suspended in rich medium supplemented with tetracycline at 20mg/ml. Induction of rsmA transcription is induced by IPTG at the concentrations given in the legend. Curves are normalized to their first OD value.

Characterisation of the leader sequences

We cloned several leader sequences that contain a ribosome-binding site (RBS) in front of a reporter gene, GFP, in order to:

• Characterise their RBS strength
• Use them for the translational control of downstream genes by the RsmA/rsmY system.

The leader sequences used were mag and fha from Pseudomonas as well as the biobrick BBa_K256003, which was used as a reference. All constructs of Figure 2 have identical promoters, GFP reporter gene and terminators and are carried by the same plasmid.

Figure 3: :Constructions used to characterise RBS included sequences strength of magA and fha leader sequences.

We used a FACSCalibur flow cytometer to measure the GFP fluorescence emitted by cells containing the constructs shown in Fig.3. Two negative controls were set up: a brick having the GFP reporter gene but no promoter ( BBa_E0840 ) and a cell culture containing no plasmid.

50 μl of LB containing cells were diluted into 500 μl of filtered PBS (OD600 of inoculum was 3± 0,3 for all samples) and then introduced into the FACS ten minutes after dilution.

The cytometer counts each particle that passes through the light beam. Therefore it is necessary to select an analysis window that corresponds to the size of the bacteria (see Fig. 3).

Figure 4: Dot plots of particle size distributions obtained with a water sample containing no bacteria (left) and a sample containing bacteria (right). 40000 events were counted. FACS measurements were realised in duplicate.

We then analysed the basal fluorescence recorded from control bacteria expressing no GFP. This allows to define two windows: the M1 window referring to basal fluorescence levels and containing fluorescence values obtained for all negative controls; the M2 window comprising fluorescence signals that are greater than the basal level. We show for each construction the average fluorescence (in red) within the window that contains most of the cells (percentages indicated in black, see Fig. 5).

Figure 5: Individual flow cytometry results (at the right) for different constructs (at the left). The red number indicates an average of GFP fluorescence. The percentage in black indicates the fraction of the bacterial population within the respective analysis window M1 or M2.

The two negative controls (cells containing no plasmid (1) or plasmid without promoter (2)) show a basal fluorescence signal as expected. Cells containing the reference brick BBa_K25003 (5) show a maximum amount of GFP fluorescence with 77 % of the cell population that fluoresce more than the negative controls. The average fluorescent signal calculated for construct 5 is 1030 (vs 2,5 (neg control)). The fluorescence signal obtained with cells containing the brick with the maga leader sequence (3) does not differ very much from the negative controls (4,4 vs 2,5). Four per cent of this cell population fluoresces more than the negative control populations.

90 % of cells containing the fha leader sequence (4) present a fluorescence signal that is higher than control cells. Their average fluorescence is 98.

Figure 6: Compilation of the flow cytometer measurements. 1and 2 are the negative controls, 3 : maga leader sequence, 4: fha leader sequence, 5 : reference biobrick BBa_K256003.

Figure 6 summarises the cytometry results. We can see that both leader sequences mag and fha allow the translation of the gfp mRNA. They can therefore be used for further characterisation of the system. The GFP fluorescence signal is much higher using the fha leader sequence when compared to mag. Figure 7 focuses on the RBS strength of those two gene leader sequences, and compares them to the strongest RBS of the part registry: BBa_0034. Mag has got very week RBS binding site strength, whereas fha is stronger (10% of the maximum value obtained for BBa_0034).

Figure 7: Relative strength of maga (BBa_K545006) and fha (BBa_K545005) leader sequences compared to BBa_K25003 RBS. The latter is the strongest RBS used to compare RBS sequences of the registry.

Effect of rsmA and rsmY on the mag and fha reporter genes

After having established the RBS strength of fha and mag leader sequences using the GFP reporter gene constructs, we quantified the translational inhibition effect of the RsmA protein and the relieve of this inhibition in presence of the rsmY RNA. In order to do these experiments, E. coli cells were co-transformed with a combination of 3 different plasmids containing:

A leader sequence or the reference RBS upstream of GFP (constructs 3, 4 and 5 on Fig. 4)
A leader sequence or RBS plus an other plasmid containing rsmA (figure 7)
A leader sequence or RBS plus rsmA plus rsmY (figure 7)

Figure 7: The IPTG-inducible rsmA gene is cloned in plasmid pSB3C5 and the Tet-inducible rsmY in plasmid pSB4K5.

The GFP fluorescence obtained from the constructs with fha, mag and reference RBS were analysed in the absence of RsmA and rsmY (single transformants) in the presence of RsmA (double transformants) and in the presence of both RsmA and rsmY (triple transformants). Note that in our experiments rsmA and rsmY were constitutively expressed because the repressors LacI and TetR were absent from our strains.

As expected, no inhibitory effect of RsmA was observed when the reference RBS ( BBa_0034 ) was used as this leader sequence does not have a binding site for RsmA (data not shown).

In contrast, RsmA decreased the GFP fluorescence level when mag or fha leader sequences were provided upstream of GFP and this decrease could be partially relieved in the presence of rsmY for fha (Figs 8 and 9).

Effect of rsmA and rsmY on fha-GFP constructs