Characterisation of RsmA system components

Can rsmA be transferred into E. coli?

The RsmA system has a homologous in Escherichia Coli named CsrA. We know that these two systems are extremely similar. The most different between these two regulation systems is about the targetedregulons. For example, in Pseudomonas aeruginosaRsmA regulates many virulence genes as type III secretion system (anjaBrencic and Stephen Lory). In Escherichia coli, RsmA homologous regulate metabolic network. Our goal is to integrate this translational regulation system in E. coli. We need to know whether RsmA is toxic for this chassis. The figure 1 present growth curve of DH5α carryingthe plasmid pVLT31 with or without rsmAand its Figure 1RsmA influence on DH5α growth.Bacteria from overnight culture were re-suspended in rich medium supplemented with tetracycline at 20mg/ml and also IPTG at the concentration given in the legend. Natural RBS cloned downstream the Plac promoter. Two triads can be seen. The triad containing the strains with empty plasmid shows an upper growth curve compared to the second triad caring pVLT31-rsmA. It’s important to say that the two groups start their growth not at the same value. This explains a time lag between these two groups. After normalization of all curves, no differences can be seen between them. We conclude thatRsmA overexpression has no effects on the growth of thosebacteria.

Characterisation of the leader sequences

We extracted and cloned several leader sequences that comport a ribosome-binding site (RBS). We first wanted to verify whether we could use them with a reporter gene,in order to:

• Characterise their RBS strength
• Use them for further testtomeasure the effect of rsmAlaterrsmA +rsmY transcription.

We first tested the following constructions with a FACSCalibur flow cytometer:

We used the brick BBa_K256003 as a reference to compare our leader sequences to. Fha1 and maga leader sequences are cloned to the same components as BBa_K256003, and carried by the same plasmid. We used two negative controls:

• A brick similar to BBa_K5450010 that differs only by the absence of promoter
• A cell culture containing no plasmid

Cell were incubate one night at 37°C, 250rpm, during 14h. They were re-suspended tree hours before the test. Optic density at 600 nm was 3± 0,3 for all samples 15mn before the test. 50 μl of LB containing cellsand medium were diluted into 500 μl of filtered in 0,2 nm PBS, and tube were loader into the FACS.

The cytometer count each particle that pass through the light beam; It is necessary to select an analysis gate that corresponds to the bacteria size. We can then see what is the natural fluorescence of our control bacteria. This allows defining the windows of basal signal (M1) and positive signal (M2). We look at the average fluorescence within windows that comport the most cells.

The two negatives controls (WT and without promoter) show a very little fluorescent signal as expected. The reference brick BBa_K25003 shows an impressive amount of GFP with about 77 % of the cell population that fluoresce more than the control.Its average fluorescent signal is 1030 vs 2,5(wild type). The signal from the brick containing maga leader sequence (BBA_K545006) doesn’t differ very much from the negative controls: four per cent of the cell population fluoresce more than the controls, at a very low level even. The fluorescence mean is 4,4 vs 2,5 from the WT control (window M1).

Part BBa_K545010 containing the fha leader sequence induce a fluorescence signal higher than the control for 90 % of the cell population. The average signal (M2 window) is 154 vs 2,5(WT) and 1029 (BBa_K25003).

Construction of a Toggle Switch test

In order to test if our system could work, we construct a toggle switch test based on Gardner's work [1].

For realized this toggle, we used 4 primary bricks :

• pTet : BBa_R0040
• RBS-LacI-oo-pLac : BBa_Q04121
• RBS-GFP : BBa_E0240
• RBS-TetR composed of RBS BBa_B0034 and TetR BBa_C0040

First we put RBS-GFP behind RBS-TetR, and Q04121 behind pTet: both size are around 1 500 bp. The following gel shows that both constructions were at the expected size. Construction were confirmed by sequencing.

Figure 1: First step of cloning gel

In a last step of cloning, we put RBS-tetR-RBS-GFP behind pTet-Q04121. The size is around 3 000 bp. The following gel shows that constructions was at the expected size. In addition to this test, transformation of bacteria have grown on plate with IPTG to block them in the fluorescence way. And some of the bacteria were fluorescent. Construction was also confirmed by sequencing.

Figure 2: Last step of cloning gel

Figure 3: Fluorescence test picture

To test this system, bacterias had grown in an aTc preculture to block them in a nonfluorescent way. This bacteria were put in a 96 wells plate with a two dimensionnal gradient (aTc and IPTG). After 10 hours of acquisition, we obtained the following curve :

We can get from this graph that between an aTc concentration of 50ng/mL and 150 ng/mL there is a switch after 3 hours of experiment. However, we can see more fluorescence than expected. In fact, the GFP protein has an half life of 10 hours and it was impossible to get no fluorescence.

We also did an experiment on bacterias which had grown in an IPTG preculture. But we did'nt see a switch because IPTG block bacterias in the fluorescence way. Because of the half life of GFP, it was possible to detect a switch only with bacteria which had grown with aTc.

To go further, it will be very interesting to put an LVA tag on GFP in order to control its degradation. In this case we will be abble to see the switch in both case and with more magnitude.

MerR-del : K545103.
MerR (BBa_K346001) without RBS Well 1: Positive control (4K5) Well 2: Negative control (Without DNA) Well 3, 4, 5, 6, 7: MerR-del (expected at 740pb, obtain around 700-800pb) Confirm by sequencing

pMerT-GFP: K545002 Original parts: K346002, E0240 PCR Well 1 and 2: Failed cloning Well 3: pMerT-GFP (expected at1200pb obtain just over 1000pb) Sequencing confirms the cloning.

pCin-CrtEBI (K545000) Originally: R0077, K274100 PCR Expected at 3500pb, obtain between 3000 and 4000pb Confirmed by sequencing

pConst-CinR (K545004) Originally J23119, B0034 C0077 Restriction gel of pConst-CinR Well 1, 2: failed cloning Well 3, 4: pConst-CinR(expected at 950pb) Confirmed by sequencing

pLac-CinR (S04607) Originally R0011, E0240 pConst-GFP (K545001) Originally J23119, E0240 Restriction gel Well 1: pLac-CinR (expected at 950pb) Well 2: pConst-CinR (expected at 900pb) Confirmed by sequencing

RBS-CinR (S04602) Originally B0034, C0077 PCR Well 1: RBS-CinR (expected at 1000) Confirmed by sequencing

RBS-TetR (S04603) Originally B0034, C0040 PCR RBS-TetR (expected at 950pb) Confirmed by sequencing