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Team:Grenoble/Projet/Results
From 2011.igem.org
Post-transcriptional regulation
The RsmA system has a homologous in Escherichia Coli named CsrA. We know these two system are extremely closed on
structural and functional sides. The most difference between this two regulation systems is on target regulon.
For example, in Pseudomonas aeruginosa Rsma regulate many virulence genes as type III secretion system
(anja Brencic and Stephen Lory). In Escherichia coli, RsmA homologous regulate metabolic network.
Our goal is to integrate this translational regulation system in the toggle switch. We need to know whether it influence
the bacteria’s life.
Figure 1 RsmA influence on DH5α growth. Bacteria from overnight culture were reseeding in rich medium supplemented with tetracycline at 20mg/ml and also IPTG at the concentration given in the legend.
Figure 1 present growth curve of DH5α carries into a plasmid pVLT31 with or without rsmA and 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 carries pVLT31-rsmA. But it’s important to say that the two groups start their growth not at
the same value. That explains a time lag between these two groups. After normalization of all curves, no differences
between these two kind of bacteria could be seen. We conclude that the RsmA overexpression hasn’t effects on the growth of bacteria.
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.
The RsmA system has a homologous in Escherichia Coli named CsrA. We know these two system are extremely closed on structural and functional sides. The most difference between this two regulation systems is on target regulon. For example, in Pseudomonas aeruginosa Rsma regulate many virulence genes as type III secretion system (anja Brencic and Stephen Lory). In Escherichia coli, RsmA homologous regulate metabolic network. Our goal is to integrate this translational regulation system in the toggle switch. We need to know whether it influence the bacteria’s life.
Figure 1 present growth curve of DH5α carries into a plasmid pVLT31 with or without rsmA and 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 carries pVLT31-rsmA. But it’s important to say that the two groups start their growth not at the same value. That explains a time lag between these two groups. After normalization of all curves, no differences between these two kind of bacteria could be seen. We conclude that the RsmA overexpression hasn’t effects on the growth of bacteria.
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
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.
