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Team:Grenoble/Project/Main Results
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<a href="#4">Issues encountered</a> • | <a href="#4">Issues encountered</a> • | ||
<a href="#5">Coming up next</a> | <a href="#5">Coming up next</a> | ||
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| - | This part will present the pre-regional Jamboree main results. | + | This part will present the pre-regional Jamboree main results. --> |
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<h1 id="4">Issues encountered</h1> | <h1 id="4">Issues encountered</h1> | ||
| - | We have not yet constructed that part. We had some problem with the ligation procedure. Another explanation could be that our bacteria are transformed with the right construction leading to an over-expression of Adenylate cyclase which is lethal: this can be attributed to the fact that overproduction of cAMP is lethal to | + | We have not yet constructed that part. We had some problem with the ligation procedure. Another explanation could be that our bacteria are transformed with the right construction leading to an over-expression of Adenylate cyclase which is lethal: this can be attributed to the fact that overproduction of cAMP is lethal to <i>E. coli</i> possibly due to an accumulation of methylglyoxal <a href="#ref">[1]</a> <a href="#ref">[2]</a>. |
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| - | As we have had issues constructing | + | As we have had issues constructing paraBAD_gfp_cyaA using the standard restriction/ligation procedure, we are going to try another approach. We want to insert it on pSB3C5 using the Gibson Assembly procedure (we have the brick but we are failing the insertion).</br> |
Once this construction is achieved, we will transform BW25113 <i>ΔcyaA E. Coli</i> strain to test the designed amplification feed-forward loop using the same protocol as for the AND gate. | Once this construction is achieved, we will transform BW25113 <i>ΔcyaA E. Coli</i> strain to test the designed amplification feed-forward loop using the same protocol as for the AND gate. | ||
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| - | In order to estimate the functionality of our new riboswitch, we are planning to transform one strain of BW25113 with | + | In order to estimate the functionality of our new riboswitch, we are planning to transform one strain of BW25113 with plac_fha1_mcherry (on a low-copy plasmid) and in a second strain the previous construct plus plac_RBS_rsmA (medium-copy plasmid). In a last strain we will add the third partner (plac_rsmY on a high-copy plasmid) to estimate its inhibition release action. To characterize this we will use flow cytometry experiments.</p> |
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<h1 id="ref">References</h1> | <h1 id="ref">References</h1> | ||
<ul><li><b>[1]</b> <a href="http://jb.asm.org/content/119/2/357.abstract">Accumulation of Toxic Concentrations of Methylglyoxal by Wild-Type Escherichia coli K-12, Rollie S. Ackerman, Nicholas R. Cozzarelli and Wolfgang Epstein</a><br/> | <ul><li><b>[1]</b> <a href="http://jb.asm.org/content/119/2/357.abstract">Accumulation of Toxic Concentrations of Methylglyoxal by Wild-Type Escherichia coli K-12, Rollie S. Ackerman, Nicholas R. Cozzarelli and Wolfgang Epstein</a><br/> | ||
| + | <br/> | ||
<li><b>[2]</b> <a href="http://mic.sgmjournals.org/content/151/3/707.abstract">Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour | <li><b>[2]</b> <a href="http://mic.sgmjournals.org/content/151/3/707.abstract">Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour | ||
Jan Weber†, Anke Kayser‡ and Ursula Rinas</a><br/> | Jan Weber†, Anke Kayser‡ and Ursula Rinas</a><br/> | ||
Latest revision as of 16:52, 8 March 2013
Main results
Simple amplification loop • Why an AND gate ? • Biological results • Issues encountered • Coming up nextSimple amplification loop
In a first place, biologists and modellers worked on a simple amplification loop.
The modellers proved that a simple amplification loop could not work. There is no state where GFP is not expressed.
Why an AND gate ?
The team thought that an AND gate could be our solution. It could add a biological noise filter and resolve the problem of the continuous expression of GFP.
Modelling results confirmed that built system will work.
Biological results
Biological AND gate test.
Issues encountered
We have not yet constructed that part. We had some problem with the ligation procedure. Another explanation could be that our bacteria are transformed with the right construction leading to an over-expression of Adenylate cyclase which is lethal: this can be attributed to the fact that overproduction of cAMP is lethal to E. coli possibly due to an accumulation of methylglyoxal [1] [2].Coming up next
In the future weeks (according to ours results) we will try to make the culture of our transformed bacteria in a medium complemented with glucose and acetate.
A possible track would be to add another regulation system on the adenylate cyclase production with the riboswitch RsmA/rsmY.
As we have had issues constructing paraBAD_gfp_cyaA using the standard restriction/ligation procedure, we are going to try another approach. We want to insert it on pSB3C5 using the Gibson Assembly procedure (we have the brick but we are failing the insertion). Once this construction is achieved, we will transform BW25113 ΔcyaA E. Coli strain to test the designed amplification feed-forward loop using the same protocol as for the AND gate.
We also plan to test our engineered membrane receptor (TapZ) in BW25113 ΔEnvZ E. Coli strain. To test it, we are going to use an mCherry reporter under the promotion of pOmpC.
In order to estimate the functionality of our new riboswitch, we are planning to transform one strain of BW25113 with plac_fha1_mcherry (on a low-copy plasmid) and in a second strain the previous construct plus plac_RBS_rsmA (medium-copy plasmid). In a last strain we will add the third partner (plac_rsmY on a high-copy plasmid) to estimate its inhibition release action. To characterize this we will use flow cytometry experiments.
References
- [1] Accumulation of Toxic Concentrations of Methylglyoxal by Wild-Type Escherichia coli K-12, Rollie S. Ackerman, Nicholas R. Cozzarelli and Wolfgang Epstein
- [2] Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour
Jan Weber†, Anke Kayser‡ and Ursula Rinas
