Team:Grenoble/Biology/AND gate

From 2012.igem.org

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<h1>Biological "AND" gate</h1>
<h1>Biological "AND" gate</h1>
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<a href="#pro">Promoter paraBAD</a> •
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<a href="#cyc">cyclic Adenosine MonoPhosphate</a> •
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<a href="#gen">Gene transcription</a> •
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<a href="#char">A new characterization of the paraBAD promoter</a> •
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<a href="#ccl">Conclusion</a>
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<section>
<section>
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<h2>Promoter paraBAD</h2>
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<h2 id="pro">Promoter paraBAD</h2>
In order to develop our device we needed a biological "AND" gate.
In order to develop our device we needed a biological "AND" gate.
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We found a promoter which can be activated by 2 molecules : CRP-cAMP complex and the AraC protein.
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We found a promoter which can be activated by 2 molecules : CRP-cAMP complex and the AraC protein.<br/>
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The paraBAD promoter has two states; in absence of L-arabinose the paraBAD promoter is repressed by AraC, whereas the
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<br/>
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the paraC promoter is activated (unless an excess of AraC is present) <a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#10">[Schemes]</a>.
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The paraBAD promoter has two states:
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</br>
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<center><img src="https://static.igem.org/mediawiki/2012/7/70/ParaBAD_1.png"/>&nbsp;&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/3/3b/ParaBAD_2.png"/></center>
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<center><a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#10">[Schemes]</a></center>
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<br/>
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<li>In absence of L-arabinose the paraBAD promoter is repressed by AraC, whereas the paraC promoter is activated (unless an excess of AraC is present).</li>
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<br/>
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<li> In presence of L-arabinose and the CRP-cAMP complex, the promoter is activated thus enabling the transcription of the downstream elements.</li>
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<center><img src="https://static.igem.org/mediawiki/2012/7/70/ParaBAD_1.png"/></center>
 
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</br>
 
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</br>
 
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In presence of L-arabinose and the CRP-cAMP complex, the promoter is activated thus enabling the transcription of the downstream elements.
 
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<center><img src="https://static.igem.org/mediawiki/2012/3/3b/ParaBAD_2.png"/></center>
 
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This AND gate provides a filter to biological noise.
This AND gate provides a filter to biological noise.
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Check out our main results page to for the AND gate characterization.
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Check out our <a href="https://2012.igem.org/Team:Grenoble/Project/Main_Results">main results page</a> to for the AND gate characterization.
</section>
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<h2>Cyclic Adenosine Monophosphate</h2>
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<h2 id="cyc">cyclic Adenosine MonoPhosphate</h2>
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In order to make our AND work we need to produce cyclic Adenosine Monophosphate (cAMP).
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In order to make our AND gate work we need to produce cyclic Adenosine Monophosphate (cAMP).
cAMP is produce by adenyl cyclase (encoded by the cyaA gene).  
cAMP is produce by adenyl cyclase (encoded by the cyaA gene).  
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It is an enzyme which catalyses the conversion of ATP to 3’,5’-cAMP.
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It is an enzyme which catalyses the conversion of ATP to 3’-5’-cAMP.<br/>
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<br/>
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In <i>Escherichia coli</i>, cAMP is involved in carbon catabolite repression  
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In <i>E. coli</i>, cAMP is involved in carbon catabolite repression  
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[1]</a> and binds to the cAMP receptor protein (CRP).  
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[1]</a> and binds to the cAMP receptor protein (CRP).  
The corresponding complex (CRP-cAMP) is a transcriptional factor controlling the expression  
The corresponding complex (CRP-cAMP) is a transcriptional factor controlling the expression  
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of more than 220 operons <a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[2]</a>.  
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of more than 220 operons <a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[2]</a>. <br/>
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It has been known for a long time that E. coli actively exports cAMP  
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<br/>
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It has been known for a long time that <i>E. coli</i> actively exports cAMP  
into the growth medium <a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[3]</a><a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[4]</a>.
into the growth medium <a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[3]</a><a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[4]</a>.
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<section>
<section>
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<h2>Gene transcription</h2>
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<h2 id="gen">Gene transcription</h2>
The transcription of the <i>cyaA</i> gene and the <i>crp</i> gene is negatively regulated by CRP-cAMP
The transcription of the <i>cyaA</i> gene and the <i>crp</i> gene is negatively regulated by CRP-cAMP
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[5]</a>
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[5]</a>
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[6]</a>.
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[6]</a>.
</br>
</br>
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The translation of adenylate cyclase mRNA is ineffective as to prevent  
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<br/>The translation of adenylate cyclase mRNA is ineffective to prevent  
excessive synthesis of adenylate cyclase.  This can be attributed to  
excessive synthesis of adenylate cyclase.  This can be attributed to  
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the fact that overproduction of cAMP is lethal to Escherichia coli  
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the fact that overproduction of cAMP is lethal to <i>E. coli</i>
possibly due to an accumulation of methylglyoxal
possibly due to an accumulation of methylglyoxal
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[7]</a>
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[7]</a>
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[8]</a>.
<a href="https://2012.igem.org/Team:Grenoble/Biology/AND_gate#1">[8]</a>.
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As we want to use paraBAD like an AND Gate and because high a concentration of cAMP is lethal for <i>E. Coli</i> we make our device in a BW25113 <i>&Delta;cyaA</i>.
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<br/><br/>As we want to use paraBAD like an AND Gate and because high a concentration of cAMP is lethal for <i>E. Coli</i> we make our device in a BW25113 <i>&Delta;cyaA</i>.
</section>
</section>
<section>
<section>
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<h2>A new characterization of the paraBAD promoter</h2>
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<h2 id="char">A new characterization of the paraBAD promoter</h2>
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<p>In order to characterize the paraBAD promoter we used a transcriptional fusion of the promoter and gfpmut2 from Alon on pUA66 in BW25113 &Delta;cyaA.<br/>
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In order to characterize the paraBAD promoter we used a transcriptional fusion of the promoter and gfpmut2 from Alon on pUA66 in BW25113 <i>&Delta;cyaA.</i><br/>
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We followed the fluorescent expression of the GFP versus different concentrations of arabinose AND cAMP over time. We made this experiment in different growth media:<br/>
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We followed the fluorescent expression of the GFP versus different concentrations of arabinose AND cAMP over time (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test">protocol</a>). <br/>
 +
<br/>We made this experiment in different growth media:
<ul>
<ul>
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<li>M9 complement with 0.1% of acetate (3)</li>
<li>M9 complement with 0.1% of acetate (3)</li>
</ul>
</ul>
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<br/>
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Those different media did not affect bacterial growth.
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See the <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols">protocol</a>.</p>
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<br/>
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<p>Those different media did not affect bacterial growth.<br/>
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<b>NB : You can click on each figure to have a larger view !</b></p>
<b>NB : You can click on each figure to have a larger view !</b></p>
<br/>
<br/>
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</section>
</section>
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<section>
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<h2 id="ccl">Conclusion</h2>
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<p>With those different experiments we demonstrated that paraBAD works as expected. It will be activated only if there is arabinose and cAMP in the medium. It appears that both cAMP concentration and time response required for a half expression of GFP depends on the medium. As we want a quick response as well as a satisfying sensivity, these analysis shows that the best medium for our device to come would be the M9 medium complemented with 0.1% of glycerol (second medium).</p>
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</section>
<section>
<section>
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of nutrients. Nat Rev Microbiol, 6(8), 613–24.</a></li>
of nutrients. Nat Rev Microbiol, 6(8), 613–24.</a></li>
<br/>
<br/>
-
<li><b>[2]</b> <a ref="http://www.ncbi.nlm.nih.gov/pubmed/18974181"target="_blank">Keseler, I. M., Bonavides-Martinez, C., Collado-Vides, J., Gama-Castro, S., Gunsalus, R. P., Johnson, D. A., Krummenacker, M., Nolan, L. M., Paley, S., Paulsen, I. T., Peralta-Gil, M., Santos-Zavaleta, A., Shearer, A. G., & Karp, P. D. 2009. EcoCyc: a comprehensive view of Escherichia coli biology. Nucleic Acids Res, 37(Database issue), D464–70.</a>
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<li><b>[2]</b> <a ref="http://www.ncbi.nlm.nih.gov/pubmed/18974181"target="_blank">Keseler, I. M., Bonavides-Martinez, C., Collado-Vides, J., Gama-Castro, S., Gunsalus, R. P., Johnson, D. A., Krummenacker, M., Nolan, L. M., Paley, S., Paulsen, I. T., Peralta-Gil, M., Santos-Zavaleta, A., Shearer, A. G., & Karp, P. D. 2009. EcoCyc: a comprehensive view of Escherichia coli biology. Nucleic Acids Res, 37(Database issue), D464–70.</a></li>
</br>
</br>
<li><b>[3]</b> <a ref="http://www.ncbi.nlm.nih.gov/pubmed/227841" target="_blank">Goldenbaum, P. E., & Hall, G. A. 1979. Transport of cyclic adenosine 3’,5’-monophosphate across
<li><b>[3]</b> <a ref="http://www.ncbi.nlm.nih.gov/pubmed/227841" target="_blank">Goldenbaum, P. E., & Hall, G. A. 1979. Transport of cyclic adenosine 3’,5’-monophosphate across

Latest revision as of 19:19, 8 March 2013

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

Biological "AND" gate

Promoter paraBAD • cyclic Adenosine MonoPhosphate • Gene transcription • A new characterization of the paraBAD promoter • Conclusion

Promoter paraBAD

In order to develop our device we needed a biological "AND" gate. We found a promoter which can be activated by 2 molecules : CRP-cAMP complex and the AraC protein.

The paraBAD promoter has two states:
    
[Schemes]

  • In absence of L-arabinose the paraBAD promoter is repressed by AraC, whereas the paraC promoter is activated (unless an excess of AraC is present).

  • In presence of L-arabinose and the CRP-cAMP complex, the promoter is activated thus enabling the transcription of the downstream elements.

    • This AND gate provides a filter to biological noise. Check out our main results page to for the AND gate characterization.

    cyclic Adenosine MonoPhosphate

    In order to make our AND gate work we need to produce cyclic Adenosine Monophosphate (cAMP). cAMP is produce by adenyl cyclase (encoded by the cyaA gene). It is an enzyme which catalyses the conversion of ATP to 3’-5’-cAMP.

    In E. coli, cAMP is involved in carbon catabolite repression [1] and binds to the cAMP receptor protein (CRP). The corresponding complex (CRP-cAMP) is a transcriptional factor controlling the expression of more than 220 operons [2].

    It has been known for a long time that E. coli actively exports cAMP into the growth medium [3][4].

    Gene transcription

    The transcription of the cyaA gene and the crp gene is negatively regulated by CRP-cAMP [5] [6].

    The translation of adenylate cyclase mRNA is ineffective to prevent excessive synthesis of adenylate cyclase. This can be attributed to the fact that overproduction of cAMP is lethal to E. coli possibly due to an accumulation of methylglyoxal [7] [8].

    As we want to use paraBAD like an AND Gate and because high a concentration of cAMP is lethal for E. Coli we make our device in a BW25113 ΔcyaA.

    A new characterization of the paraBAD promoter

    In order to characterize the paraBAD promoter we used a transcriptional fusion of the promoter and gfpmut2 from Alon on pUA66 in BW25113 ΔcyaA.
    We followed the fluorescent expression of the GFP versus different concentrations of arabinose AND cAMP over time (protocol).

    We made this experiment in different growth media:
    • M9 complement with 0.03% of glucose and 0.03% of acetate (1)
    • M9 complement with 0.1% of glycerol (2)
    • M9 complement with 0.1% of acetate (3)
    Those different media did not affect bacterial growth. NB : You can click on each figure to have a larger view !


    medium (1)
    medium (2)
    medium (3)

    In a first place, we tested the robustness of the AND gate.


    medium (1)
    medium (2)
    medium (3)

    As you can see on this figure even after few hours there is no fluorescent expression if one activator is absent.
    Now for our system we studied the half-expression of GFP versus the cAMP concentration at 1.6% of arabinose.


    medium (1)
    medium (2)
    medium (3)

    As you can see in the two first media we have the half expression of GFP at 0.4mM of cAMP whereas in the third medium its concentration is: 0.8mM. We want a sensitive detector, therefore the third medium conditions are not optimum.

    NB : if there is no arabinose we do not have any GFP expression.

    medium (1)
    medium (2)
    medium (3)

    One of ours imperative is : a fast response. Consequently, we need to know the time it takes to reach the half expression.


    medium (1)
    medium (3)

    In this figure we can see that GFP expression is faster in the medium with glycerol.

    Conclusion

    With those different experiments we demonstrated that paraBAD works as expected. It will be activated only if there is arabinose and cAMP in the medium. It appears that both cAMP concentration and time response required for a half expression of GFP depends on the medium. As we want a quick response as well as a satisfying sensivity, these analysis shows that the best medium for our device to come would be the M9 medium complemented with 0.1% of glycerol (second medium).

    References

    Retrieved from "http://2012.igem.org/Team:Grenoble/Biology/AND_gate"