Difference between revisions of "Part:BBa K763003"

(New page: __NOTOC__ <partinfo>BBa_K763003 short</partinfo> When there is no ammonia in the media the RNA-polimerase binds to RpoN Promoter and the transcription starts. Why? In gram-negative bact...)
 
 
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<partinfo>BBa_K763003 short</partinfo>
 
<partinfo>BBa_K763003 short</partinfo>
  
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This construction contains two parts:
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<ol>
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    <li> The transcription factor-binding site inside the promoter and
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    <li> the coding sequence, which contains a synthetic fluorescent (yellow) protein. We chose as the promoter sequence the one of the glnA gene.
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    There is long evidence that this promoter is regulated by nitrogen concentration [1]. Moreover, the promoter is not the canonical one (sigma70) but an alternative one (sigma54) [2,3].
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</ol>
  
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When there is no ammonia in the media the RNA-polimerase binds to RpoN Promoter and the transcription starts. Why?
 
  
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In gram-negative bacteria, transcriptional activation in response to some external stimuli (absence of nitrogen, for example) often involves the alternative sigma factor, Sigma 54. This factor, also called RpoN or Sigma N, was originally identified as the sigma factor for nitrogen-controlled genes. Sigma 54 works in conjunction with members of the NtrC (Nitrogen regulatory protein C) superfamily of transcriptional activators.
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[[Image:Nitrogeno biobricks.png|400px|right]]
  
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“RpoN Promoter” contains a binding site which is recognized by the RNA-polimerase when Sigma 54 is binding to this protein. The polimerase binds to RpoN promoter which is present on genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions (aprox. 2% of the E. coli genome appears to be under NtrC control).  
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When is the protein synthesized? In order to obtain the yellow fluorescent protein a condition should to be met. That condition is related to nitrogen-starvation. The less nitrogen there is, the more expression you get!<br><br>
 +
The molecular mechanism underlying this phenomenon is as follows: in gram-negative bacteria, transcriptional activation in response to some external stimuli (absence of nitrogen, high UV-dosis, for example) often involves the alternative sigma factor, sigma54. This factor, also called RpoN or sigma N, was originally identified as the sigma factor for nitrogen-controlled genes. sigma54 works in conjunction with members of the NtrC (Nitrogenregulatoryprotein C) superfamily of transcriptional activators. In our case, when ammonia levels are low the bacterium undergoes some metabolic changes. Within these changes, there are some related to nitrogen assimilation and processing, so our construction, which responds to low ammonia levels, increases its transcription.<br><br>
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How did we deal with this construction?We developed a nitrogen-gradation tube experiment, which means that we added different (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> concentrations to each tube, so we can measure a gradation of the protein fluorescence. We used a synthetic medium for this.<br><br>
  
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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===Characterization===
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<br>
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In order to characterize our nitrogen-sensitive construction, we carried out an experiment as follows: our <i>E.coli</i> strain carrying the ZsYellow1 gene under the control of the glnA promoter was grown on LBA medium (until an OD of 1.5) in order the bacteria to have sufficient amounts of proteins and nitrogen compounds. Then, cells were pelleted and resuspended in a medium lacking nitrogen. After that, values of OD and fluorescence intensity were measured at different times. As a control, we resuspended one of the aliquots in medium containing 10 g/L of ammonium sulphate.
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As expected according to the <html><a href="http://2012.igem.org/Team:Valencia_Biocampus/Molecular#NITROGEN-REGULATED_PROMOTER"><b>underlying molecular mechanism</b></a></html>, nitrogen starvation induces the expression of the fluorescent protein:<br>
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<table align="center" border="0.01" bordercolor="#FFFFFF" style="background-color:#FFFFFF">
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    <tr>
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        <td><html><img src="https://static.igem.org/mediawiki/parts/e/e2/60M_nitrogen_biobricks.png" width="300" height="250" BORDER=0</a></html></td>
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        <td><html><img src="https://static.igem.org/mediawiki/parts/b/bd/150_nitrogeno_biobricks.png" width="300" height="250" BORDER=0</a></html></td>
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</tr>
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    <tr><td><b>Figure 4.</b> Fluorescence intensity (FI) normalized by<br> the optical density (OD) of the culture. Measures were<br> taken 60 min after the resuspension in a medium<br> containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).</td>
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        <td><b>Figure 5.</b> Fluorescence intensity (FI) normalized by<br> the optical density (OD) of the culture. Measures were<br> taken 150 min after the resuspension in a medium<br> containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).</td>
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    </tr>
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</table>
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<br><br>
  
 
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Latest revision as of 14:26, 25 September 2012

pGlnA + Gene encoding ZsYellow1

This construction contains two parts:

  1. The transcription factor-binding site inside the promoter and
  2. the coding sequence, which contains a synthetic fluorescent (yellow) protein. We chose as the promoter sequence the one of the glnA gene. There is long evidence that this promoter is regulated by nitrogen concentration [1]. Moreover, the promoter is not the canonical one (sigma70) but an alternative one (sigma54) [2,3].


Nitrogeno biobricks.png

When is the protein synthesized? In order to obtain the yellow fluorescent protein a condition should to be met. That condition is related to nitrogen-starvation. The less nitrogen there is, the more expression you get!

The molecular mechanism underlying this phenomenon is as follows: in gram-negative bacteria, transcriptional activation in response to some external stimuli (absence of nitrogen, high UV-dosis, for example) often involves the alternative sigma factor, sigma54. This factor, also called RpoN or sigma N, was originally identified as the sigma factor for nitrogen-controlled genes. sigma54 works in conjunction with members of the NtrC (Nitrogenregulatoryprotein C) superfamily of transcriptional activators. In our case, when ammonia levels are low the bacterium undergoes some metabolic changes. Within these changes, there are some related to nitrogen assimilation and processing, so our construction, which responds to low ammonia levels, increases its transcription.

How did we deal with this construction?We developed a nitrogen-gradation tube experiment, which means that we added different (NH4)2SO4 concentrations to each tube, so we can measure a gradation of the protein fluorescence. We used a synthetic medium for this.


Characterization


In order to characterize our nitrogen-sensitive construction, we carried out an experiment as follows: our E.coli strain carrying the ZsYellow1 gene under the control of the glnA promoter was grown on LBA medium (until an OD of 1.5) in order the bacteria to have sufficient amounts of proteins and nitrogen compounds. Then, cells were pelleted and resuspended in a medium lacking nitrogen. After that, values of OD and fluorescence intensity were measured at different times. As a control, we resuspended one of the aliquots in medium containing 10 g/L of ammonium sulphate.

As expected according to the underlying molecular mechanism, nitrogen starvation induces the expression of the fluorescent protein:

Figure 4. Fluorescence intensity (FI) normalized by
the optical density (OD) of the culture. Measures were
taken 60 min after the resuspension in a medium
containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).		 Figure 5. Fluorescence intensity (FI) normalized by
the optical density (OD) of the culture. Measures were
taken 150 min after the resuspension in a medium
containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 689
  • 1000
    COMPATIBLE WITH RFC[1000]