Team:Brasil-SP/Results/CharacterizationAssemblies

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   <h1>Characterization Assemblies</h1>
   <h1>Characterization Assemblies</h1>
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   <p> Apart from the main genetic circuit we also assembled others for characterization purposes, such as the validation of the promoters and tunning of our threshold setter concentration, the QteE.</p>
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   <p> Apart from the main genetic circuit we also assembled others for characterization purposes, such as the validation of the promoters and tunning of our threshold setter concentration, the QteE. Click on the circuit images to acess the lab forms.</p>
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<div align=center><h2> DI circuit</h2>
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<a class="map_link_f" id="map_link_0_f" title="" href="https://static.igem.org/mediawiki/2014/7/72/DI.pdf">DI</a>
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<h2> KV Characterization</h2>
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<div align=center><h2> KV Characterization</h2></div>
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<div align=center><img src="https://static.igem.org/mediawiki/2014/8/8a/KV_-_Assembly_Measurement.png"></div>
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<p>This construction is a simple assembly of the of the Lac promoter BBa_K143015 and the GFP BBa_E0840. The Plac was synthesized by our team using PCR (check out the <a href=” https://static.igem.org/mediawiki/2014/3/35/PCR_for_synthesis.pdf”>PCR for Synthesis Protocol</a>). Since there is no LacI being produce, apart from the low levels the <em>e. coli</em> does, the output expected is a significant GFP fluorescence. The result obtained corresponded matched our expectations as shown in the graph above.</p>
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<div align="justify"><p><strong>What we expected to see</strong></p>
 
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<p><strong>DI</strong>: Here we expected no flourescence at all since the PcomE promoter would have no phosphorilated ComE to activate LasR expression.</p>
 
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<p><strong>KX</strong>: In this construction LasR was being producessed constitutively while there was no QteE expression. Because of that the LasR would be able to induce the GFP expression with no expression barrier.</p>
 
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<p><strong>KXIV</strong>: This circuit has both LasR and QteE being generated at the same rate, that's because they are under the control of the same promter. What we wanted to verify was wheter the LasR could induce GFP expression whem in the same molar concentration as QteE.</p>
 
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<p><strong>What actually happened</strong>: After measuring the construction above, the results were inconsistent. KX assembly, which was supposed to have a higher fluorescence compared to the other two, did not display such behavior. Some of the problems we might have had in our construction:
 
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<li>The lasI gene, which produces the HSL needed for proper folding and indicer ability of the LasR protein did not produce it correctly, impossibilitating the induction of the PlasR promoter.</li>
 
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<li>The PlasR promoter is just not working properly.</li>
 
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<li>Any false positive in the confirmation of the assemblies could have harmed the circuit's biological cascade.</li>
 
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<a class="map_link_e" id="map_link_3_e" title="" href="https://static.igem.org/mediawiki/2014/9/95/KX.pdf">KX</b></a>
<a class="map_link_e" id="map_link_3_e" title="" href="https://static.igem.org/mediawiki/2014/9/95/KX.pdf">KX</b></a>
<a class="map_link_e" id="map_link_4_e" title="" href="https://static.igem.org/mediawiki/2014/5/5b/BIV.pdf"></b></a>
<a class="map_link_e" id="map_link_4_e" title="" href="https://static.igem.org/mediawiki/2014/5/5b/BIV.pdf"></b></a>
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<div align="justify"><p><strong>What we expected to see</strong></p>
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<p><strong>DI</strong>: Here we expected no flourescence at all since the PcomE promoter would have no phosphorilated ComE to activate LasR expression.</p>
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<p><strong>KX</strong>: In this construction LasR was being producessed constitutively while there was no QteE expression. Because of that the LasR would be able to induce the GFP expression with no expression barrier.</p>
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<p><strong>KXIV</strong>: This circuit has both LasR and QteE being generated at the same rate, that's because they are under the control of the same promter. What we wanted to verify was wheter the LasR could induce GFP expression whem in the same molar concentration as QteE.</p>
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<p><h2> KX Characterization</h2></p>
<p><h2> KX Characterization</h2></p>
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<img src="" width="200" height="auto">
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<p>Besides we have obtained a low number of fluorescence cells their fluorescence was expressive. The low rate of fluorescence cells might be related to antibiotic problems. It would cause the bacteria do not replicate the plasmid with the assembly because it became nonessestial and hence, only few bacteria expressed GFP.</p>
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<img src="https://static.igem.org/mediawiki/2014/7/72/KXbrasilssp.png" width="600" height="auto">
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<img src="https://static.igem.org/mediawiki/2014/2/2c/KXstatisticsbrasilsp.png" width="600" height="auto">
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<p><h2> KXIV Characterization</h2></p>
<p><h2> KXIV Characterization</h2></p>
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<p>In this assembly, LasR e QteE are constitutive and we expected to use it as a control for an equivalent assembly in which QteE is not under control of a constitutive promoter but an inducible promoter designed for high expression in <em>Bacillus subtilis</em> <a href="http://parts.igem.org/Part:BBa_K143015">(BBa_K143015)</a>.</p>
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<img src="https://static.igem.org/mediawiki/2014/1/1f/KXIVbrasilsp.png" width="600" height="auto">
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<img src="https://static.igem.org/mediawiki/2014/a/a6/KXIVstatistics.png" width="600" height="auto">
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<p>We displayed data in logarithmic scale, then geometric mean is generally chosen to analysis. As we expected, KX GFP expression levels were higher, as follows</p>
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<img src="https://static.igem.org/mediawiki/2014/5/5c/KXKXIVDIbrasilsp.png" width="600" height="auto">
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<p><strong>Observation:</strong>There is no control displayed in the graph above because geometric mean is zero as there is no fluorescence in FL1-H, only autofluorescence.</p>
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<h3>References</h3>
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SPIDLEN, J.  et al. Data File Standard for Flow Cytometry, Version FCS 3.1. Cytometry Part A, v. 77A, n. 1, p. 97-100, Jan 2010. ISSN 1552-4922. Disponível em: < <Go to ISI>://WOS:000273384700012 >.
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Latest revision as of 00:34, 18 October 2014

ResultsBRASILSP.png

Characterization Assemblies

Apart from the main genetic circuit we also assembled others for characterization purposes, such as the validation of the promoters and tunning of our threshold setter concentration, the QteE. Click on the circuit images to acess the lab forms.

Promoter BBa_K823003

Question: Does the constitutive promoter BBa_K823003 work properly?

KI

Results: After the incubation period of the transformed E. coli a large portion of the colonies were glowing green. So the promoter does work. Moreover, this biobrick works on E. coli despite the fact it was designed for B. subtilis.

Promoter BBa_K143015

Question: How does the transcription caused by this promoter varies with the IPTG induction?

KV BIII KXVI

DI circuit

DI

KV Characterization



This construction is a simple assembly of the of the Lac promoter BBa_K143015 and the GFP BBa_E0840. The Plac was synthesized by our team using PCR (check out the PCR for Synthesis Protocol). Since there is no LacI being produce, apart from the low levels the e. coli does, the output expected is a significant GFP fluorescence. The result obtained corresponded matched our expectations as shown in the graph above.

Tunning of the QteE Threshold

Question:What are the concentration of QteE needed to hamper the LasR induction of the promoter PlasR?

This is the most difficult task of our project. Tunning the production of QteE so that we establish the correct threshold for the discretization of the Cystatin C level in serum. To attack this challenge we designed 3 circuits so that we could plot a calibration curve. In this circuits we put the transcription of the LasR and QteE under two differnt promoters, the Pveg (BBa_K823003) and PlasR (BBa_K143015).





KII CII KIV KXI






KIII CII AIV KX






KIII CII AIV KX

What we expected to see


DI: Here we expected no flourescence at all since the PcomE promoter would have no phosphorilated ComE to activate LasR expression.

KX: In this construction LasR was being producessed constitutively while there was no QteE expression. Because of that the LasR would be able to induce the GFP expression with no expression barrier.

KXIV: This circuit has both LasR and QteE being generated at the same rate, that's because they are under the control of the same promter. What we wanted to verify was wheter the LasR could induce GFP expression whem in the same molar concentration as QteE.


KX Characterization

Besides we have obtained a low number of fluorescence cells their fluorescence was expressive. The low rate of fluorescence cells might be related to antibiotic problems. It would cause the bacteria do not replicate the plasmid with the assembly because it became nonessestial and hence, only few bacteria expressed GFP.





KXIV Characterization

In this assembly, LasR e QteE are constitutive and we expected to use it as a control for an equivalent assembly in which QteE is not under control of a constitutive promoter but an inducible promoter designed for high expression in Bacillus subtilis (BBa_K143015).






We displayed data in logarithmic scale, then geometric mean is generally chosen to analysis. As we expected, KX GFP expression levels were higher, as follows


Observation:There is no control displayed in the graph above because geometric mean is zero as there is no fluorescence in FL1-H, only autofluorescence.

References

SPIDLEN, J. et al. Data File Standard for Flow Cytometry, Version FCS 3.1. Cytometry Part A, v. 77A, n. 1, p. 97-100, Jan 2010. ISSN 1552-4922. Disponível em: < ://WOS:000273384700012 >.

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This project is part of iGEM 2014

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