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Team:Brasil-SP/Results/CharacterizationAssemblies
From 2014.igem.org
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<h2>Tunning of the QteE Threshold</h2> | <h2>Tunning of the QteE Threshold</h2> | ||
| - | <p><strong>Question</strong>:What are the concentration of QteE needed to hamper the LasR induction of the promoter PlasR?</p> | + | <p><strong>Question</strong>:What are the concentration of QteE needed to hamper the LasR induction of the promoter PlasR?</strong></p> |
<p>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).</p> | <p>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).</p> | ||
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<br><br> | <br><br> | ||
| - | <p font-size="16px">< | + | <p font-size="16px"><strong>What we expedted to see</strong></p> |
<br> | <br> | ||
| - | <p>< | + | <p><strong>DI</strong>: Here we expected no flourescence at all as the PcomE promoter woud have no phosphorilated ComE to activate LasR expression.</p> |
| - | <p>< | + | <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> |
| - | <p>< | + | <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 wnated to verify was wheter the LasR could induce GFP expression whem in the same molar concentration as QteE.</p> |
| + | <br> | ||
| + | |||
| + | <p><strong>What actually happened</strong>: After measuring the construction above the result we had was inconsistents. The KX assembly which was supposed to ave a higher fluorescence whem compared to the other two did not display such behavior. Some are the problems we might have had with our construction: | ||
| + | <ul> | ||
| + | <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> | ||
| + | <li>The PlasR promoter is just not working properly.</li> | ||
| + | <li>Any false positive in the confirmation of the assemblies could have harmed the circuit's biological cascade.</li> | ||
<br> | <br> | ||
Revision as of 05:32, 16 October 2014
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?
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).
What we expedted to see
DI: Here we expected no flourescence at all as the PcomE promoter woud 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 wnated to verify was wheter the LasR could induce GFP expression whem in the same molar concentration as QteE.
What actually happened: After measuring the construction above the result we had was inconsistents. The KX assembly which was supposed to ave a higher fluorescence whem compared to the other two did not display such behavior. Some are the problems we might have had with our construction:
- 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.
- The PlasR promoter is just not working properly.
- Any false positive in the confirmation of the assemblies could have harmed the circuit's biological cascade.
