Team:INSA Toulouse/contenu/lab practice/results/general inducer

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<p class="texte">Characterize the general inducer system using the couple TetR-pTet without and with aTc. Reminder: TetR is an inducer that binds to pTet promoter and thus, represses expression the downstream system. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to TetR and invert the operation (inhibits expression of mRFP in this case).</p>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<h3 class="title3">General inductor</h3>
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  <p class="texte">The couple TetR-pTet system construction is a first step to characterize the entire system. A constitutive promoter (BBa_J23116) was assemble to tetR and then assemble to pTet-rbs-RFP-term (Bba_I13521). pTet is a regulator constitutively ON that expresses the mRFP1 protein. TetR is an inducer that binds to pTet promoter and thus, represses expression the downstream system. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to tetR and invert the operation (inhibits expression of mRFP in this case).<br>
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After 18 hours, clones containing this assembly (BBa_J23116-TetR-pTet-RFP) show a leaky basal expression of mRFP. We suppose that the promoter was too weak to express TetR in large quantity. Assembly of a stronger promoter could improve the system and lock the response to an ON/OFF response. <br>
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Besides, an experience was done to analyze the effect of the aTc inducer. Result show a visible induction of the red fluorescent protein expression by addition of aTC (60 ng/mL).</p>
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  <h2 class="title2">Conception</h2>
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<p class="texte">A constitutive promoter is bind to tetR and then is assembled to pTet-rbs-RFP-term (<a href="http://parts.igem.org/Part:BBa_I13521" target="_blank">Bba_I13521</a>). </p>
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  <h2 class="title2">Result</h2>
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<p class="texte">We didn't managed to get the contruction under a strong promoter but we suceeded to obtain the previous assembly with a weak promoter (<a href="http://parts.igem.org/Part:BBa_J23116" target="_blank">BBa_J23116</a>). <br>
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  <br><img src="https://static.igem.org/mediawiki/2013/d/d4/General_inducer_notebook_-_680px.png" /><br><br>
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After 18 hours, clones containing this assembly (BBa_J23116-TetR-pTet-RFP) show a leaky basal expression of mRFP. We suppose that the promoter was too weak to express TetR in large quantity.
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Besides, an experience was done to analyze the effect of the aTc inducer. Result show a visible induction of the red fluorescent protein expression by addition of aTc (60 ng/mL).<br>
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  <br><img src="https://static.igem.org/mediawiki/2013/e/e1/Induct_gen_pfaible.png" /></p>
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  <h2 class="title2">Discussion</h2>
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<p class="texte">The <a href="http://parts.igem.org/Part:BBa_K1132022" target="_blank">construction</a> with the weak promoter is available on the registry. An intermediate construction was assembled without any promoter and is ready to be cloned to a strong promoter: <a href="http://parts.igem.org/Part:BBa_K1132021" target="_blank">BBa_K1132021</a>. Assembly of a stronger promoter could improve the system and lock the response to an ON/OFF response. </p>
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Latest revision as of 00:32, 5 October 2013

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Results - General Inducer Characterization

Objective

Characterize the general inducer system using the couple TetR-pTet without and with aTc. Reminder: TetR is an inducer that binds to pTet promoter and thus, represses expression the downstream system. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to TetR and invert the operation (inhibits expression of mRFP in this case).

Conception

A constitutive promoter is bind to tetR and then is assembled to pTet-rbs-RFP-term (Bba_I13521).

Result

We didn't managed to get the contruction under a strong promoter but we suceeded to obtain the previous assembly with a weak promoter (BBa_J23116).



After 18 hours, clones containing this assembly (BBa_J23116-TetR-pTet-RFP) show a leaky basal expression of mRFP. We suppose that the promoter was too weak to express TetR in large quantity.

Besides, an experience was done to analyze the effect of the aTc inducer. Result show a visible induction of the red fluorescent protein expression by addition of aTc (60 ng/mL).

Discussion

The construction with the weak promoter is available on the registry. An intermediate construction was assembled without any promoter and is ready to be cloned to a strong promoter: BBa_K1132021. Assembly of a stronger promoter could improve the system and lock the response to an ON/OFF response.

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