Team:INSA-Lyon/Project/Stage3/Strategy/construction1

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<h3>Construction 1</h3>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Strategy" class="cteal"> < back </a></li>
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<h2> Construction for the regulation by arabinose </h2>
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<p> Constitutive production of LuxR and LuxI proteins. Igem parts of luxR and luxI genes are digested and then ligated in the pSB1T3 tetracylcin resistant plasmid. This first construction is then digested by XbaI and PstI. In parallel, igem part of the constitutive promoter is digested by SpeI and PstI. Finally the products are ligated together. The construction obtained can produce constitutively LuxR and LuxI proteins in normal growth conditions (LB 37°C)</p>
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<br><p>We can find below the details schemas of the three constructions requisite for this regulation
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Strategy/construction1#lux">LuxR and LuxI construction</li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Strategy/construction1#hsl">LuxR and HSL construction</li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Strategy/construction1#pbad">pBad and AraC construction</li>
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<h3><font color="purple">Constitutive production of LuxR and LuxI proteins</font></h3><br><br>
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<p>Igem parts of luxR and luxI genes are digested and then ligated in the pSB1T3 tetracylcin resistant plasmid. This first construction is then digested by XbaI and PstI. In parallel, igem part of the constitutive promoter is digested by SpeI and PstI. Finally the products are ligated together. The construction obtained can produce constitutively LuxR and LuxI proteins in normal growth conditions (LB 37°C).</p><br /><br />
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<p style="text-align:center; font-size:0.9em;"><em> Figure 1 : Constitutive production of LuxR and LuxI proteins</em></p><br />
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<h3><font color="purple">Regulation by LuxR/HSL </font></h3><br><br>
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<p>The LuxR/HSL regulated promoter controls PHB synthesis. This synthesis is encoded by an operon containing three genes, phaC, phaA and phaB. The following construction is realized :</p><br /><br /><br />
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<p style="text-align:center; font-size:0.9em;"><em> Figure 2 : Synthesis under the control of LuxR/HSL regulated promoter</em></p><br /><br />
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<h3><font color="purple">Regulation under pBad and AraC protein</font></h3><br><br>
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<p>First, two phasin proteins extracted from igem database and an intein protein are ligated to form the phasin-intein construction, kept in collection for a future ligation. A part of this phasin-intein construction is placed after a constitutive promoter in order to test the construction validity by fluorescence quantification. </p>
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<p>Pbad/araC has to be placed upstream the cI gene and the phasin-intein construction,the productions of cI repressor and phasin and intein proteins are dependant of arabinose addition in medium.
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<br /><p style="text-align:center; font-size:0.9em;"><em> Figure 3 : Phasin-intein construction</em></p>
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<br /><p style="text-align:center; font-size:0.9em;"><em> Figure 4 : Construction under Pbad araC control </em></p>
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Latest revision as of 22:20, 27 October 2010





Construction for the regulation by arabinose



We can find below the details schemas of the three constructions requisite for this regulation

  1. LuxR and LuxI construction
  2. LuxR and HSL construction
  3. pBad and AraC construction

Constitutive production of LuxR and LuxI proteins



Igem parts of luxR and luxI genes are digested and then ligated in the pSB1T3 tetracylcin resistant plasmid. This first construction is then digested by XbaI and PstI. In parallel, igem part of the constitutive promoter is digested by SpeI and PstI. Finally the products are ligated together. The construction obtained can produce constitutively LuxR and LuxI proteins in normal growth conditions (LB 37°C).





Figure 1 : Constitutive production of LuxR and LuxI proteins

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Regulation by LuxR/HSL



The LuxR/HSL regulated promoter controls PHB synthesis. This synthesis is encoded by an operon containing three genes, phaC, phaA and phaB. The following construction is realized :

Figure 2 : Synthesis under the control of LuxR/HSL regulated promoter



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Regulation under pBad and AraC protein



First, two phasin proteins extracted from igem database and an intein protein are ligated to form the phasin-intein construction, kept in collection for a future ligation. A part of this phasin-intein construction is placed after a constitutive promoter in order to test the construction validity by fluorescence quantification.

Pbad/araC has to be placed upstream the cI gene and the phasin-intein construction,the productions of cI repressor and phasin and intein proteins are dependant of arabinose addition in medium.





Figure 3 : Phasin-intein construction








Figure 4 : Construction under Pbad araC control






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