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Team:INSA-Lyon/Project/Stage3/Strategy/construction1
From 2010.igem.org
Construction for the regulation by arabinose
We can find below the details schemas of the three constructions requisite for this regulation
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
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
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
