Team:ULB-Brussels/Project/WetLab
From 2014.igem.org
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<h3><font color="#000050"> 1. Positive selection using RFP:: 2A::phoA </font></h3> | <h3><font color="#000050"> 1. Positive selection using RFP:: 2A::phoA </font></h3> | ||
- | <p>Alkaline phosphatase (phoA) is a periplasmic enzyme whose activity is easily detectable, even at low level, on chromogenic substrate (XP-medium (5-bromo-4-chloro-3-indolyl phosphate)). It is usually used to study protein secretion, but we will use it as a molecular marker for the activity of the 2A peptide. If phoA is correctly synthetized and exported in the periplasm, the substrate acquires a characteristic blue color. | + | <p>Alkaline phosphatase (phoA) is a periplasmic enzyme whose activity is easily detectable, even at low level, on chromogenic substrate (XP-medium (5-bromo-4-chloro-3-indolyl phosphate)). It is usually used to study protein secretion, but we will use it as a molecular marker for the activity of the 2A peptide. If phoA is correctly synthetized and exported in the periplasm, the substrate acquires a characteristic blue color [<b>8,10</b>]. |
<p>In order to assess the efficacity of the 2A peptid, we need to design a plasmid containing 2 molecular markers (the red fluorescent protein (RFP) and phoA separated by a 2A peptide (RFP::2A::phoA). After cloning this plasmid in bacteria lacking the phoA gene in their genome (those bacteria were obtained from E.coli Keio Knockout collection) and after growth on chromogenic and selective XP-medium, we should be able to observe 4 types of results: </p>$\hspace{0.25cm}$ | <p>In order to assess the efficacity of the 2A peptid, we need to design a plasmid containing 2 molecular markers (the red fluorescent protein (RFP) and phoA separated by a 2A peptide (RFP::2A::phoA). After cloning this plasmid in bacteria lacking the phoA gene in their genome (those bacteria were obtained from E.coli Keio Knockout collection) and after growth on chromogenic and selective XP-medium, we should be able to observe 4 types of results: </p>$\hspace{0.25cm}$ | ||
1. Colourless colonies and blue medium <br>$\hspace{0.25cm}$ | 1. Colourless colonies and blue medium <br>$\hspace{0.25cm}$ | ||
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The first control will be colonies expressing the construction RFP::phoA, which should confirm that neither RFP nor phoA are active when 2A is not functional. Therefore, we would observe a blue medium with red colonies only when there is cleavage of the peptide 2A.</p> | The first control will be colonies expressing the construction RFP::phoA, which should confirm that neither RFP nor phoA are active when 2A is not functional. Therefore, we would observe a blue medium with red colonies only when there is cleavage of the peptide 2A.</p> | ||
The second control is actually a control experiment, which will be done by insertion of proline::phoA sequences into bacteria lacking the phoA gene, and growth on chromogenic and selective XP-medium. </p> | The second control is actually a control experiment, which will be done by insertion of proline::phoA sequences into bacteria lacking the phoA gene, and growth on chromogenic and selective XP-medium. </p> | ||
- | <p>Indeed, the N-terminal extremity of phoA is a signal sequence that allows it to be translocated into the bacterial periplasm, where it will be folded in its active form. | + | <p>Indeed, the N-terminal extremity of phoA is a signal sequence that allows it to be translocated into the bacterial periplasm, where it will be folded in its active form [<b>8,10</b>]. It means that the addition of the prolin from the 2A peptide to the N-extremity of phoA [<b>7</b>] could possibly disrupt the translocation process. If such a phenomenon should occur, our positive screening would reveal only negative results for phoA, even when the 2A peptide works correctly. We must thus design this control experiment in order to check that the translocation of phoA in the periplasm occurs even if we add a prolin on its N-extremity. |
The control experiment should produce one of the following results: </p>$\hspace{0.25cm}$ | The control experiment should produce one of the following results: </p>$\hspace{0.25cm}$ | ||
- Colonies on blue medium <br>$\hspace{0.25cm}$ | - Colonies on blue medium <br>$\hspace{0.25cm}$ | ||
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<h3><font color="#000050"> 2. Negative selection using RFP::2A::ccdB </font></h3> | <h3><font color="#000050"> 2. Negative selection using RFP::2A::ccdB </font></h3> | ||
<p>This second screening will serve as an independent test of the first screening. | <p>This second screening will serve as an independent test of the first screening. | ||
- | CcdB is a prokaryotic toxin affecting the DNA gyrase, which leads to the death of the cell. | + | CcdB is a prokaryotic toxin affecting the DNA gyrase, which leads to the death of the cell. [<b>12</b>] After growth on selective medium, the construction RFP::2A::ccdB expressed by our bacteria should lead to 3 kinds of results: </p>$\hspace{0.25cm}$ |
1. Red colonies <br>$\hspace{0.25cm}$ | 1. Red colonies <br>$\hspace{0.25cm}$ | ||
2. Colourless colonies <br>$\hspace{0.25cm}$ | 2. Colourless colonies <br>$\hspace{0.25cm}$ | ||
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<h3><font color="#000050"> 1. Analysis of the improvement in the quantity of the product </font></h3> | <h3><font color="#000050"> 1. Analysis of the improvement in the quantity of the product </font></h3> | ||
- | <p>The quantification of yield improvement shall be done by spectrophotometry in collaboration with F. Delvigne from the University of Liège (ULg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors. | + | <p>The quantification of yield improvement shall be done by spectrophotometry in collaboration with F. Delvigne from the University of Liège (ULg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors [<b>11</b>]. We will compare the GFP production yield of a common E.coli bacterium and the one of an E.coli expressing the Mighty Coli system (one plasmid containing the ccdB gene, and the other containing the construction GFP::2A::ccdA).</p> |
<h3><font color="#000050"> 2. Analysis of the efficiency to reduce the heterogeneity of population </font></h3> | <h3><font color="#000050"> 2. Analysis of the efficiency to reduce the heterogeneity of population </font></h3> | ||
- | <p>If we have the time, it will be done with the collaboration F. Delvigne from the University of Liège (Ulg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors. | + | <p>If we have the time, it will be done with the collaboration F. Delvigne from the University of Liège (Ulg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors [<b>11</b>].</p> |
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<h3> <font color="#000050"> 1. Analysis of the improvement in the quantity of the product </font></h3> | <h3> <font color="#000050"> 1. Analysis of the improvement in the quantity of the product </font></h3> | ||
- | <p>The quantification of yield improvement shall be done by spectrophotometry in collaboration with F. Delvigne from the University of Liège (Ulg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors. | + | <p>The quantification of yield improvement shall be done by spectrophotometry in collaboration with F. Delvigne from the University of Liège (Ulg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors [<b>11</b>]. We will compare the GFP production yield of a common yeast and the one of a yeast expressing the Mighty Coli system (one plasmid containing the Kid gene, and the other containing the construction GFP::P2A::Kis).</p> |
<h3> <font color="#000050"> 2. Analysis of the improvement in its quality </font></h3> | <h3> <font color="#000050"> 2. Analysis of the improvement in its quality </font></h3> |
Revision as of 16:32, 13 October 2014
$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}} \newcommand{\Stabi}{\small Stabi}$ $\newcommand{\EColi}{\small E.coli} \newcommand{\SCere}{\small S.cerevisae}\\[0cm] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\PI}{\small PI}$ $\newcommand{\Igo}{\Large\mathcal{I}} \newcommand{\Tgo}{\Large\mathcal{T}} \newcommand{\Ogo}{\Large\mathcal{O}} ~$
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