Team:ULB-Brussels/Project/WetLab
From 2014.igem.org
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Those two screening will be carried on independently, and we hope that their results will be coherent with each other.</p> | Those two screening will be carried on independently, and we hope that their results will be coherent with each other.</p> | ||
- | <h3>1. Positive selection using RFP:: 2A::phoA</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. (Hoffman and Wright 1985; van Geest and Lolkema 2000) | <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. (Hoffman and Wright 1985; van Geest and Lolkema 2000) | ||
<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: <br>$\hspace{0.25cm}$ | <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: <br>$\hspace{0.25cm}$ | ||
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<p>If proline::phoA bacteria colour their medium, it will mean that the alkaline phosphatase can be translocated into the periplasm even if it is fused with an additional proline on its N-extremity. The previous experiment (Screening of different phoA-like sequences) will thus be carried on. If their medium remains colourless, it means that we cannot use phoA to test the activity of the 2A peptides and we will have to re-design an experiment with other molecular markers. The control group of this control experiment will consist of bacteria lacking the phoA gene in which we will have inserted the phoA gene ourselves, and it should produce bacteria that colour their XP-medium.</p> | <p>If proline::phoA bacteria colour their medium, it will mean that the alkaline phosphatase can be translocated into the periplasm even if it is fused with an additional proline on its N-extremity. The previous experiment (Screening of different phoA-like sequences) will thus be carried on. If their medium remains colourless, it means that we cannot use phoA to test the activity of the 2A peptides and we will have to re-design an experiment with other molecular markers. The control group of this control experiment will consist of bacteria lacking the phoA gene in which we will have inserted the phoA gene ourselves, and it should produce bacteria that colour their XP-medium.</p> | ||
- | <h3>2. Negative selection using RFP::2A::ccdB</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. (Van Melderen, 2010) After growth on selective medium, the construction RFP::2A::ccdB expressed by our bacteria should lead to 3 kinds of results: <br>$\hspace{0.25cm}$ | CcdB is a prokaryotic toxin affecting the DNA gyrase, which leads to the death of the cell. (Van Melderen, 2010) After growth on selective medium, the construction RFP::2A::ccdB expressed by our bacteria should lead to 3 kinds of results: <br>$\hspace{0.25cm}$ | ||
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<h2>B. Characterization of the Mighty coli system in E.coli</h2> | <h2>B. Characterization of the Mighty coli system in E.coli</h2> | ||
- | <h3>1. Analysis of the improvement in the quantity of the product</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. (Delvigne et al., 2009) 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> | <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. (Delvigne et al., 2009) 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> |
<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. (Delvigne et al., 2009)</p> | <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. (Delvigne et al., 2009)</p> | ||
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<h2> B. Characterization of the Mighty coli system in S.cerevisiae </h2> | <h2> B. Characterization of the Mighty coli system in S.cerevisiae </h2> | ||
- | <h3> 1. Analysis of the improvement in the quantity of the product</h3> | + | <h3> <font color="#000050"> 1. Analysis of the improvement in the quantity of the product</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. (Delvigne et al., 2009) 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> | <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. (Delvigne et al., 2009) 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> 2. Analysis of the improvement in the quality of the product</h3> | + | <h3> <font color="#000050"> 2. Analysis of the improvement in the quality of the product</h3> |
<p>To evaluate the improvement in the quality of the protein production, we will use Apol1 as protein of interest. | <p>To evaluate the improvement in the quality of the protein production, we will use Apol1 as protein of interest. | ||
Indeed, this protein possesses several isoforms, each of them the resulting of a mutation of the original Apol1 gene, and the concentration of each can be easily measured. We will thus compare the relative concentrations of the isoforms of Apol1 produced by a common yeast with those of a yeast expressing the Mighty Coli system (one plasmid containing the Kid gene, and the other containing the construction Apol1::P2A::Kis).</p> | Indeed, this protein possesses several isoforms, each of them the resulting of a mutation of the original Apol1 gene, and the concentration of each can be easily measured. We will thus compare the relative concentrations of the isoforms of Apol1 produced by a common yeast with those of a yeast expressing the Mighty Coli system (one plasmid containing the Kid gene, and the other containing the construction Apol1::P2A::Kis).</p> |
Revision as of 20:59, 10 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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