Team:Baskent-Meds/Project

From 2012.igem.org

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    <p>&nbsp;    </p> 
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    <ul><li class="green"><p class="phunk"><a href="/Team:Baskent-Meds">Home</a></p>
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        <span class="style2">Where we started</span></p> 
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            <p><a href="/Team:Baskent-Meds/Project">Project</a></p> 
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            <p class="style2">Our lovely project</p>
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            <p><a href="/Team:Baskent-Meds/Notebook">Notebook</a></p>
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            <p><a href="/Team:Baskent-Meds/Results">Results</a></p> 
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            <p class="style2">Yeah, that's all, for now </p>
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            <p><a href="/Team:Baskent-Meds/Gallery">Gallery</a></p>
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            <p class="style2">Our little time capsule</p>
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            <p><a href="/Team:Baskent-Meds/Team">Team</a></p>
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<div id="ana" style="position:absolute; left:-25px; top:-100px; width:1000px; height:1000px; z-index:1; background-color: #FFFFFF; layer-background-color: #FFFFFF; border: 1px none #000000;">
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      <p align="center" class="style10" style="margin: 0.4em 0px 0.5em; line-height: 1.5em; font-size: 18px; color: rgb(0, 0, 0); font-family: 'normal Arial', Helvetica, sans-serif; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); ">&nbsp;</p>
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<div id="Sxmenu" style="position:absolute; left:25px; top:16px; width:1000px; height:200px; z-index:1">
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      <p align="center" class="style10" style="margin: 0.4em 0px 0.5em; line-height: 1.5em; font-size: 24px; color: rgb(0, 0, 0); font-family: 'Gill Sans MT', Helvetica; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); "><strong>Transformation of<span class="Apple-converted-space"> </span><em>Escherichia coli</em><span class="Apple-converted-space"> </span>In Order To Develop</strong></p>
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      <p align="center" class="style10" style="margin: 0.4em 0px 0.5em; line-height: 1.5em; font-size: 24px; color: rgb(0, 0, 0); font-family: 'Gill Sans MT', Helvetica; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); "><strong><em>Legionella pneumophila</em><span class="Apple-converted-space"> </span>Sensing Bacteria</strong></p>
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      <p align="center" style="margin: 0.4em 0px 0.5em; line-height: 1.5em; color: rgb(0, 0, 0); font-family: 'Gill Sans MT', Helvetica; font-size: 18px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); "><strong> </strong></p>
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<!--menu bar basladi -->
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      <p style="margin: 0.4em 0px 0.5em; line-height: 1.5em; color: rgb(0, 0, 0); font-family: 'Gill Sans MT', Helvetica; font-size: 18px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); ">Our aim, as the team “Baskent_Meds”, is developing bacteria which can recognize<span class="Apple-converted-space"> </span><em>Legionella pneumophila</em><span class="Apple-converted-space"> </span>specifically at species level by legionella quorum sensing (lqs), and respond to that recognition by producing anti-Legionella peptide which is produced by some<em>Staphylococcus</em><span class="Apple-converted-space"> </span>strains.<span class="Apple-converted-space"> </span><em>lqs</em><span class="Apple-converted-space"> </span>gene locus is responsible for quorum sensing mechanism in<em><span class="Apple-converted-space"> </span>Legionella pneumophila</em>. On the<span class="Apple-converted-space"> </span><em>lqs</em><span class="Apple-converted-space"> </span>locus there are three genes;<span class="Apple-converted-space"> </span><em>lqsA</em><span class="Apple-converted-space"> </span>(encoding autoinducer synthase),<span class="Apple-converted-space"> </span><em>lqsR</em><span class="Apple-converted-space"> </span>(encoding response regulator), and<span class="Apple-converted-space"> </span><em>lqsS</em><span class="Apple-converted-space"> </span>(encoding a sensor kinase). LqsA is a pyridoxal-5′-phosphate-dependent enzyme that catalyses the production of the signaling molecule 3-hydroxypentadecane-4-one (LAI-1; Legionella autoinduer-1), a novel member of α-hydroxyketone signalling molecules (Spirig et al., 2008). In this comprehensive and long-termed project, our initial objectives were; generating competent<span class="Apple-converted-space"> </span><em>E. coli</em><span class="Apple-converted-space"> </span>cells in which we are planning to clone<span class="Apple-converted-space"> </span><em>lqs</em><span class="Apple-converted-space"> </span>gene locus (the gene locus responsible for quorum sensing mechanism in<em><span class="Apple-converted-space"> </span>Legionella pneumophila</em>), and optimizing transformation procedures. pNT-1 (Tiaden et al., 2007), a pUCBM20-derivative, bearing full<span class="Apple-converted-space"> </span><em>lqs</em><span class="Apple-converted-space"> </span>gene cluster, and pTS-2 (Spirig et al., 2008), a pMMB207C-derivative expression vector, bearing<span class="Apple-converted-space"> </span><em>LqsA</em><span class="Apple-converted-space"> </span>gene were kindly provided by Prof. Dr. Hubert Hilbi.</p>
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      <p style="margin: 0.4em 0px 0.5em; line-height: 1.5em; color: rgb(0, 0, 0); font-family: 'Gill Sans MT', Helvetica; font-size: 18px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); ">Initially,<span class="Apple-converted-space"> </span><em>E. coli</em><span class="Apple-converted-space"> </span>competent cell groups (JM109, DH5α and XL1-Blue) were formed by exerting calcium chloride precipitation. pNT-1 and pTS-2 were transferred into competent<span class="Apple-converted-space"> </span><em>E. coli</em><span class="Apple-converted-space"> </span>cells using heat shock transformation method, and clones were selected with ampicillin and chloramphenicol, respectively. Plasmid isolations were performed from transformants by QIAprep Spin Miniprep Kit (Qiagen), and visualized by agarose gel electrophoresis. Our recent studies are designing our construct with BioBricks, and amplifying<span class="Apple-converted-space"> </span><em>LqsR</em><span class="Apple-converted-space"> </span>and<span class="Apple-converted-space"> </span><em>LqsS</em>by gene specific primers having restriction enzyme cut sites as small adaptors. We are going to clone these genes by digesting with these restriction enzymes followed by ligation to one of the iGEM vectors. After cloning these genes, we are planning to clone anti-Legionella peptide nucleotide sequence under<span class="Apple-converted-space"> </span><em>lqs</em><span class="Apple-converted-space"> </span>control. In order to check response of transformant<span class="Apple-converted-space"> </span><em>E. coli</em><span class="Apple-converted-space"> </span>we are planning to clone pTS-2 to BL21(DE3)<span class="Apple-converted-space"> </span><em>E. coli</em><span class="Apple-converted-space"> </span>for the constitutive expression and production of the LAI-1 without the handling of the pathogen strains in our laboratory.</p>
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      <p style="margin: 0.4em 0px 0.5em; line-height: 1.5em; color: rgb(0, 0, 0); font-family: 'Gill Sans MT', Helvetica; font-size: 18px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); ">By the end of our experiments, we aim to obtain sensor<span class="Apple-converted-space"> </span><em>E. coli</em><span class="Apple-converted-space"> </span>cells which respond to<span class="Apple-converted-space"> </span><em>Legionella pneumophila<span class="Apple-converted-space"> </span></em>quorum sensing by recognizing LAI-1. Our expression construct will also enable production of the anti-Legionella peptide in response to LAI-1. Since the peptide has specific antibacterial activity on<span class="Apple-converted-space"> </span><em>Legionella</em><span class="Apple-converted-space"> </span>spp., the achievement of the project is the destruction of<span class="Apple-converted-space"> </span><em>Legionella pneumophila<span class="Apple-converted-space"> </span></em>by transformant<span class="Apple-converted-space"> </span><em>E. coli</em><span class="Apple-converted-space"> </span>sensor cells.</p>
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<li><a href="/Team:Baskent-Meds" title="BaskentMeds main page">Home</a></li>
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<a href="/Team:Baskent-Meds/Project" title="Our Project Abstract">Project</a>
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<li><a href="/Team:Baskent-Meds/Notebook" title="Our Beloved Notebook!">Notebook</a></li>
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<li><a href="/Team:Baskent-Meds/Results" title="Still Working on this">Results</a></li>
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  <p align="center" class="style10"><strong> Transformation of <em>Escherichia coli</em> In Order To Develop </strong></p>
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  <p align="center" class="style10"><strong><em> Legionella pneumophila</em> Sensing Bacteria </strong></p>
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  <p align="center"><strong> &nbsp;</strong></p>
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  <p> Our aim, as the team &ldquo;Baskent_Meds&rdquo;, is developing bacteria which can recognize <em>Legionella pneumophila</em> specifically at species level by legionella quorum sensing (lqs), and respond to that recognition by producing anti-Legionella peptide which is produced by some <em>Staphylococcus</em> strains. <em>lqs</em> gene locus is responsible for quorum sensing mechanism in<em> Legionella pneumophila</em>. On the <em>lqs</em> locus there are three genes; <em>lqsA</em> (encoding autoinducer synthase), <em>lqsR</em> (encoding response regulator), and <em>lqsS</em> (encoding a sensor kinase). LqsA is a pyridoxal-5&prime;-phosphate-dependent enzyme that catalyses the production of the signaling molecule 3-hydroxypentadecane-4-one (LAI-1; Legionella autoinduer-1), a novel member of &alpha;-hydroxyketone signalling molecules (Spirig et al., 2008). In this comprehensive and long-termed project, our initial objectives were; generating competent <em>E. coli</em> cells in which we are planning to clone <em>lqs</em> gene locus (the gene locus responsible for quorum sensing mechanism in<em> Legionella pneumophila</em>), and optimizing transformation procedures. pNT-1 (Tiaden et al., 2007), a pUCBM20-derivative, bearing full <em>lqs</em> gene cluster, and pTS-2 (Spirig et al., 2008), a pMMB207C-derivative expression vector, bearing <em>LqsA</em> gene were kindly provided by Prof. Dr. Hubert Hilbi. </p>
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  <p> Initially, <em>E. coli</em> competent cell groups (JM109, DH5&alpha; and XL1-Blue) were formed by exerting calcium chloride precipitation. pNT-1 and pTS-2 were transferred into competent <em>E. coli</em> cells using heat shock transformation method, and clones were selected with ampicillin and chloramphenicol, respectively. Plasmid isolations were performed from transformants by QIAprep Spin Miniprep Kit (Qiagen), and visualized by agarose gel electrophoresis. Our recent studies are designing our construct with BioBricks, and amplifying <em>LqsR</em> and <em>LqsS</em> by gene specific primers having restriction enzyme cut sites as small adaptors. We are going to clone these genes by digesting with these restriction enzymes followed by ligation to one of the iGEM vectors. After cloning these genes, we are planning to clone anti-Legionella peptide nucleotide sequence under <em>lqs</em> control. In order to check response of transformant <em>E. coli</em> we are planning to clone pTS-2 to BL21(DE3) <em>E. coli</em> for the constitutive expression and production of the LAI-1 without the handling of the pathogen strains in our laboratory. </p>
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  <p> By the end of our experiments, we aim to obtain sensor <em>E. coli</em> cells which respond to <em>Legionella pneumophila </em>quorum sensing by recognizing LAI-1. Our expression construct will also enable production of the anti-Legionella peptide in response to LAI-1. Since the peptide has specific antibacterial activity on <em>Legionella</em> spp., the achievement of the project is the destruction of <em>Legionella pneumophila </em>by transformant <em>E. coli</em> sensor cells. </p>
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Transformation of Escherichia coli In Order To Develop

Legionella pneumophila Sensing Bacteria

 

Our aim, as the team “Baskent_Meds”, is developing bacteria which can recognize Legionella pneumophila specifically at species level by legionella quorum sensing (lqs), and respond to that recognition by producing anti-Legionella peptide which is produced by someStaphylococcus strains. lqs gene locus is responsible for quorum sensing mechanism in Legionella pneumophila. On the lqs locus there are three genes; lqsA (encoding autoinducer synthase), lqsR (encoding response regulator), and lqsS (encoding a sensor kinase). LqsA is a pyridoxal-5′-phosphate-dependent enzyme that catalyses the production of the signaling molecule 3-hydroxypentadecane-4-one (LAI-1; Legionella autoinduer-1), a novel member of α-hydroxyketone signalling molecules (Spirig et al., 2008). In this comprehensive and long-termed project, our initial objectives were; generating competent E. coli cells in which we are planning to clone lqs gene locus (the gene locus responsible for quorum sensing mechanism in Legionella pneumophila), and optimizing transformation procedures. pNT-1 (Tiaden et al., 2007), a pUCBM20-derivative, bearing full lqs gene cluster, and pTS-2 (Spirig et al., 2008), a pMMB207C-derivative expression vector, bearing LqsA gene were kindly provided by Prof. Dr. Hubert Hilbi.

Initially, E. coli competent cell groups (JM109, DH5α and XL1-Blue) were formed by exerting calcium chloride precipitation. pNT-1 and pTS-2 were transferred into competent E. coli cells using heat shock transformation method, and clones were selected with ampicillin and chloramphenicol, respectively. Plasmid isolations were performed from transformants by QIAprep Spin Miniprep Kit (Qiagen), and visualized by agarose gel electrophoresis. Our recent studies are designing our construct with BioBricks, and amplifying LqsR and LqsSby gene specific primers having restriction enzyme cut sites as small adaptors. We are going to clone these genes by digesting with these restriction enzymes followed by ligation to one of the iGEM vectors. After cloning these genes, we are planning to clone anti-Legionella peptide nucleotide sequence under lqs control. In order to check response of transformant E. coli we are planning to clone pTS-2 to BL21(DE3) E. coli for the constitutive expression and production of the LAI-1 without the handling of the pathogen strains in our laboratory.

By the end of our experiments, we aim to obtain sensor E. coli cells which respond to Legionella pneumophila quorum sensing by recognizing LAI-1. Our expression construct will also enable production of the anti-Legionella peptide in response to LAI-1. Since the peptide has specific antibacterial activity on Legionella spp., the achievement of the project is the destruction of Legionella pneumophila by transformant E. coli sensor cells.

Retrieved from "http://2012.igem.org/Team:Baskent-Meds/Project"