Team:Baskent Meds/Project

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     <p align="left"><strong>Transformation of Escherichia coli In Order To Develop Legionella pneumophila Sensing Bacteria </strong></p><br>
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     <p class="style8" align="left"><strong>Transformation of Escherichia coli In Order To Develop Legionella pneumophila Sensing Bacteria</strong></p><br>
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    <p> 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.</p>
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<p>In order to reduce the infection risk, detection of environmental source and reduction of microbial load are required. Hyperchlorination of water source and permanence of height water temperature provides mid-grade success. Besides, total eradication of Legionella pneumophila from water sources is quite difficult. Since organism’s potential of infection in closed water storage areas is low, reducing the number of organisms in water source would be a sufficient precaution for control. Our recombinant bacteria are able to respond to biological load of Legionella pneumophila so, increase in number of Legionella cells leads to increased secretion of anti-legionella peptide, and that plays an important role in controlling Legionella numbers in water source.
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Legionella pneumophila cannot be cultured using conventional media, it needs mediums with cysteine and iron. Application of standard culture techniques and detection of bacteria from samples can last up to 10 days. Even identification at genus level is done on media, identification at species level is troubled and samples are generally sent to reference laboratories for this step. When detection with simultaneous quantitative polymerase chain reaction is considered, both method itself reduces required time and identification time is shortened by usage of commercial kits. However, in order this method to give accurate quantitative results at adequate titration, sensitive standardizations must be done for different types of environmental samples. This test is needed to be done in standardized reference laboratories and cost per sample is high. With our quorum sensing based system, presence of low bacterial load can be detected at species level.
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In conclusion, detection and prevention of colonization of Legionella pneumophila are substantially costly and requires long processes. Our biological system can both provide detection and prevent colonization that occurred via high bacterial load with a considerably low cost. Since is works with natural biological expression process, the system can react to environmental alterations in a small amount of time. As the system can settle itself on colonization surfaces, it is easy to use after the first inoculation. The system does not constitute any risks in means of biosafety because Escherichia coli strains which are noninfectious, present in environment and has low virulence are manipulated and developed</p>
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    <p>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.</p>
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    <p>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.
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<p><strong>Our Sponsors</strong></p>
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<a href="http://www.baskent.edu.tr" title="Başkent Üniversitesi"><img src="https://static.igem.org/mediawiki/2013/3/30/Baskentlogo.gif" width="100" height="90" ></a><a href = "http://www.tubitak.gov.tr" title="TÜBİTAK"><img src="https://static.igem.org/mediawiki/2013/b/bf/Tubitaklogo.jpg" width="100" height="90"></a><a href = "http://www.home.agilent.com/agilent/home.jspx?lc=eng&cc=TR" title="Agilent Türkiye"><img src="https://static.igem.org/mediawiki/2013/c/c3/Aglientsmszw.png" width="100" height="90"></a>
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Latest revision as of 18:23, 4 October 2013

Baskent_Meds IGEMwiki



Transformation of Escherichia coli In Order To Develop Legionella pneumophila Sensing Bacteria


In order to reduce the infection risk, detection of environmental source and reduction of microbial load are required. Hyperchlorination of water source and permanence of height water temperature provides mid-grade success. Besides, total eradication of Legionella pneumophila from water sources is quite difficult. Since organism’s potential of infection in closed water storage areas is low, reducing the number of organisms in water source would be a sufficient precaution for control. Our recombinant bacteria are able to respond to biological load of Legionella pneumophila so, increase in number of Legionella cells leads to increased secretion of anti-legionella peptide, and that plays an important role in controlling Legionella numbers in water source.
Legionella pneumophila cannot be cultured using conventional media, it needs mediums with cysteine and iron. Application of standard culture techniques and detection of bacteria from samples can last up to 10 days. Even identification at genus level is done on media, identification at species level is troubled and samples are generally sent to reference laboratories for this step. When detection with simultaneous quantitative polymerase chain reaction is considered, both method itself reduces required time and identification time is shortened by usage of commercial kits. However, in order this method to give accurate quantitative results at adequate titration, sensitive standardizations must be done for different types of environmental samples. This test is needed to be done in standardized reference laboratories and cost per sample is high. With our quorum sensing based system, presence of low bacterial load can be detected at species level.
In conclusion, detection and prevention of colonization of Legionella pneumophila are substantially costly and requires long processes. Our biological system can both provide detection and prevent colonization that occurred via high bacterial load with a considerably low cost. Since is works with natural biological expression process, the system can react to environmental alterations in a small amount of time. As the system can settle itself on colonization surfaces, it is easy to use after the first inoculation. The system does not constitute any risks in means of biosafety because Escherichia coli strains which are noninfectious, present in environment and has low virulence are manipulated and developed




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