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Team:Baskent Meds/Project
From 2013.igem.org
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| - | <p align="left"><strong> | + | <p align="left"><strong>Transformation of Escherichia coli In Order To Develop Legionella pneumophila Sensing Bacteria </strong></p> |
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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> | + | <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> |
| - | <p> | + | |
| + | <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. | ||
Revision as of 04:04, 1 August 2013
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.
