Difference between revisions of "Team:ECUST/Experiments"

 
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    <div id="bannerquote">Quorum Sensing </div>
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<h1 class="box-heading">Description</h1>
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<p>Quorum sensing is an organic chemical signal that regulates a variety of physiological activities of bacteria, such as cell movement, growth, and biofilm formation. This sensing mechanism is mediated by diffusible chemical signals called homoserine lactones. The concentration of signal molecules outside the cell increases with increasing cell density and the signal molecule itself is permeable to the cell membrane. Upon reaching the threshold concentration of the chemical signal, the quorum sensing system is activated to trigger expression or inhibition of the gene. </p>
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<img src="https://static.igem.org/mediawiki/2018/3/32/T--ECUST--quo_sen1.png" class="zoom">
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<h1 class="box-heading">Design</h1>
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<p>We used the quorum sensing system of <i>iron bacteria</i> isolated from pipeline rust. This system is homologous to the LuxR system. The <i>iron bacteria</i> itself produces the AfeR protein, which binds to 3-oxo-C12-AHL and dimerizes. This dimer binds to afeR box and activates gene transcription. Therefore, the recognition of <i>iron bacteria</i> by <i>Escherichia coli</i> can be achieved by constructing a heterologous AfeR protein of <i>iron bacteria</i> and a corresponding activated promoter in engineered <i>E. coli</i>.</p>
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<img src="https://static.igem.org/mediawiki/2018/8/8a/T--ECUST--quo_sen2.png" alt="Figure1.1 OD600 reference point tab" class="zoom">
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<img src="https://static.igem.org/mediawiki/2018/3/3c/T--ECUST--quo_sen3.png" alt="Figure1.1 OD600 reference point tab" class="zoom">
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<h1>Experiments</h1>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
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<h1 class="box-heading">Result</h1>
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<p >Gene fragment of GFP is synthesized through Genescript and it is on vector pUC57. </p>
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<img src="https://static.igem.org/mediawiki/2018/e/e4/T--ECUST--QUORUM_SENSING_F1.jpeg" class="Q1">
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<figcaption><b>Figure 1. Insert GFP(controlled by promoter with Afe-box) to vector pUC57.</b></figcaption>
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<p >The plasmid was transformed to <i>E. coli</i> DH5α and cultured at 37 °C for 12 h. Then positive bacteria is cultured in LB culture medium adding with 0.1% ampicillin. After bacteria grows to logarithmic phase, adding AHL until final concentration is 10<sup>-4</sup> ,10<sup>-5</sup>,10<sup>-6</sup> ,10<sup>-7</sup>,10<sup>-8</sup>, 10<sup>-9</sup> mol/L.  Then light absorption is measured. Excitation/Emission wavelength is 485nm/530nm. </p>
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<figcaption ><b>Figure 2. (a) Fluorescence intensity at different time induced by different concentrations of AHL.
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          (b)A three-dimensional map with log10 concentration of AHL,time and fluorescence intensity as the coordinate
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<p >When the AHL concentration is in the range of 10<sup>-7</sup>~10<sup>-8</sup> mol/L, the fluorescence insensity was jump. When the AHL concentration is above 10<sup>-7</sup> mol/L, the promoter will respond. </p>
 
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<h1 class="box-heading">Reference</h1>
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<p>1. Mol Syst Biol. 2011; 7: 521. Engineering microbes to sense and eradicate <i>Pseudomonas aeruginosa</i>, a human pathogen Nazanin Saeidi,1,* Choon Kit Wong</p>
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<h3>What should this page contain?</h3>
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<p>2. Biological Research 2005 A Lux-like quorum sensing system in the extreme acidophile <i>Acidithiobacillus ferrooxidans</i>. Mariella O. Rivas, Michael Seeger</p>
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<p>3.American Society for Microbiology Evidence for a Functional Quorum-Sensing Type AI-1 System in the Extremophilic Bacterium <i>Acidithiobacillus ferrooxidans</i> Carolina Farah1, Mario Vera</p>
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<li> Protocols </li>
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<li> Experiments </li>
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<li> Documentation of the development of your project </li>
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<h3>Inspiration</h3>
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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Latest revision as of 02:52, 18 October 2018

Quorum Sensing

Description

Quorum sensing is an organic chemical signal that regulates a variety of physiological activities of bacteria, such as cell movement, growth, and biofilm formation. This sensing mechanism is mediated by diffusible chemical signals called homoserine lactones. The concentration of signal molecules outside the cell increases with increasing cell density and the signal molecule itself is permeable to the cell membrane. Upon reaching the threshold concentration of the chemical signal, the quorum sensing system is activated to trigger expression or inhibition of the gene.

Design

We used the quorum sensing system of iron bacteria isolated from pipeline rust. This system is homologous to the LuxR system. The iron bacteria itself produces the AfeR protein, which binds to 3-oxo-C12-AHL and dimerizes. This dimer binds to afeR box and activates gene transcription. Therefore, the recognition of iron bacteria by Escherichia coli can be achieved by constructing a heterologous AfeR protein of iron bacteria and a corresponding activated promoter in engineered E. coli.

Figure1.1 OD600 reference point tab
Figure1.1 OD600 reference point tab

Result

Gene fragment of GFP is synthesized through Genescript and it is on vector pUC57.

Figure 1. Insert GFP(controlled by promoter with Afe-box) to vector pUC57.

The plasmid was transformed to E. coli DH5α and cultured at 37 °C for 12 h. Then positive bacteria is cultured in LB culture medium adding with 0.1% ampicillin. After bacteria grows to logarithmic phase, adding AHL until final concentration is 10-4 ,10-5,10-6 ,10-7,10-8, 10-9 mol/L. Then light absorption is measured. Excitation/Emission wavelength is 485nm/530nm.

Figure 2. (a) Fluorescence intensity at different time induced by different concentrations of AHL. (b)A three-dimensional map with log10 concentration of AHL,time and fluorescence intensity as the coordinate

When the AHL concentration is in the range of 10-7~10-8 mol/L, the fluorescence insensity was jump. When the AHL concentration is above 10-7 mol/L, the promoter will respond.

Reference

1. Mol Syst Biol. 2011; 7: 521. Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen Nazanin Saeidi,1,* Choon Kit Wong

2. Biological Research 2005 A Lux-like quorum sensing system in the extreme acidophile Acidithiobacillus ferrooxidans. Mariella O. Rivas, Michael Seeger

3.American Society for Microbiology Evidence for a Functional Quorum-Sensing Type AI-1 System in the Extremophilic Bacterium Acidithiobacillus ferrooxidans Carolina Farah1, Mario Vera