Difference between revisions of "Team:ECUST/Experiments"
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<h1 class="box-heading">Design</h1> | <h1 class="box-heading">Design</h1> | ||
| − | <p>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.</p> | + | <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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<figcaption><b>Figure 1. Insert GFP(controlled by promoter with Afe-box) to vector pUC57.</b></figcaption> | <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 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. </p> | + | <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 10-4 ,10-5,10-6 ,10-7,10-8, 10-9 mol/L. Then light absorption is measured. Excitation/Emission wavelength is 485nm/530nm. </p> |
<figure> | <figure> | ||
<figure class="makeresponsive" style="width: 50%;margin-left:24%;margin-right:30%;"> | <figure class="makeresponsive" style="width: 50%;margin-left:24%;margin-right:30%;"> | ||
Revision as of 23:12, 17 October 2018
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 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.
Result
Gene fragment of GFP is synthesized through Genescript and it is on 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.
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.