Difference between revisions of "Team:KU Leuven/Research/Methods"
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<p><b>Theory</b></br> | <p><b>Theory</b></br> | ||
N-acyl homoserine lactones (AHL) are small diffusible molecules used in cell-to-cell signaling by Gram-negative bacteria. <i>Chromobacterium violaceum</i> is a Gram-negative bacterium that produces the violet pigment violacein as a result of sensing AHL. AHL is produced by the autoinducer synthase CviI and released in the environment. When a quorum has been reached, AHL diffuses back into the bacteria and binds the transcriptional regulator CviR. This activates the expression of specific genes which leads to the production of violacein. </p> | N-acyl homoserine lactones (AHL) are small diffusible molecules used in cell-to-cell signaling by Gram-negative bacteria. <i>Chromobacterium violaceum</i> is a Gram-negative bacterium that produces the violet pigment violacein as a result of sensing AHL. AHL is produced by the autoinducer synthase CviI and released in the environment. When a quorum has been reached, AHL diffuses back into the bacteria and binds the transcriptional regulator CviR. This activates the expression of specific genes which leads to the production of violacein. </p> | ||
− | <p>In our project, the mutant <i>C. violaceum</i> CV026 was used to quantify the amount of N-(3-Oxohexanoyl)-L-homoserine lactone (OHHL), a specific type of AHL, produced by the LuxI enzyme our E. coli strains. The CV026 strain is <i>cviI</i> gene deficient and therefore requires exogenous addition of AHL to produce violacein. The idea is to plot a standard curve in which CV026 is induced with different concentrations of AHL. In the standard curve, results were normalized to the OD.</p> | + | <p>In our project, the mutant <i>C. violaceum</i> CV026 was used to quantify the amount of N-(3-Oxohexanoyl)-L-homoserine lactone (OHHL), a specific type of AHL, produced by the LuxI enzyme our <i>E. coli</i> strains. The CV026 strain is <i>cviI</i> gene deficient and therefore requires exogenous addition of AHL to produce violacein. The idea is to plot a standard curve in which CV026 is induced with different concentrations of AHL. In the standard curve, results were normalized to the OD.</p> |
<p>To quantify to amount of AHL produced by <i>E. coli</i>, these were grown and pelleted after which CV026 was added. After incubating for several hours, CV026 and violacein are spun down and the supernatant is removed. The pellet is resuspended in dimethyl sulfoxide and again centrifuged to separate the violacein dissolved in dimethyl sulfoxide from the cells. Absorbance of the supernatans was measured at 585 nm.</p> | <p>To quantify to amount of AHL produced by <i>E. coli</i>, these were grown and pelleted after which CV026 was added. After incubating for several hours, CV026 and violacein are spun down and the supernatant is removed. The pellet is resuspended in dimethyl sulfoxide and again centrifuged to separate the violacein dissolved in dimethyl sulfoxide from the cells. Absorbance of the supernatans was measured at 585 nm.</p> | ||
<p><b>Protocol</b></br> | <p><b>Protocol</b></br> |
Revision as of 00:02, 19 September 2015
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Methods
On this page you can find all of the methods and protocols used in the lab to obtain our results. For some techniques, we included some basic theory, since it is a prerequisite to get acquainted with the theory behind these techniques before using them. To learn more about them, click the titles below!
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be