Difference between revisions of "Team:KU Leuven/Research/Methods"
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<p><b>Theory</b></p> | <p><b>Theory</b></p> | ||
<p> N-acyl homoserine lactones (AHL) are small diffusible molecules used for bacterial cell-to-cell signaling in Gram-negative bacteria. <i>Chromobacterium violaceum</i> is a Gram-negative bacteria which 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, the AHL diffuses back into the bacteria and binds to the transcriptional regulator CviR. This activates the expression of specific genes which lead to violacein. </p> | <p> N-acyl homoserine lactones (AHL) are small diffusible molecules used for bacterial cell-to-cell signaling in Gram-negative bacteria. <i>Chromobacterium violaceum</i> is a Gram-negative bacteria which 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, the AHL diffuses back into the bacteria and binds to the transcriptional regulator CviR. This activates the expression of specific genes which lead to violacein. </p> | ||
− | <p>In our project, the mutant <i>C. violaceum</i> CV026 is used to quantify the amount of OHHL, a specific type of AHL which is produced by luxI. This strain is deficient CviI and therefore requires exogenous addition of AHL to produce violacein. The idea is to achieve a standard curve where the <i>C. violaceum</i> CV026 is induced with different amounts of AHL. In this standard curve we also take into account how long we grow the bacteria and make a correction for the optical density. In the end, we obtained an absorbance value divided by the optical density.</p> | + | <p>In our project, the mutant <i>C. violaceum</i> CV026 is used to quantify the amount of N-(3-Oxohexanoyl)-L-homoserine lactone (OHHL), a specific type of AHL which is produced by luxI. This strain is deficient CviI and therefore requires exogenous addition of AHL to produce violacein. The idea is to achieve a standard curve where the <i>C. violaceum</i> CV026 is induced with different amounts of AHL. In this standard curve we also take into account how long we grow the bacteria and make a correction for the optical density. In the end, we obtained an absorbance value divided by the optical density.</p> |
<p>To begin quantifying the samples, the samples are grown until a certain optical density. Then the cells are spun down whereafter the <i>C. violaceum</i> CV026 is added. After incubating them for several hours, the <i>C. violaceum</i> CV026 and violacein are spun down and the supernatant is removed. The pellet is resuspended in dimethyl sulfoxide whereafter the cells are spun down. Because the violacein prefers to solve in dimethyl sulfoxide, the supernatants was used to measure the absorbance at 585 nm.</p> | <p>To begin quantifying the samples, the samples are grown until a certain optical density. Then the cells are spun down whereafter the <i>C. violaceum</i> CV026 is added. After incubating them for several hours, the <i>C. violaceum</i> CV026 and violacein are spun down and the supernatant is removed. The pellet is resuspended in dimethyl sulfoxide whereafter the cells are spun down. Because the violacein prefers to solve in dimethyl sulfoxide, the supernatants was used to measure the absorbance at 585 nm.</p> | ||
<p><b>Protocol</b></p> | <p><b>Protocol</b></p> |
Revision as of 17:34, 18 September 2015
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