Difference between revisions of "Team:KU Leuven/Research/Methods"
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<div id="togglethreehalf" > | <div id="togglethreehalf" > | ||
<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 | + | <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 | + | <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>To begin quantifying the samples, the samples are grown until a certain optical density.Then the cells are | + | <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, we used the supernatans to measure the absorbance at 585 nm.</p> |
<p><b>Protocol</b></p> | <p><b>Protocol</b></p> | ||
<p><u>Make a standard curve</u><br><br> | <p><u>Make a standard curve</u><br><br> | ||
− | The goal is to measure the absorbance of violacein versus the concentration of | + | The goal is to measure the absorbance of violacein versus the concentration of OHHL around 0.04 mM.</p> |
<dl> | <dl> | ||
<dd>1. Make a OHHL stock solution of 10 mM.</dd> | <dd>1. Make a OHHL stock solution of 10 mM.</dd> | ||
− | <dd>2. Make a dilution | + | <dd>2. Make a dilution series of OHHL in LB medium. Take into account that this will further be diluted when adding the <i>C. violaceum</i> CV026. </dd> |
<dd>3. Add <i>C. violaceum</i> CV026 in this way that the volume of the cells is 10% of the end volume. | <dd>3. Add <i>C. violaceum</i> CV026 in this way that the volume of the cells is 10% of the end volume. | ||
</dd> | </dd> | ||
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<tr> | <tr> | ||
<th>End concentration </br>(in mM)</th> | <th>End concentration </br>(in mM)</th> | ||
− | <th>Volume of 10 mM OHHL stock </br>(in | + | <th>Volume of 10 mM OHHL stock </br>(in mL)</th> |
<th>Volume LB medium </br> (in mL)</th> | <th>Volume LB medium </br> (in mL)</th> | ||
− | <th>Volume of </br><i>C. violacein</i> CV026</th> | + | <th>Volume of </br><i>C. violacein</i> CV026 (in mL)</th> |
</tr> | </tr> | ||
<tr class="lightrow"> | <tr class="lightrow"> | ||
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<dl> | <dl> | ||
<dd>1. Incubate <i>Chromobacterium violaceum</i> CV026 for 16-18h.</dd> | <dd>1. Incubate <i>Chromobacterium violaceum</i> CV026 for 16-18h.</dd> | ||
− | <dd>2. Take 1 mL of the sample that you would like to quantify. Measure the | + | <dd>2. Take 1 mL of the sample that you would like to quantify. Measure the OD(600 nm) and centrifuge till the cells are down (max speed 15000 rpm, 10 min). If you want to include the amount of AHL inside the cell, you should lyse the cells in advance.</dd> |
− | <dd>3. Inoculate the <i>C. violaceum</i> CV026 to OD(600 nm) = 0.1 in air-lid culture tubes containing LB and LB supplemented with 1 mL | + | <dd>3. Inoculate the <i>C. violaceum</i> CV026 to OD(600 nm) = 0.1 in air-lid culture tubes containing LB and LB supplemented with 1 mL supernatans of your sample at 27°C (150 rev/min agitation) for 24 h in a shaking incubator. </dd> |
</dl> | </dl> | ||
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<p><u>Quantification</u><br><br></p> | <p><u>Quantification</u><br><br></p> | ||
<dl> | <dl> | ||
− | <dd>1. Centrifuge 1 ml culture (10 min at | + | <dd>1. Centrifuge 1 ml culture (10 min at 13000 rev/min) to precipitate the insoluble violacein.</dd> |
<dd>2. Discard supernatant (culture) and add 1 ml of dimethyl sulfoxide to the pellet.</dd> | <dd>2. Discard supernatant (culture) and add 1 ml of dimethyl sulfoxide to the pellet.</dd> | ||
<dd>3. Vortex the solution vigorously for 30 s to completely solubilize violacein and centrifuge at 13 000 rev/min for 10 min to remove the cells. </dd> | <dd>3. Vortex the solution vigorously for 30 s to completely solubilize violacein and centrifuge at 13 000 rev/min for 10 min to remove the cells. </dd> |
Revision as of 21:28, 17 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