The overall goal of the following experiment is to use dietary means to alter the fatty acid composition of sea elegance in order to study fatty acid function and regulation. This is achieved by culturing the nematode food source in the presence of unsaturated fatty acids. Next sea elegance are fed the supplemented bacteria allowing for uptake of the fatty acid and its incorporation into the worm.
Lipids then changes in sea elegance. Fatty acid composition are confirmed by gas chromatography mass spectrometry analysis. Finally, after feeding on unsaturated fatty acids, the worms are scored for sterility.
Results are obtained that show the physiological effects of altered fatty acid composition through development behavior, and changes in gene expression. This method can help answer key questions in the sea elegance development and physiology fields because we can use both genetic manipulation and dietary contributions to alter fatty acid composition and monitor the consequences. Generally, individuals new to this method must be careful and cautious because it is difficult to obtain consistent results due to the sensitivity of unsaturated fatty acids to oxygen and high temperatures.
To prepare one liter of fatty acid supplemented medium, measure out the following components into a flask. Add millipore water up to 800 milliliters and autoclave the medium along with an empty glass bottle for each fatty acid concentration to be tested as well as appropriate graduated cylinders while the medium is cooling at 55 degrees Celsius. Prepare the working stock solution of the fatty acid sodium salt by carefully breaking open the glass vial so as not to get glass particles into the powder and weighing out enough to make a 100 millimolar stock solution add the necessary amount of purified water.
Next, using argon or nitrogen gas, purge the solution to prevent fatty acid oxidation. Then cap the vial and store the vial in the dark while the fatty acid, sodium salt is dissolving. When the agri has cooled to 55 degrees Celsius at the following, then near a flame, transferred the solution into a sterile graduated cylinder.
Before adding sterile water up to one liter, transfer the medium back to the flask and continue to mix. Use a sterile graduated cylinder to transfer an aliquot of medium into one autoclaved bottle for each fatty acid concentration to be tested. Then keeping the bottles warm on a stir plate.
Stir in the appropriate concentration of fatty acid stock solution before using a sterile 25 milliliter pipette to pour the plates after the plates have solidified cover and store it room temperature in a well-vetted area in the dark. Two days later, use e coli OP 50 to seed the plates, then incubate the plates in a dark environment at room temperature while the bacterial lawn is drying. Prepare a synchronized population of L one larvae by treating Ravi Hermaphrodites with alkaline hypochlorite solution.
Gently rock the GR hermaphrodites in this solution until the adult worms dissolve. Then centrifuge at low speed to pellet the eggs. Use M nine buffer to wash the eggs three times.
Then in a 15 milliliter tube, resuspend them in five milliliters of M nine buffer and rock overnight. Add the L one larvae to OP 50 seeded plates that have been supplemented with 0.3 millimolar di homo gammalin acid or DGLA and incubate them at 20 degrees Celsius for three days or until the larvae reach the adult stage. Use a dissecting microscope to score adult worms for sterility.
Successful germ loss will appear as a clear uterus devoid of eggs. Alternatively, score worms by picking them into a drop of M nine buffer on a watch glass. Then use one milliliter of 0.2 nanograms per milliliter dappy solution to flood the watch glass and incubate for about five minutes.
After the incubation, pick the worms into a drop of mounting medium on a slide before covering with a cover slip, seal the cover slip and store at four degrees Celsius in the dark overnight or view immediately under a fluorescent microscope to produce fatty acid methyl esters or fames. To quantify fatty acid uptake, collect 500 to 1000 adult worms by using water to wash them off of the plates and transfer them to siliconized. 13 by 100 millimeter glass screw top tubes.
Let worms settle by gravity and then use a glass past pipette to remove as much water as possible. Add one milliliter of 2.5%sulfuric acid in methanol and heat the tubes in a water bath at 70 degrees Celsius for one hour after cooling for one minute under the hood, extract the frames by adding 1.5 milliliters of water and 0.25 milliliters of hexane. Recap the tubes and shake vigorously.
Then centrifuge the tubes in a tabletop clinical centrifuge for one minute to separate the hexane from the aqueous solvent. Transfer the top hexane layer to a gas chromatography or GC vial insert within a GC vial. To analyze by gc, inject one to two microliters of fames onto a polar capillary gas chromatography column suitable for fame analysis.
Set the injector at 250 degrees Celsius with a flow rate of 1.4 milliliters per minute. Program the oven for an initial temperature of 130 degrees Celsius held for one minute. Then ramp the temperature 10 degrees Celsius per minute until it reaches 190 degrees Celsius, and then ramp it again at five degrees Celsius per minute up to 210 degrees Celsius, and then hold for an additional minute to ensure that uptake of DGLA has occurred.
Analyze frames by flame ionization detection or mass spectrometry or MS detection using authentic standards for the identification of the sea elegance. Fatty acids supplementation of the sea elegance diet is limited by the ability of the bacterial food source to uptake and incorporate fatty acids into the bacterial membrane to determine the ability of e coli OP 50 to assimilate various fatty acids into its membranes. OP 50 was plated onto media with no supplement, 0.1 millimolar and 0.3 millimolar concentrations of steric acid, sodium ate, and sodium DGLA as seen in this figure Fame analysis by GCMS demonstrates that unsaturated fatty acids, including OLEATE and DGLA incorporate into OP 50 at higher levels than the saturated fatty acid steric acid.
In addition, measuring the amounts of fatty acids in adult stage worms reveals that supplementation with saturated fatty acids has no effect on the relative amount of saturated fatty acids in worm tissues. However, supplementation with unsaturated fatty acids increased the relative amounts of these fats taken together. These data indicate that the relative accumulation of supplemented fatty acids in see elegance correlates directly with the relative accumulation of fatty acids in the dietary e coli.
As we previously demonstrated, this graph illustrates the dose response of DGLA induction of sterility in sea elegance. The concentration of DGLA in worm lipids in which 50%of the population will be sterile is approximately 12%And interestingly, the response to DGLA can be altered by genetic mutations in the worms. Based on a recent finding that the insulin growth factor dependent stress pathways can influence DGLA induced germ cell destruction.
We supplemented the diet of worms containing deleterious mutations in either the DAF two insulin IGF receptor or the DAF 16 FOXO transcription factor With DGLA, A scoring of sterility in each strain showed that DAF two mutants were fertile with little to no induced germ cell loss compared to wild type worms at both DGLA concentrations. In contrast, DGLA supplemented worms with inactive vox O displayed a higher percentage of sterile worms compared to wild type when fed on plates containing 0.15 millimolar of the fatty acid. Following this procedure, researchers can perform numerous assays with the supplemented sea elegance, such as gene expression studies, behavioral assays, or developmental or reproductive analyses.
These assays will reveal roles for specific fatty acids in these processes. After watching this video, you should have a good understanding of how to consistently prepare fatty acid supplemented media that can be used to alter the fatty acid composition of sea elegance.
