The overall goal of the following experiment is to detail an efficient methodology employed to phylo Genetically characterize the metabolically active bacteria in the gut of an insect. This is achieved by first feeding C 13 labeled glucose to the insect to isotopically enrich the DNA of the metabolically active bacteria in their gut. The next step is to extract the DNA of the bacterial community present in the gut.
The third step is to separate out the DNA of the metabolically active species, which incorporated the C 13. The DNA is then phylogenetically identified and quantified for the species present by pyro sequencing. Thus identifying the metabolically active bacterial species comprising the community present in the insect gut.
So the gut of multicellular organism is an ingenious invention to exploit the metabolic capacities of microorganisms to dissolve or to solubilize complex organic matter so that it can finally serve as nutrients for the insect host. To understand the system, we have to analyze first who is out there, and second who has which of metabolic activities. If we have these data, we can reconstruct the flux of metabolites and degradation products between the degrading microorganisms and the insect host.
This method can help us to answer key questions in the microbial ecology field, particularly in the manner that these microbes are interacting with their host. In that sense, we could know more and clarify and define what are the functions these organisms are serving for their host. Though this method can provide insight into the general active gut bacteria.
By tracking the ucle glucose, it can be also performed to other carbon source, such as labeling the cellose to identify active bacteria involved in the digestion process of the ocrant nutrient. Maintain op liters, eggs and Petri dishes add room temperature until they hatch. Then transfer the newly hatched larvae to a clean plastic box and rear them on an artificial diet at 23 to 25 degrees Celsius under a long day regime with 16 hours of illumination and eight hours of darkness after a few days transfer nine to 15 healthy second instar larvae from the rearing box to individual Petri dishes containing fresh C 13 glucose.
Spiked artificial diet, at least three replicates of three larvae each are needed per treatment. Use an artificial diet providing the same amount of native glucose as a natural food source like cotton for feeding the control group. Over the next 24 hours.
Frequently renew the artificial diets. Begin by transferring the larvae with soft forceps to a pool of distilled water to remove large debris. Then transfer to a new clean dish to anesthetize and kill the insects.
Place them on ice and then drop the temperature to negative five degrees Celsius in a cooler. Then disinfect the dead insects with 70%ethanol and immerse them in PBS for the dissection. The tools must also be cleaned with 70%ethanol.
Begin with incisions in the cuticle along the right and left sides. Commence on the head and finish on the anus. Remove the entire exoskeleton, the malian tubules, and the fat body.
Then replace the buffer before opening the gut with the gut isolated from the other tissues. Process the whole gut or sections for gut midgut or hind gut. Depending on the scientific question.
Transfer the gut tissues without PBS to a small sterile tube and store them in the freezer. Prior to the DNA extraction, dry the samples at 45 degrees Celsius in a vacuum concentrator device for 30 to 90 minutes, depending on the size of the tissue. Next, crush the dried tissue using a sterile plastic Pele to extract the genomic DNA using a kit for soil DNA extraction.
Then determine the concentration of the final eluded DNA using a spectrophotometer. Verify a successful extraction of the microbial metagenomic DNA from the insect gut by preparing a diagnostic PCR assay using bacterial general primers 27 F and 1, 492 R.For diagnosis. Run five microliters of each PCR product on a 1%agros gel at 300 volts and 115 milliamps for about 20 minutes.
The correct size of the amplicons is 1.5 kilobases. Pull the extracted DNA corresponding to each experimental replicate together. Keeping those with and without C 13 glucose separate.
Normalize the DNA concentrations to ensure each sample contributes equally to its pool. A final concentration of 500 to 5, 000 nanograms of DNA is suitable for the ultracentrifugation process. Double check the anticipated concentration with the spectrophotometer in a sterile 15 milliliter screw cap tube.
Set up the gradient medium. Combine the quantified DNA with 4.8 milliliters of cesium chloride solution. Then top off the volume with six milliliters of gradient buffer.
Carefully transfer the mixtures into 5.1 milliliter ultracentrifuge tubes. Using a syringe and needle fill the tubes to the base of their neck and avoid pumping or forming any air bubbles. Include at least one blank gradient with no DNA in each run.
To act as a reference gradient, select pairs of tubes and balance them to within 10 milligrams. Apply a tube topper to each tube and ensure that they are leak free by inverting the tube and applying moderate pressure by hand. Use a near vertical rotor with eight wells.
Carefully seal the rotor wells and load the rotor into the ultracentrifuge according to the manufacturer's instructions. Spin the DNA down at 50, 000 RPM at 20 degrees Celsius under a vacuum for 40 hours. Select maximum acceleration and the deceleration without braking.
Two days later, carefully remove the tubes from the centrifuge rotor using forceps. Place them in a rack without disturbing the formed gradients and process the tubes immediately. First equilibrate an HPLC using a flow rate of 850 microliters per minute.
Then attach the ultracentrifuge tube to a clamp stand and pierce the bottom of the tube with a 23 gauge one inch needle attached to a syringe. So carefully pierce the top of the tube with a control miner to prevent disturbing the form, the gradient, and the practice beforehand to ensure a successful PSA motion. Next, insert the needle attached to the pump tubing into the top of the tube where there is no fluid and collect drops from the bottom directly into sterile 1.5 milliliter Micro fuge tubes collect a fraction every 30 seconds for 425 microliters per fraction.
In this example, 12 fractions are collected from each gradient. Next, measure the density of each separated fraction on an analytical balance. Proceed with DNA recovery and pyro sequencing by consulting the text protocol for 24 hours.
10 millimolar C 13 glucose was supplemented in the diet of ssop liters.Larva. The same amount of normal glucose was added to the food of the control insects. Next DNA was extracted from the bacterial community of the insect gut.
On an electrophoresis gel, a strong band of genomic DNA at the top confirmed a successful DNA extraction, a diagnostic PCR. Using bacterial universal primers showed the presence of bacterial DNA quantitative pyro sequencing was performed directly on the representative SIP gradients to reveal the species lineage and their relative abundance. A total of 120, 045 high quality reads were generated with an average length of 404 OTs.
The pyro sequencing profile showed an increased abundance of certain species, including enterococcus and panto in the labeled sample compared to that in the control group. Therefore, those bacteria are considered to be metabolically active and merit further study. So while attempting this procedure, it is important to remember to involve a proper control in the CYP analysis.
So which would subtract the impact of the background, the contamination in the hair flash and ensure the bacteria diversity and abundance appearing or disappearing are not artifact of the mass itself. Following this procedure or the methodologies like fluorescence in situ hybridization could be used and perform in order to answer additional questions. In that case will be looking after the localization and arrangement of the bacteria inside the gut of the insect.
