TITLE: Extraction of flagellum basal body complexes from Buchnera aphidicola, an endosymbiont of aphids
AUTHORS: mschepers 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-style:italic;">Buchnera aphidicola</span><span> is an intracellular bacterial symbiont of aphids and maintains a small genome of only 600 kbps. </span><span style = "font-style:italic;">Buchnera</span><span> is thought to maintain only genes relevant to the symbiosis with its aphid host. Curiously, the </span><span style = "font-style:italic;">Buchnera</span><span> genome contains gene clusters coding for flagellum basal body structural proteins and for flagellum type III export machinery. These structures have been shown to be highly expressed and present in large numbers on </span><span style = "font-style:italic;">Buchnera</span><span> cells. No recognizable pathogenicity factors or secreted proteins have been identified in the </span><span style = "font-style:italic;">Buchnera</span><span> genome, and the relevance of this protein complex to the symbiosis is unknown. Here, provide a procedure for isolating the </span><span style = "font-style:italic;">Buchnera </span><span>flagellum basal body proteins from the cellular membrane of </span><span style = "font-style:italic;">Buchnera</span><span>. This will facilitate studies of the structure and function of the </span><span style = "font-style:italic;">Buchnera</span><span> flagellum, and its role in this model symbiosis.</span></div><style>
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[STEPS]
?. [Liberating Buchnera from Aphids]
Grow aphids from birth to fourth instar (10 days) and harvest from plants. For this protocol, we used 3-5g live aphids.
?. [Liberating Buchnera from Aphids]
Load aphids into sterile fine mesh tea infuser or other sterile straining device.
?. [Liberating Buchnera from Aphids]
Submerge into 0.5% NaClO solution for 1 min.
?. [Liberating Buchnera from Aphids]
Transfer and submerge into sterile MQ H2O at 4oC for 1 minute.
?. [Liberating Buchnera from Aphids]
Repeat step 4 (Transfer and submerge into sterile MQ H2O for 1 minute).
?. [Liberating Buchnera from Aphids]
Remove aphids from strainer, placing in a mortar kept at 4oC. Add 20mL buffer A + EDTA (pH 7.5) and homogenize aphids w/ pestle.
?. [Liberating Buchnera from Aphids]
Pour homogenized aphids into sterile vacuum filter equipped with a 100uM nylon filter. Collect homogenate on ice.
?. [Liberating Buchnera from Aphids]
Remove nylon filter and place aphid filter cake back into mortar. Repeat homogenization with an additional 20mL buffer A.
?. [Liberating Buchnera from Aphids]
Repeat filtration with new 100mM nylon filter, combining filtrate.
?. [Liberating Buchnera from Aphids]
Centrifuge 100uM filtrate for 10 min at 1500g at 4oC in a pre-cooled centrifuge.
?. [Liberating Buchnera from Aphids]
Discard supernatant. Gently resuspend pellet in 40mL cold buffer A with sterile spatula or wide-bore pipette tip. Do not vortex.
?. [Liberating Buchnera from Aphids]
Vacuum filter resuspension through a 20uM nylon filter. Collect filtrate on ice.
?. [Liberating Buchnera from Aphids]
Immediately continue filtration through a 11uM nylon filter, collecting filtrate on ice.
?. [Liberating Buchnera from Aphids]
Filter through a 5uM nylon filter, collecting filtrate on ice.
?. [Liberating Buchnera from Aphids]
Centrifuge 5 uM filtrate for 30 minutes at 1500g at 4C in a pre-cooled centrifuge.
?. [Liberating Buchnera from Aphids]
Discard supernatant. Gently resuspend pellet in 100 mL sterile sucrose solution. Do not vortex.
?. [Extracting Flagellum complexes from Buchnera]
Optional: Use light microscope to check integrity of Buchnera cells. Cells are spherical, averaging 3uM in size.
?. [Extracting Flagellum complexes from Buchnera]
Begin to gently stir on ice.
?. [Extracting Flagellum complexes from Buchnera]
Add 5mL lysozyme (2 mg/mL, freshly prepared in H2O at 4oC).
?. [Extracting Flagellum complexes from Buchnera]
Add 10mL 100mM EDTA solution pH 7.5 and incubate on ice gently spinning for 40 min.
?. [Extracting Flagellum complexes from Buchnera]
Add 10mL 10% Triton X-100 (in H2O) to lyse cells. Solution should turn from turbid to clear.
?. [Extracting Flagellum complexes from Buchnera]
Once solution is transparent, add 100mM MgSO4 and 1 mg/ml RNase-free DNase. Incubate at room temperature, gently stirring, allowing DNases to degrade cellular DNA. Solution should become less viscous after 30 min, this step can be extended to an overnight incubation at 4oC if DNases require more time.
?. [Extracting Flagellum complexes from Buchnera]
Raise pH of the solution to pH 10 with 1N NaOH.
?. [Extracting Flagellum complexes from Buchnera]
Spin solution at 5000g in a pre-cooled 4C centrifuge three times, saving the supernatant and decanting into a new tube each time.
?. [Extracting Flagellum complexes from Buchnera]
After three spins, transfer supernatant into a polyallomer centrifuge tube and spin at 30,000g for 1h at 4C in a pre-cooled centrifuge.
?. [Extracting Flagellum complexes from Buchnera]
Carefully decant or draw off supernatant and discarded. Cover pellet with TET (10mM Tris-HCl, 5mM EDTA, 0.1% Triton X-100, pH 8.0) and leave overnight at 4C to soften protein pellet. Buchnera protein is ready for downstream analyses.
?. [Required Reagents]
NaClO solution: 0.5%; 83.5 mL 6% NaCLO in 916.5 mL sterile H2OBuffer A: 25mM KCl, 35mM Tris base, 10mM MgCl2, 250mM anhydrous EDTA in 950 mL sterile H2O. Add pellets of KOH until anhydrous EDTA is dissolved/solution becomes clear. Adjust pH to 7.5. Autoclave, then add 50 mL filter-sterilized 2.5M sucrose.Sucrose solution: 0.5M sucrose, 100mM Tris-HCl. Raise pH to 8 and filter sterilize.Lysozyme solution: Prepare 2 mg/mL egg white lysozyme in sterile H2O.EDTA solution: 100mM anhydrous EDTA in sterile H2O. Add KOH until EDTA is dissolved/solution becomes clear. Adjust pH to 7.5.Triton X-100 solution: 10% w/v Triton X-100 detergent in sterile H2O.MgSO4 solution: 100mM MgSO4 in sterile H2O.RNase-free DNase: 1 mg/mL in sterile H2O.NaOH, 1N: Dissolve NaOH pellets in sterile H2O.TET Buffer: 10mM Tris-Hcl, 5mM EDTA, 0.1% w/v Triton X-100. Add KOH or NaOH to dissolve anhydrous EDTA, then adjust pH to 8.0.