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MB Sample ID: SA247743
| Local Sample ID: | None_Azathioprine_R1 |
| Subject ID: | SU002563 |
| Subject Type: | Bacteria |
| Subject Species: | Veillonella parvula |
| Taxonomy ID: | 29466 |
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Collection:
| Collection ID: | CO002556 |
| Collection Summary: | All strain experiments were done in the Xavier lab. Strains and growth conditions: we utilized the following strains of Veillonella for growth and metabolic analysis; Veillonella parvula SKV38 [DR071], Veillonella parvula SKV38 xdh::cat* [DR214], Veillonella parvula SKV38 pucD::cat* [DR213]. All strains were first streaked on an agar plate with SK media (composition: yeast extract 10 gL-1, casitone 10 gL-1, NaCl 2 gL-1, K2HPO4 0.4 gL-1) supplemented with 50 mM lactate and 40 mM KNO3 (SKLN medium) and antibiotics if required. From this agar plate, a single colony was selected and inoculated in 5 mL SKLN media and grown for 24 hours. Next, overnight cells from this inoculum were grown using a 1/50 inoculum of the overnight culture, on either SK, SK + 50 mM lactate (SKL), SK + 40 mM nitrate (SKN), and SKLN. Supernatants were collected at the mid-exponential phase (OD600~0.4) and collected for metabolomic analyses (HILIC-pos metabolite profiling method described above). For analysis of the metabolism of the IBD drugs, cells were prepared as described above, and mid-exponential phase cells were collected, washed twice with sterile SK, and resuspended in 100 µl of SK. A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Additionally, sterile SK was prepared in an identical fashion to serve as a control for the experiment. Cells were incubated in a plate reader (4 wells/treatment) with low shaking and 37ºC for about ~5 h, and when the OD600 reached 0.4 cells were collected. For collection, 700 µl of the suspensions were centrifuged at 21,000 g for 2 min, and 400 µl of the supernatant harvested and stored at -20 ºC for analysis. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day. |
| Sample Type: | Culture Media |
