Summary of Study ST001125
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000754. The data can be accessed directly via it's Project DOI: 10.21228/M8709G This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001125 |
| Study Title | WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus grown BHI liquid media (part I) |
| Study Summary | Lipid profiling was applied to WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus grown BHI liquid media revealing a greater variety of Bacteroides-derived sphingolipids than previously recognized, including ceramide phosphoinositol and deoxy-sphingolipids. |
| Institute | Broad Institute of MIT and Harvard |
| Department | Gastrointestinal Unit, Center for the Study of Inflammatory Bowel Disease, University Medical Center Groningen |
| Last Name | Avila-Pacheco |
| First Name | Julian |
| Address | 415 Main Street |
| jravilap@broadinstitute.org | |
| Phone | 617-714-8264 |
| Submit Date | 2019-01-15 |
| Num Groups | 4 |
| Total Subjects | 8 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2019-03-06 |
| Release Version | 1 |
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Treatment:
| Treatment ID: | TR001201 |
| Treatment Summary: | In-frame deletion of the serine palmitoyl transferase gene (spt) in B. thetaiotaomicron Δtdk was generated using a counter-selectable allelic exchange procedure (Koropatkin et al., 2008). Briefly, a 2 kb fragment concatenating the 700 bp upstream sequence, the first 291bp of the SPT gene (BT0870) followed by a stop, and the 1 kb downstream sequences of SPT was synthesized by IDT. The in-frame deletion in spt encodes only the first 97 amino acids of the B. thetaiotaomicron Spt protein. This 2 kb fragment was amplified using primer pairs BT0870_Up700b_BamHI _F and BT0870_Down1kb_NotI_R and ligated into the suicide vector pExchange_tdk (obtained from Dr. Harry Brumer, UBC). The resulting pExchange_tdk_BT0870_DEL vector was electroporated into E. coli S17-1 λ pir and then conjugated into B. thetaiotaomicron VPI-5482. Single recombinants were selected on BHI agar plates containing gentamicin and erythromycin, cultured in liquid BHI medium overnight without antibiotics and then plated onto BHI agar plates containing 200 μg/mL 5-fluoro-2-deoxyuridine (FUdR). Single colonies of SPT deletion candidates were confirmed by PCR using the diagnostic primers BT0870_Up1kb_F and BT0870_Down1150b_R. The B. ovatus spt deletion mutant was generated in a similar fashion. Briefly, ~900 bp fragments upstream and downstream of the B. ovatus SPT (BACOVA_02588) were cloned and fused using primer pairs ΔSPT Xba1-UPF (5’AGTCACGACGTTGTAAAACGACGGCCAGT-3’), BamH1 UPR-(5’- GGCGTAATCATGGTCATAGCTGTTTCCTG-3’), EcoR1-DNF (5’- GTTGTAAAACGACGGCCAGT-3’) and HindIII-DNR (5’-GGCGTAATCATGGTCATAGC-3’), respectively, and ligated into pExchange_tdk. The resulting pExchange_tdk_BACOVA_02588_DEL vector was electroporated into E. coli S17-1 λ pir and then conjugated into B. ovatus ATCC 8483. Single recombinants were selected on BHI agar plates containing gentamicin and erythromycin, cultured in liquid BHI plus medium [BHI lemented with 5% heat inactivated fetal bovine serum, 1% vitamin 500 K1-hemin solution (Becton Dickinson), 1% trace mineral supplement (ATCC), 1% vitamin supplement (ATCC), 2.9 mM (+)-cellobiose (Becton Dickinson), 2.9 mM maltose (Hardy Diagnostics), 5.8 mM D-(−)- Fructose (Sigma) and 2.8 mM L-Cysteine hydrochloride monohydrate (Sigma)] overnight without antibiotics, and then plated onto BHI plus agar plates containing 200 μg/mL FUdR. Single colonies of SPT deletion candidates were screened and confirmed by PCR using the diagnostic primers BACOVA_02588 _Up1kb_F and BACOVA_02588 _Down1kb_R. |
| Cell Storage: | -80C |
| Cell Growth Container: | anaerobic chamber (Coy Laboratory Products) with an atmosphere of 20% CO2, 5% H2, and 75% N2 at 37°C. |
| Cell Media: | Brain Heart Infusion (BHI, Becton Dickinson) medium supplemented with 1% Vitamin K1-Hemin Solution (VitK/Hemin, Becton Dickinson), or on BHI agar (Becton Dickinson) supplemented with 1% VitK/Hemin. |
