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MB Sample ID: SA078040
| Local Sample ID: | BOSPT_1 |
| Subject ID: | SU001186 |
| Subject Type: | Bacteria |
| Subject Species: | Bacteroides thetaiotaomicron VPI-5482;Bacteroides ovatus ATCC 8483 |
| Taxonomy ID: | 226186;411476 |
| Genotype Strain: | WT (VPI-5482), SPT K.O. (BT_0870), (ATCC_8483), SPT K.O. (BACOVA_02588) |
| Age Or Age Range: | 411476 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Wild type Bacteroides strains and serine palmitoyltransferase (SPT) knockouts |
| Subject Comments: | Grown in BHI Media (Bacteroides tethaiotamicron, Bateroides ovatus : VPI-5482/ATCC_8483) |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001186 |
| Subject Type: | Bacteria |
| Subject Species: | Bacteroides thetaiotaomicron VPI-5482;Bacteroides ovatus ATCC 8483 |
| Taxonomy ID: | 226186;411476 |
| Genotype Strain: | WT (VPI-5482), SPT K.O. (BT_0870), (ATCC_8483), SPT K.O. (BACOVA_02588) |
| Age Or Age Range: | 411476 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Wild type Bacteroides strains and serine palmitoyltransferase (SPT) knockouts |
| Subject Comments: | Grown in BHI Media (Bacteroides tethaiotamicron, Bateroides ovatus : VPI-5482/ATCC_8483) |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| BOSPT_1 | SA078040 | FL011880 | Bacteroides ovatus | Species |
| BOSPT_1 | SA078040 | FL011880 | SPT K.O. (BACOVA_02588) | Strain |
Collection:
| Collection ID: | CO001180 |
| Collection Summary: | WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus were grown in 5 mL of BHI liquid media supplemented with vitamin K and hemin. After 24 hrs, cultures were normalized by OD600 and centrifuged at 8,000 rpm for 10 min to pellet cells. Resulting pellets were washed and further centrifuged twice to remove residual media. Resulting pellets were resuspended in 5 mL isopropanol + 0.1 ng/μL C24:0 PC, incubated at RT for 1 hr, and centrifuged for 10 min at 10,000g. From each sample, 2 μL was injected. |
| Sample Type: | Bacterial cells |
| Storage Conditions: | -80℃ |
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. |
Sample Preparation:
| Sampleprep ID: | SP001194 |
| Sampleprep Summary: | Bacterial cell pellets from 5ml overnight BHI cultures were resuspended in 5 mL isopropanol + 0.1 ng/μL C24:0 PC, incubated at RT for 1 hr, and centrifuged for 10 min at 10,000g. |
Combined analysis:
| Analysis ID | AN001850 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Shimadzu Nexera X2 |
| Column | Waters Acquity BEH C8 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Focus |
| Ion Mode | POSITIVE |
| Units | abundance |
Chromatography:
| Chromatography ID: | CH001338 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters Acquity BEH C8 (100 x 2.1mm,1.7um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001711 |
| Analysis ID: | AN001850 |
| Instrument Name: | Thermo Q Exactive Focus |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.1 (Thermo Fisher) and Progenesis QI (Nonlinear dynamics). Retention time, m/z, MS/MS fragmentation were used to confirm the identity of metabolite using authentic reference standards when available (level 1 identifications), in addition to monitoring the product ions produced and the retention time and mass of species belonging to the each lipid class (level 2-3 annotation). |
| Ion Mode: | POSITIVE |
