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MB Sample ID: SA078041

Local Sample ID:BOSPT_2
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)

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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.
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