pSAM_Bt
(Plasmid #112497)

• Purpose: Transposon mutagenesis vector for Bacteroides thetaiotaomicron. Compatible with Insertion Sequencing (INSeq).
• Depositing Labs: Andrew Goodman, Jeffrey Gordon
• Publication: Goodman et al Cell Host Microbe. 2009 Sep 17;6(3):279-89. doi: 10.1016/j.chom.2009.08.003.

Backbone

• Vector backbone: pKNOCK_Cm
• Backbone manufacturer: Alexeyev, M.F. (PMID 10337469)
• Backbone size w/o insert (bp): 1860
• Total vector size (bp): 4582
• Modifications to backbone: Construction of mutagenesis vector pSAM (Sequencing-Adapted Mariner). The E. coli ß-lactamase gene was amplified by PCR from pUC19 (New England Biolabs) using primers MluI Ap 5’ and MluI Ap 3’ (see PMID 19748469, Table S19 for a list of all primers used in this study), and subcloned as an MluI fragment into pKNOCK_Cm (Alexeyev, 1999). Platinum Pfx DNA polymerase (Invitrogen) was used for PCR. The mariner transposon was constructed by PCR amplification of the ermG fragment from pGERM (Salyers et al., 2000) using primers ErmGm 5’ and ErmGm 3’, and introduced into pKNOCK_Ap as a KpnI/NotI fragment to create pMar1m. An alternate construct pMar1, lacking MmeI sites in the transposon inverted repeats, was constructed by PCR amplification of the ermG fragment from pGERM with primers ErmG 5’ and ErmG 3’. The gene encoding Himar1C9 transposase (Lampe et al., 1999) was cloned from pBADC9 (kindly provided by J. Mougous, University of Washington), using primers NdeI Himar1C9 5’ and NotI Himar1C9 3’, as an NdeI/Not fragment to create pSAM. The 300bp region upstream of the Bacteroides thetaiotaomicron VPI-5482 rpoD gene (BT1311) was PCR-amplified using primers Bt1311 prom 5’ and Bt1311 prom 3’ and mobilized as an NdeI/BamHI fragment to create pMar3m_Bt1311. To introduce the Illumina P7 sequencing adapters into the transposon, the ermG fragment of pMar3m_Bt1311 was PCR-amplified with P7 5’ and P7 3’ and re-cloned into the vector backbone. An E. coli rrnB T1/T2 transcriptional terminator cassette was amplified from pFW11 (Whipple, 1998) (kindly provided by Simon Dove, Harvard Medical School) using primers XbaI Term 5’ and PstI Term 3’ and cloned as an XbaI/PstI fragment to create pSAM_Bt. Intermediate and final clones were verified by bi-directional sequencing.

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): S-17 λ pir
• Copy number: Unknown

Gene/Insert 1

• Gene/Insert name: INSeq-adapted mariner transposon
• Insert Size (bp): 1344

Gene/Insert 2

• Gene/Insert name: Himar1C9
• Insert Size (bp): 1047

Resource Information

• Supplemental Documents:
  • pSAM_Bt.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry