Staphylococcus aureus subsp. aureus USA300_FPR3757

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The NTML Screening Array

The Nebraska Transposon Mutant Library (NTML) Screening Array for phenotype screens consists of 1,920 transposon (Tn) mutants of the NTML collection arrayed in five 384-well microtiter plates. It is intended to be a resource for performing high-throughput phenotypic screens to identify candidate genes for future research. Each well contains 150 µl of a single transposon mutant frozen in 25% glycerol. Unlike the collection at NARSA, these mutant strains have not had their Tn-insertion site re-sequenced to confirm the identity of each mutation. Secondary-site mutations may have occurred during the generation of the library; hence we recommend that array users confirm each mutant selected from a screen, and that users also transfer the mutation into a fresh background by bacteriophage-mediated transduction. Finally, to ensure the quality of this resource for the research community and to reduce the number of passages from user to user, we ask that this library not be reproduced and distributed to other investigators in any way (both in total or as single isolates), as these mutant strains can be obtained for free through NARSA by visiting this NTML website and placing your order. Please download the "NTML 384-well Array well identification" reference document for a listing of the mutant strains in each well of the NTML Screening Array.

A valuable genetic toolbox developed by the CSR at UNMC to enhance the utility of the NTML is now available from NARSA

Important information is provided below regarding a genetic toolbox to enhance the utility of the NTML. This system uses allelic exchange via homologous recombination to replace any of the bursa aurealis Tn insertions. All exchanges eliminate the erythromycin resistance and gfp genes of the NTML mutants. Fluorescent reporter genes come in "F" and "R" versions in order to be correctly chosen based on transposon insertion orientation. For details on this system, please see the Bose et al. 2013 publication that describes this system in detail. In the table below you will find a short description of the system with links to plasmid maps and plasmid sequence files.

This toolbox is available through NARSA and includes all the exchange plasmids listed below in S. aureus RN4220 as well as pJB38 (in DH5α), the temperature-sensitive allelic exchange plasmid that is the parent plasmid of the toolbox's plasmid constructs. pJB38 is useful for the chromosomal mutagenesis of any non-essential gene in S. aureus.

A protocol for using this system can be found here

Ordering information and plasmid map and DNA sequence files for the NTML genetic toolbox available from NARSA

NE number Replacement Plasmid map file (download link) Plasmid sequence file (download link)
NE3000 Toolbox containing all plasmids below All files All files
NE3001 Not for Tn exchange pJB38 pJB38
NE3002 Unmarked pTnT pTnT
NE3003 Spectinomycin resistance pSPC pSPC
NE3004 Kanamycin resistance pKAN pKAN
NE3005 Tetracycline resistance pTET pTET
NE3006 Superfolder GFP reporter pGFP-F pGFP-F
NE3007 Superfolder GFP reporter pGFP-R pGFP-R
NE3008 EYFP reporter pYFP-F pYFP-F
NE3009 EYFP reporter pYFP-R pYFP-R
NE3010 ECFP reporter pBFP-F pBFP-F
NE3011 ECFP reporter pBFP-R pBFP-R
NE3012 DsRed.T3(DNT) reporter pRFP-F pRFP-F
NE3013 DsRed.T3(DNT) reporter pFRP-R pFRP-R
NE3014 eqFP650 reporter pFP650-F pFP650-F
NE3015 eqFP650 reporter pFP650-R pFP650-R

To order any of these tools please contact:

NARSA Contract Administrator
Phone: +1 703-480-2532
E-mail: carolopiela [at] eurofins [dot] com