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.
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.
|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|
|NE3006||Superfolder GFP reporter||pGFP-F||pGFP-F|
|NE3007||Superfolder GFP reporter||pGFP-R||pGFP-R|