The 96x96 clone Mycobacterium bovis BCG Pasteur transposon insertion (Tn) library
In February 2018, a large transposon insertion mutant library of the Mycobacterium bovis BCG (Bacille Calmette Guerin) Pasteur 1721 vaccine strain (Master et al, 2008) has been transferred from VIB-UGent to the BCCM/ITM Mycobacteria Collection situated at the Institute of Tropical Medicine in Antwerp.
The transposon used to construct this >8000 clones library (96x96-wells) is derived from the mariner element Himar1 which is shown to efficiently transpose in bacteria (Rubin et al 1999).
Transposon mutagenesis followed by transposon deep sequencing has proven to be a useful tool in gene-essentiality studies in M. tuberculosis. For the construction of this unique Tn mutant library, the lab of Prof. Dr. Nico Callewaert (VIB Medical Biotechnology Center, UGent) optimized the Illumina library preparation protocol and also implemented an additional Cartesian pooling step. The combined approach of Cartesian Pooling – Coordinate Sequencing (CP-CSeq) reports both on the identity as well as on the physical location of sequence-tagged mutants in the well-plate clone collection.
By implementing the combined CP-CSeq method, an almost comprehensive set of genome-wide mutant strains is created in less than the time required to generate even a single mutant with classical techniques.
With M. bovis BCG being 99% genetically identical to M. tuberculosis, it is a highly suitable and safer alternative for genetic studies. Moreover, although generated almost a century ago, M. bovis BCG is still the only licensed vaccine against tuberculosis.
Although an injection with BCG offers good protection in infants, it is not very effective for adults. Therefore the need for a new and better vaccine against TB grows stronger every day.
The generation of this Tn mutant library can thus be of great value for the tuberculosis research community as these transposon mutants can be directly used for hypothesis testing and validation studies, both in vaccine research and in drug development programs.
One of the mutants from the collection with a disruption in the SapM locus (sapM::Tn) has already been shown to be a better vaccine candidate against M. tuberculosis than the parental M. bovis BCG strain in mice (Festjens et al, 2011). Nevertheless, further improvements through additional mutations will probably be required to progress into clinical trials.
A small glimpse into the 2x96x96 clone M. bovis BCG Danish Tn library
More recently, the lab of Prof. Dr. Nico Callewaert has transferred a second transposon insertion library, derived from the M. bovis BCG Danish 1331 strain (first WHO reference strain, NIBSC 07/270), to BCCM/ITM (Borgers et al., 2019). This new ‘Danish’ library is twice as big as the first ‘Pasteur’ library (2x96x96 wells, 15,320 traceable mutants) and targets 83% of all non-essential M. tb homologues. Moreover, it was made with a derivative of the optimized Himar1 transposon (incorporating a SacB gene and FRT- and ISceI-sites flanking the antibiotic resistance cassette) to afterwards allow the creation of unmarked transposon mutants (Borgers et al., 2020).
The single transposon mutants of both libraries are now available at BCCM/ITM and can be provided to any researcher upon request. To select the mutants you require, please follow this link1 towards the Excel file of the Pasteur library and/or this link² towards the Excel file of the Danish library. Both Excel files contain a list of every transposon-insertion mutant present in the libraries together with its assigned location.
In case you place an order, BCCM/ITM will send out the solid agar plates containing the grown colonies. A comprehensive protocol will be provided explaining how to obtain clonally pure mutants from hereon.
Remark: the clonal purification step is crucial, as many of the stocks in the library are not completely clonally pure; probably due to the in-bulk creation of the library and the significant clumping behavior of BCG.
Borgers, K, Ou, J, Zheng, P, Tiels, P, Van Hecke, A, Plets, E, Michielsen, G, Festjens, N, Callewaert, N & Lin, Y. (2019). Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine. BMC Genomics 20, 561. https://doi.org/10.1186/s12864-019-5909-5
Borgers, K., Vandewalle, K., Van Hecke, A., Michielsen, G., Plets, E., van Schie, L., ... & Callewaert, N. (2020). Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource. Msystems, 5(4).
Festjens, N, Bogaert, P, Batni, A, Houthuys, E, Plets, E, Vanderschaeghe, D, Laukens B, Asselbergh B, Parthoens E, De Rycke R, Willart MA, Jacques P, Elewaut D, Brouckaert P, Lambrecht BN, Huygen K, Callewaert N. (2011). Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis. EMBO molecular medicine, 3: 222-234.
Master SS, Rampini SK, Davis AS, Keller C, Ehlers S, Springer B, Timmins GS, Sander P, Deretic V. (2008). Mycobacterium tuberculosis prevents inflammasome activation. Cell Host Microbe 3:224–232.
Rubin, EJ, Akerley, BJ, Novik, VN, Lampe, DJ, Husson, RN, Mekalanos, JJ. (1999). In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proceedings of the National Academy of Sciences, 96: 1645-1650.
Vandewalle K, Festjens N, Plets E, Vuylsteke M, Saeys Y, Callewaert N. (2015). Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing. Nature Communications 11:1-7.
1Link Excel Pasteur:
List containing all transposon insertion events in the M. bovis BCG Pasteur mutant library.
² Link Excel Danish
List containing all transposon insertion events in the M. bovis BCG Danish mutant library.