| Catalog Number | Kit Size |
| D070101 | 10 reactions |
Genomic Integration Kit (CamR)
Efficient mutagenesis of all types of gram-negative bacterial strains
Applications
- Insert chloramphenicol-resistance marker gene into the genome of bacterial species
- Efficiently generate gene knock-outs in vivo
- Generate exhaustive single-insertion transposon mutant libraries
- Map the transposon insertion sites within genomic DNA using the primers included in the kit
Benefits
- Simple: directly electroporate Transposition complexes (CamR) into your favourite bacteria
- Efficient: generate thousands of mutants containing transposon integration from a single reaction
Genomic Integration Kit (CamR) for genomics applications
Genomic Integration Kit (CamR) has been designed for efficient mutagenesis of all types of gram-negative bacterial strains. Kit contains Mu DNA Transposition Complexes (CamR), which are stable complexes formed between hyperactive v.3 MuA Transposase (in vivo Integrator) and Cat-Mu Transposon (CamR). Cat-Mu Transposon (CamR) contains the marker gene cat, which confers resistance to chloramphenicol, and is designed for all types of gram-negative bacteria.
Genomic Integration Kit is easy to use, simply electroporate Mu DNA Transposition Complexes (CamR) into your bacterial strain of interest. To select the clones with transposon integration, grow bacterial clones on selective (chloramphenicol) media.
The transposon insertion clones can be screened using a phenotypic screen, genomic DNA sequencing or PCR. The kit contains Cat-Mu Transposon -specific forward and reverse primers, which can be used for determining the transposon integration sites, for example, by direct sequencing of microbial genomic DNA without cloning.
Electroporation of Mu DNA transposition complexes has been used successfully with many bacterial species, including gram-negatives, such as coli, Salmonella enterica, Erwinia carotovora, Yersinia enterocolitica, Erwinia carotovora, Pseudomonas aeruginosa (Lamberg et al. 2002, Li et al. 2007). The efficiency of genomic integration is influenced mostly by the competence status of a given strain.
Figure 1 (click to enlarge) Genome engineering with Genomic Integration Kit (CamR). Genomic Integration Kit contains Mu DNA Transposition Complexes (transpososomes). Transpososomes are stable and remain inactive in the absence of divalent ions. Upon encountering Mg2+ ions inside the host cell, the complex executes integration of transposon DNA into the genomic DNA. Transposon (Tn) contains an antibiotic resistance (AbR) selection marker. From the selected clones, sequencing can be carried out bi-directionally using primers specific for the transposon (included in the kit).
References
- Lamberg A, Nieminen S, Qiao M, Savilahti H (2002) Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage Mu. Appl Environ Microbiol 68:705-712
- Li, Y., Xia, H., Bai, F., Xu, H., Yang, L., Yao, H., Zhang, L., Zhang, X. et al. 2007. Identification of a new gene PA5017 involved in flagella-mediated motility, chemotaxis and biofilm formation in Pseudomonas aeruginosa. FEMS Microbiol Lett 272, 188–195.