Genomic Integration Kit (EmR) for Gram-positive bacteria
Efficient mutagenesis of Gram-positive bacterial strains
| Catalog Number | Product Name | Kit Size | Price | |
|---|---|---|---|---|
| D070301 | Bacterial Genomic Integration Kit (Gram-positive, EmR) | 10 reactions | 520 € (VAT 0%) | Buy Now |
Applications
- Insert erythromycin-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 (EmR) into your favourite bacteria
- Efficient: generate thousands of mutants containing transposon integration from a single reaction
Overview
Bacterial Genomic Integration Kit (Gram-positive, EmR) has been designed for efficient mutagenesis of all types of Gram-positive bacterial strains. Kit contains Mu DNA Transposition Complexes (EmR), which are stable complexes formed between hyperactive v.3 MuA Transposase (in vivo Integrator) and Em-Mu Transposon (EmR). Em-Mu Transposon (EmR) contains the marker gene ermB, which confers resistance to erythromycin, and is designed for Gram-positive bacteria.
Genomic Integration Kit is easy to use, simply electroporate Mu DNA Transposition Complexes (EmR) into your bacterial strain of interest. To select the clones with transposon integration, grow bacterial clones on selective (erythromycin) media.
The transposon insertion clones can be screened using a phenotypic screen, genomic DNA sequencing or PCR. The kit contains Em-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-positives, such as Staphylococcus aureus, Streptococcus pyogenes, and Clostridium perfringens (Pajunen et al. 2005, Lanckriet et al. 2009). 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. 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).
Documents
- Product info for Bacterial Genomic Integration Kit (Gram-positive, EmR)
- Em-Mu (EmR) Transposon sequence
References
- Pajunen MI, Pulliainen AT, Finne J, Savilahti H (2005) Generation of transposon insertion mutant libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes. Microbiology 151:1209-1218
- Lanckriet A, Timbermont L, Happonen LJ, Pajunen MI, Pasmans F, Haesebrouck F, Ducatelle R, Savilahti H, Van Immerseel F (2009) Generation of single-copy transposon insertions in Clostridium perfringens by electroporation of phage Mu DNA transposition complexes. Applied and Environmental Microbiology 75:2638–2642.