Functional Genomics & RNAi

T7 RNA Polymerase


TargeTron Vector pAR1219 (T2076) is a pBR322-based vector that expresses T7 RNA Polymerase under control of the IPTG inducible lac UV51 promoter and is intended for use with the TargeTron Gene Knockout System (TA0100). Many TargeTron System plasmids use the T7 promoter for intron expression. By co-transforming plasmid pAR1219 with the TargeTron pACD4 plasmid, the T7 promoter can be used to express the intron and to disrupt the chromosomal genes in alternative hosts such as Salmonella typhimurium and Shigella flexneri.2 Additionally, chromosomal gene disruptions in non-DE3 strains of E. coli can also be performed using pAR1219 with the pACD4 intron expression plasmids.




TargeTron Vector

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References

  1. Davanloo,P. et al., Proc. Natl. Acad. Sci. USA, 81, 2035-2039 (1984)
  2. Karberg, M. et al., J. Biol. Chem., 273 (36), 22884-22891 (1998)

 

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Disruption of lacZ and tonA in E. coli

Data illustrating the disruption of lacZ and tonA in E. coli HB101 using TargeTron® Vector pACD4K-C (included in the TargeTron Gene Knockout System - TA0100) and the T7 helper plasmid pAR1219 (T2076).

Figure 1. Mutation of E. coli HB101 using TargeTron vector pACD4K-C and T7 helper plasmid pAR1219. Targetron vector pACD4K-C was re-targeted to disrupt the lacZ and tonA genes. Mutants were selected on kanamycin plates. Intron isnertions were detected by colony PCR using an intron primer (3p-exon-1, 5'-TACGCAGCGGTATTTTTCGATCAG) and gene specific primers for lacZ and tonA. All colonies screened showed the expected insertion.


Figure 2. White phenotype of HB101 lacZ disruptants created using the pACD4K-C TargeTron plasmid with T7 helper plasmid pAR1219.



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