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Functional Genomics & RNAi

Other Organisms FAQ


TargeTron® Gene Knockout System FAQs
TargeTron® Design Site FAQs


Adapting the TargeTron® Gene Knockout System to Other Organisms FAQs

  1. Will the pACD4-K-C vector included in the TargeTron Gene Knockout System work in other Gram-negative bacteria, such as Vibrio, without having to do any subcloning?
    The system has not yet been validated in Vibrio, but if your organism is normally compatible with E. coli plasmids (T7 promoter and origin of replication), then it is likely that the TargeTron system would work for Vibrio. To use the system in Vibrio or another organism that does not have a source of T7 RNA polymerase, the plasmid would need to be co-transfected with TargeTron Vector pAR1219 (T2076), a TargeTron plasmid which encodes an IPTG-inducible T7 RNA polymerase. Similar experiments were done by Michael Karberg in Shigella and Salmonella (Karberg et. al. 2001. Group II Introns as Controllable Gene Targeting Vectors for Genetic Manipulation of Bacteria. Nature Biotechnology. 19, 1162-1167 1.52 Mb pdf).

  2. What is the best way to engineer a new promoter?
    Subcloning the 3 kb DNA encoded group II intron and the Intron Encoded Protein (IEP) into the host specific plasmid, generating an alternative promoter during a second round of PCR or co-transforming a plasmid encoding the T7 RNA polymerase (TargeTron Vector pAR1219, Product Code T2076) with the current TargeTron vector are all options for driving expression of the TargeTron system in different organisms.

    Subcloning:

    In TA0100 kit. Works with E. coli, Salmonella, and Shigella.

    Primary recommendation for sub-cloning intron components is a double digestion with HindIII - PshAI (works for different varieties, pACD4-C, pACD4K, pACD4K-C-loxP).


    Generating an alternate promoter by second round PCR:
    The PCR product from the initial PCR reaction can be diluted 100-fold and used as a template to insert an alternative promoter to express the intron. This will be required for non-DE3 strains. Alternative promoters may require induction by agents other than IPTG, or no induction at all. The E. coli spc constitutive promoter has been successfully used without induction. For the spc promoter, modify the existing protocol (included in the TargeTron Gene Knockout System User Guide) by taking the transformation from the 1 hr 37 °C incubation, diluting 100 µl into 3 ml of LB-chloramphenicol-glucose, and grow overnight at 30 °C. After overnight growth, spin down the cells and resuspend in 150 µl of LB. Plate the entire 150 µl onto a 25 µg/ml kanamycin plate. Using the lacZ control reaction as a comparison, the spc promoter has shown lower insertion efficiencies than the T7 promoter in E. coli, but has routinely produced insertional mutants. The protocol is included in the TargeTron Gene Knockout System User Guide.

    Co-transfecting with pAR1219 which provides a source of T7 RNA polymerase:

Non-DE3 cell (e.g. DH5α, GC5, HB101, Salmonella, Sigella, etc.)



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TargeTron® Gene Knockout System