Julius B. Lucks/Bibliography/Podhajska-Gene-40-1985

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Notes on [1]

cited-by: Ham-BiotechBioengineering-94-2006, [2]

  • inducible gene expression system based on lambda Int/attP/attB
    • OFF phase facing away from gene and blocked by strong terminator
    • ON phase has promoter inverted
    • heat pulse used to switch from OFF to ON
  • use lambda_xis^-cIts875 prophage
    • transiently de-repress to induce Int
    • Int causes promoter inversion between inverted P'OP phage att site and normal Delta_P Delta_PO pseudo-bacterial att site
    • inverted promoter controls expression of desired gene (here galK), and tandemly cloned lambda N gene
    • N acts on NutL placed downstream of promoter to antiterminate any termination signals in the att site
  • entire promoter-inversion module can be put on any plasmid as 1.3 kb AvaI-ClaI fragment
  • lambda Int
    • using direct orientation of att, does genomeic insertions or excisions
    • using inverse orientation of att, does inversions
  • inversion seen in other systems
    • control of flagella in Salmonella
    • inverting G region of Mu
    • see (Simon and Hersxowitz - 1985)

Details

  • att site (POP')
    • 492 bp HindIII-BamHI fragment of lambda (Weisberg, Landy - 1983)
    • placed in inverted P'OP orientation
  • att Delta_PODelta_P'
    • 116bp truncated version of lambda attP that only has elements functionally in common with bacterial attBOB'
    • serves as role of attB site
  • since inverted attP, action of Int is inversion rather than excision
    • see (Mizuuchi - 1980, Pollock and Nash - 1983, Reyes - 1979)
  • Xis inactivated so doesn't catalyze excision of the inverted product (which is substrate for Int+Xis excision)
  • terminators in P'OP region are overcome when N is expressed
  • Fig 2 - looks like fully induced in about 2 hours after 10 min exposure to 42 C
    • almost no basal expression when not induced

Other ways to do this

  • orient attP and attB directly with a terminator in between and use Int to excise this region
    • see (Backman - 1984)

References

  1. Podhajska AJ, Hasan N, and Szybalski W. Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module. Gene. 1985;40(1):163-8. PubMed ID:3005124 | HubMed [Podhajska-Gene-40-1985]
    Notes
  2. Ham TS, Lee SK, Keasling JD, and Arkin AP. A tightly regulated inducible expression system utilizing the fim inversion recombination switch. Biotechnol Bioeng. 2006 May 5;94(1):1-4. DOI:10.1002/bit.20916 | PubMed ID:16534780 | HubMed [Ham-BiotechBioengineering-94-2006]
    Notes
All Medline abstracts: PubMed | HubMed