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

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. DOI:10.1016/0378-1119(85)90038-1 |

   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 |

   Notes