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

= Notes on Podhajska-Gene-40-1985 = cited-by: Ham-BiotechBioengineering-94-2006, Ham-BiotechBioengineering-94-2006


 * 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) Julius B. Lucks/Bibliography