UTILIZATION OF A MINI-DLAC TRANSPOSABLE ELEMENT TO CREATE AN ALPHA-COMPLEMENTATION AND REGULATED EXPRESSION SYSTEM FOR CLONING IN PSEUDOMONAS-AERUGINOSA

Citation
Rr. Karkhoffschweizer et Hp. Schweizer, UTILIZATION OF A MINI-DLAC TRANSPOSABLE ELEMENT TO CREATE AN ALPHA-COMPLEMENTATION AND REGULATED EXPRESSION SYSTEM FOR CLONING IN PSEUDOMONAS-AERUGINOSA, Gene, 140(1), 1994, pp. 7-15
Citations number
46
Categorie Soggetti
Genetics & Heredity
Journal title
GeneACNP
ISSN journal
03781119
Volume
140
Issue
1
Year of publication
1994
Pages
7 - 15
Database
ISI
SICI code
0378-1119(1994)140:1<7:UOAMTE>2.0.ZU;2-N
Abstract
A lac-based alpha-complementation and expression system was developed for use in molecular cloning in Pseudomonas aeruginosa. A bacteriophag e D3ll2-based mini-Dlac transposable element, containing the lacI(q)-r egulated lazZ Delta M15 gene next to a selectable marker, was construc ted. Mixed D3112 lysates were used to transduce P. aeruginosa PAO1, an d derivatives containing randomly inserted chromosomal copies of the m ini-Dine element were obtained. Transformation of the PAO1::mini-Dlac transductants with the broad-host-range vector, pUCP19, led to the for mation of blue colonies on indicator medium in the presence of inducer . In contrast, transformants harboring the pUCP19 derivative pCDO, con taining the catechol-2,3-dioxygenase (C230)-encoding xy/E gene under I nc promoter control, were white on the same medium. Expression of xy/E was tightly controlled by single-copy mini-Dine-encoded inc repressor and in induced cultures was increased more than 100-fold over that ob served in uninduced cultures. The usefulness of the system for molecul ar cloning in P. aeruginosa was demonstrated by ligating size-fraction ated PAO1 chromosomal fragments into pUCP19, followed by transformatio n of the newly isolated PAO1::mini-Dlac host. All randomly chosen whit e colonies contained recombinant plasmids, with inserts of the correct size range, while blue colonies contained pUCP19 alone. The functiona lity of the system was also shown in another frequently studied strain , PA103.