Stable Tagging of Bacteria with Firefly Luciferase Gene for Environmental Monitoring

Gram-negative strains engineered for in situ applications must meet a number of practical requirements for safe and efficient performance. These include not only the absence of traits that may give them an advantage over nonengineered strains but also the ability to retain the acquired genotype and phenotype in the absence of selection. Use of firefly lu-ciferase addresses the first of these concerns, since D-luciferin is expected to be absent from all prokaryotic cells and the enzyme is unlikely to provide any advantage over the wild type. Stable inheritance of the marker gene, in the absence of selective pressure, can be achieved by chromosomal marking using the mini-Ti5 system.44 A Hindlll fragment containing ony the XPR promoter and the luc genes was isolated and cloned into pUC18Not. The NotI fragment with the luc gene was cloned into the unique NotI site of pUT/mini-Tn5 Sm/Sp and into pUTKm. The resulting plasmids, pTCR210 and pTCR240, were selected for studies of transposition and luciferase expression in various gram-negative bacteria.36 To tag bacteria without the use of antibiotic resistance genes, the Km resistance gene may be replaced with the lPR::luc fusion. To attenuate the expression in the environment, where the marked strain must survive, and to allow sensitive detection, a DNA fragment containing the repressor gene laclq and a PR::lucOR fusion has been introduced on a suicide plasmid. PTEB510 (Fig. 5.5) is a representative of constructs able to express high luciferase levels only when induced by IPTG. Transposition frequencies were suitable for marking purposes and the levels of luminescence were sufficient for detection by the standard methods.

I End O End

Sm/Sp Ni lac/1 Pire luc.OR Nl U pTEB510

Te Ni Nl

H tomvMwaiiwiiii PTEB520

I End O End

Sm/Sp Ni lac/1 Pire luc.OR Nl U pTEB510

Te Ni Nl

H tomvMwaiiwiiii PTEB520

Nl Nl pTEB50

Fig. 5.5. Construction of suicide orange click beetle luciferase plasmids used for marking gramnegative bacteria. The selective markers are flanked by Tn5 19-bp terminal ends (I end and O end). The IS50R tnp gene devoid of NotI sites (tnp*) is oriented divergently from the I end. Restriction sites: E, EcoR1; Nt> NotI; Xb, XbaI; H, HindIII; B, BamHI; S, SaH; P, PstI; Bg, BgHI. Reprinted with permission from VĂ¡zquez ME, Cebolla A, Palomares, AJ. FEMS Microbiol Lett 1994; 121:11-18.

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