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Workplan Meeting
Timeline: Day 35
In depth presentations of preliminary reports from reviewers concerning engineering, hazard and production issues
Description of Review Coverage and Example of Commitee Report
Days 36-49
Reviewers critically examine the application and amplified the information supplied with literature data. Preliminary conclusions about the hazard involved and the extent of exposure are reached.
Example:
Summary of hazard report PMN95-1601
Pseudomonas fluorescens HK9 (sal+ phenotype) was modified to contain a 116 kb recombinant bioluminescent reporter plasmid (pUTK21) for naphthalene and PAH (polycyclic aromatic hydrocarbons) degradations. The constructed PMN strain is designated as P.f. HK44 (nal+, sal+, and Tcr phenotype) and was first describrf in a 1990 publication (King et al). Plasmid pUTK21 was developed by transposon (Tn4431, 15 kb)) insertion of a promoterless lux gene cassette (luxCEABE) from Vibrio fischeri MJ_1 into a large natural naphthalene catabolic plasmid pKA1 (101 kb) in P.f. strain 5R. The transposition function and tetracycline resistance gene on Tn4431 were derived from a natural transposon (Tn1721) which resides in a plasmid in E. coli D1021. Plasmid pKA1 is a NAH7_like plasmid and it can degrade phenanthrene and anthracene as well as napthalene. In pKA1 the upper and lower catabolic regions share extensive homology with NAH7 plasmid (83 kb) whose naphthalene catabolic genes are organized into two operons, nah and sal. Other genes on pKA1 are not known according to Sayler. The lux transposon (Tn4431) was inserted into pKA1 in the nahG (salicylate hydroxylase) gene which is the first gene in the sal operon (lower pathway). Thus, the PMN strain has a defective plasmid_borne sal pathway, but the chromosomal sal pathway one remains functional. The nahG::lux transcriptional fusion on pUTK21 produces truncated salicylate hydroxylase of about 100 amino acids.
The light producing PMN strain is to be produced by fermentation. It is to be used for a research on biomediation of PAH contaminated Manufacture Gas Plant (MGP) soil for analysis of microbial biodegradative activity. The study will be carried out in a set of semi_contained soil lysimeters in the field by the University of Tennessee and DOE's Oak Ridge National Laboratory. The project is funded by DOE. Four major differences between the PMN strain P.f. HK44 and the parent strain P.f. HK9 are: 1) the PMN strain is a recombinant strain containing intergeneric coding DNA sequences from E. coli and Vibrio fischeri while the parent is a natural isolate; 2) the PMN strain has nah and sal pathways while the parent strain has only sal pathway; 3) the PMN strain produces visible light upon induction with salicylate (the metabolite of napthalene) while the parent strain does not; and 4) the PMN strain is tetracycline resistant while the parent strain is not. Plasmid pUTK21 in the PMN strain and the transposon residing on pUTK21 are capable of transferring to other microorganisms.
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