DETECTION AND BEHAVIOR STUDY OF SOME GENETICALLY ENGINEERED MICROORGANISMS RELEASED IN SOIL USING PLATE COUNTING, GENE TRANSFER ASSAY AND SPECIFIC PCR METHODS

The objective of this study was monitoring and behavior study of genetically engineered microorganisms (GEMs) that might be accidentally or deliberately released into the environments. Three methods (plate count, gene transfer and specific PCR) were used with four genetically engineered E. coli strains (A:pJan25, B: pJan25, pGWB533 and pGWB404) harboring antibiotics resistance genes with additional gfp and gus genes as a molecular markers. The GEMs were added to soil microcosms at 10⁷ cells/g soil and incubated at room temperature for 35 days. After intervals time 0, 7, 14, 21, 28 and 35 days, bacterial cells were recovered and CFU/g were estimated. The number of  viable cells were decreased from 10⁷ to 10³ after 28 days with A: pJan25 and pGWB533 and reached to zero at 35 days. Strain B: pJan25 was survived up to 35 days (10³ CFU/g), while pGWB404 was disappeared after 21 days. Comparing with sterilized soil, it was found that the viable cells were alive up to 35 days (CFU/g was 10⁴).The variation of CFU and presence of a viable cells in soil microcosm may due to the effect of indigenous microbial populations and the type of strain.                                       
Specific PCR was applied on the random selected colonies at 14, 21 and 28 days only, the target genes was gfp and gus. The results  showed  the presence of both genes in all tested colonies. This indicated that the tested GEMs could be maintained their constructed genes at long incubation time.
Horizontal gene transfer was also assayed using conjugation under laboratory and soil microcosm conditions to confirm that GEMs genes were transferred to other organisms andto monitor the persistence of GEMs genes in soil. The gene transfer was started at 14 days in sterilized soil and 21 days in soil microcosm. The conjugation frequency under laboratory and sterilized soil conditions was higher than under soil microcosm condition. The results showed that the used GEMs were able to transferred  three genes to recipient cells. This indicated that these genes were plasmid harboring and it were transferred to a recipient