Author : Saifur Rohman, M.

Summary : Genetically engineered microorganisms (GEM) have been widely used in various environments. However, there is still limited information regarding the behavior of GEM in the environment, particularly in competition with other microorganisms. Previous studies concerning competition of GEM have been established, but most of them have been done in environments which support function of carried genes (specific environments). Accordingly, the objective of this research was to study the competitiveness of GEM in non-specific environments supplied with rich and poor nutrition.

Three strains of genetically engineered Escherichia coli, namely: E. coli R6.0, R6.3, and R6.4, were used as GEM models. E. coli R6.0, R6.3, and R6.4 harbor plasmids pEOR, pEOR 6.3, and pEOR 6.4, respectively. Plasmid pEOR was constructed in a previous study for expressing chemically synthesized open reading frame (artificial ORF). Plasmids pEOR 6.3, and pEOR 6.4 were constructed from plasmid pEOR inserted with artificial ORF in its KplI-BamHI site. The ORF of pEOR 6.3 could be expressed while the ORF of pEOR 6.4 could not. Competition experiment was done by co-culturing the three strains of E. coli in batch culture system. The media used in the competition experiment were Luria-Bertani (LB) and M9 for rich and poor environments, respectively. The expression of the artificial ORF was induced by adding isoprophyl-thio-ß-D-galactopyranoside (IPTG) into the culturing media. Samples for competition analyses were taken from lag, log, stationary, and prolonged stationary phases. Competitiveness was quantified by enumerating the proportion of each strain after co-culturing in the co-culture.

The results showed that E. coli R6.3 had the smallest proportion when it was co-cultured with its competitors (R6.0 and R6.4), whether in LB or M9 media, with or without IPTG addition. The proportion of E. coli R6.3 and R6.4 in M9 medium was smaller than in LB medium. It can be concluded that the size of plasmid and the expression of artificial ORF had reduced the competitiveness of the GEM, although it was cultured in the rich nutrition environment. The energy burden of GEM became much higher when it was cultured in poor nutrition environment.