The negative effect of induction with IPTG on plasmid segregation identified in this study was already mentioned in the literature [14], [29] and [30]. Marí et al. [29] found that when they used vectors pYMK5 and pYMK7, which contain brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) genes, respectively, plasmid stability declined in the presence of the inducer (1 mM IPTG) in E. coli, with or without the antibiotics ampicillin and kanamycin. Data on the stability of plasmid pED-GnRH3 (obtained from vector pET28a), transformed in E. coli, indicate that plasmid segregation is far more dependent on induction than the

presence or absence of kanamycin, and that after 10 h cultivation in non-induced

cultures, plasmid stability was as high as 95% with antibiotics and 90% without them. However, stability levels in induced cultures were far lower after 10 h induction, dropping as low SB431542 nmr as 15% with antibiotics and 10% without them [30]. If one looks at the values for Φ obtained in the experiments at the center point ( Table 1), one might think that the value obtained in experiment 6 (CP) is an outlier since it differs from the trend seen for all the other Φ values from the replications performed at the center point. An outlier is defined as an experimental point that would seem not to fit into a particular distribution pattern of probabilities defined by the vast majority of the other experimental points [18]. However, the identification see more of outliers is a controversial issue and the elimination of a putative outlier could result in a misinterpretation of the data. For this reason, the effects of the variables on the plasmid-bearing cells (Φ) GBA3 were analyzed both taking account of and discarding the Φ value obtained from experiment 6 (CP), resulting in

the same conclusions about the effects. Also, it can be perceived from the Φ values (fraction of plasmid-bearing cells) ( Table 1) that the behavior of the Φ values was not linear, which was confirmed by the low value of the linear adjustment coefficient (R2). As it is only possible to assess linear regression coefficients for each variable when analyzing central composite design, the low R2 indicates that the linear model does not adjust well to the data. According to the studied ranges, in order to obtain lower plasmid segregation levels, 0.1 mM IPTG should be used. These data do not rule out the possibility of there being an optimal point lower than 0.1 mM IPTG that would still assure minimum plasmid segregation and good protein expression levels. The results of the statistical analysis showed that according to the Student’s t-test, the mean CFU/mL values obtained from the experiments were equivalent, meaning that for most of the data they were statistically equivalent (within a 95% confidence level), as can be seen from Fig. 3.