Long-term stability of the antiobiotic-free selection

To verify if the plasmid stability of the antibiotic-free selection system is comparable to an appropriate antibiotic selection system, E. coli carrying the plasmid BBa_K1465401 was cultivated for 40 h in LB media, containing normal LB, LB supplemented with 5 mM D-alanine and LB containing 30 mg/L chloramphenicol. After reaching the stationary phase of the cultivation the culture was diluted to an OD₆₀₀ of 0.0001 and then diluted again 1:1000. Volumes of 10 µl, 20 µl and 50 µl of the dilution were spread onto the corresponding media to estimate the ratio between red colonies (harboring the plasmid) and white colonies (loss of the plasmid). The cultivation with 5 mM D-alanine should probably lead to the loss of the plasmid, because it is not necessary for bacterial growth in case of D-alanine supplementation. As shown in Figure 10, white cells occur after 17 h of cultivation. After an incubation of about 12 h on LB plates, the first phenotypically identification which intends a loss of the plasmid BBa_K1465401 took place after 29 h. In contrast to that, there was no formatioin of white colonies within the antibiotic-free selection system. In comparison to the selection with chloramphenicol, we can suggest that the plasmid stability might be the same for 52 h. A more accurate quantification would be achieved with a long-term cultivation to investigate plasmid loss in one of the selection media. Usually E. coli is not cultivated for such a long time, which means that the data should be sufficient enough. Yet, the investigation of the plasmid stability using the antibiotic-free selection system can be improved by the fluorescence measurement of a reporter protein to quantify the plasmid stability more precisely.

Figure 10: Long-term stability of the plasmid BBa_K1465401 by calculation of the ratio between white colonies (loss of the plasmid) and red colonies (harboring the plasmid).