Long-term stability of the antiobiotic-free selection

To verify if the plasmid stability of the antibiotic-free selection system is comparable to that of an appropiate antibiotic, 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. When stationary phase of the cultivation was reached the cultivation was diluted to OD₆₀₀ of 0.0001 and then diltued again 1:1000. From this dilution volumes of 10 µl, 20 µl and 50 µl were plated out on 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 led to the loss of the plasmid, because it is not neccessary for bacterial grwoth when D-alanine is supplemented. And as shown in Figure 10 below the first white cells occurs after 17 h of cultivation. As they were incubated on LB for about 12 h, the first phenotypically identication for an loss of the plasmid BBa_K1465401 took place after 29 h. In contrast there where no perfomonce of white colonies within the antibiotic-free selection comparable to the selection with Chloramphenicol suggesting that the plasmid stability might be the same for 52 h. A more accurate quantification would be archieved by a longer duration of the long-term cultivation to investigate if there is also a loss of the plasmid after some time in on of the selection media, but usually E. coli is not cultivated for such a long time, so that the data should be sufficient enough. Yet the investigation of the plasmid stability of the antibiotic-free selection system can be improved by flourescence measurement of a reporter to quantify the plasmid stability in this period of time more precisely.

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