Transformation process

In order to further investigate the transformation process and to prove if the higher transformation efficiency is due to the temporary storage of D-alanine and to the lacking inhibition by an appropriate antibiotic the bacterial growth of the E. coli strain DH5α Δalr ΔdadX was analyzed during the incubation in the SOC media. Therefore, small volumes of 40 µl were continuously plated in a time interval of 15 min. The samples were spread in parallel onto normal LB-Agar for an antibiotic-free selection and onto LB containing 30 mg/L chloramphenicol for the classical selection with antibiotic.
As demonstrated in Figure 9 the higher transformation efficiency can be explained by the lacking inhibition of a corresponding antibiotic, because just after the transformation there are no colonies observable with antibiotic-selection. In contrast to that there are positive red colonies on the LB plates. This might be due to the quick over-inoculation directly after the transformation. The chloramphenicol acetyltransferase, which normally prevents the antibacterial effect of chloramphenicol to bind to the 50S ribosomale subunit, is not been translated and functionally expressed so far. Thus the chloramphenicol stops the bacterial growth. Whereas, cells spread onto LB for the antibiotic-free selection still accumulate some D-alanine from the SOC media, which enables a short-term growth without the complementation of the alanine racemase from the plasmid BBa_K1465401. A stop of bacterial cell division could be also possible until the alanine racemase is functionally expressed and the cell can start growing.

Figure 9: Colony forming during the transformation by using antibiotic-free selection (black) or antibiotic-selection with chlormaphenicol (red) for the positive red colonies carrying the plasmid BBa_K1465401. It can be demonstrated that the antibiotic-free selection is more efficient and requires less incubation time.

In addition to the higher transformation efficiency of the antibiotic-free selection system, it can be observed that the incubation time can be reduced by the antibiotic-free selection. A high transformation efficiency can be already observed shortly after the transformation. Besides the transformation efficiency does not increase with the incubation time, because the raising colony forming units in both curves are due to the bacterial growth in the SOC media. Another important aspect is the quotient of false-positives to identify the most suitable moment for a screening with the lowest possible portion of false colonies, phenotypically white colonies in this case. The quotient of false-positive white colonies was always about 2 %. An incubation from 30 to 60 min is advisable for the antibiotic-free selection, because afterwards the higher cfu is only due to the bacterial growth in SOC media and therefore due to the higher risk of contamination not advisable.