Transformation process

For the further investigation of the transformation process and to prove if the higher transformation efficiency is corresponding to the temporary storage of D-alanine and due to the lacking inhibition by an appropriate antibiotic the bacterial growth of the E. coli strain DH5α Δalr ΔdadX during incubation in the SOC-Media was analyzed. Therefore small volumes of 40 µl were continuous plated out in a time interval of 15 min. The samples were streaked out in parallel on normal LB-Agar for an antibiotic-free selection and on LB containing 30 mg/L Chloramphenicol for the classical selection via 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 via antibiotic-selection, but on there a positive red colonies on the LB-plate. This might be due to the quick over-inoculation directly after the transformation so that the Chloramphenicol acetyltransferase, which normally prevents the antibacterial effect of Chloramphenicol to bind to the 50S ribosomale subunit, has not been translated and functionally expressed so far, so that the Chloramphenicol stops the bacterial growth. Meanwhile the cell streaked out on normal LB for the antibiotic-free selection have still accumulate some D-alanine from the SOC-Media so that they probably can grow some time without the complementation of the alanine racemase from the plasmid BBa_K1465401. Also a stop of bacterial cell division is possible until the alanine racemase is functionally expressed and the cell can start growing.

Figure 9: Colonie forming during the Transformation by using antibiotic-free selection (black) or antibiotic-selection with Chlormaphenicol (red) for the positive red colonies harboring the plasmid BBa_K1465401. It could be demonstrated that the antibiotic-free selection is more efficient and requires less incubation time.

Beside the higher transformation efficiency of the antibiotic-free selection it can be observed, that the incubation time can be reduced by the antibiotic-free selection, because already shortly after the transformation there is a high transformation efficiency. Besides the transformation efficiency do 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 ideal moment for a screening with the lowest portion of false colonies, phenotypically white colonies in this case. Actually the quotient of false-positive white colonies was always about 2 % so that an incubation from 0 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 because of the higher risk of contamination not advisable.