Summary

It could be demonstrated, that the antibiotic-free selection system by the D-alanine auxotrophic strain DH5α Δalr ΔdadX is not only possible, but even more efficent according to the transformation efficiency with the plasmid BBa_K1465401 then classical approaches. In addition the novel system enables a shorter incubation in SOC media after the transformation to reach comparable transformation efficencies to chloramphenicol. It was demonstrated that this system can be used for molecular cloning of plasmids with normal size like BBa_I13522 with a total size of 4100 bp. Furthermore the selection via the complementation of the alanine racemase is suitable for long-term cultivations and guarantees a plasmid stability comparable to the antibiotic system with chloramphenicol.

Figure 12: Comparision of the transformation efficiency for the classical selection with Chloramphenicol (left) or antibiotic-free on normal LB-media (right).

Nevertheless there are remaining challenges for the future to optimize the system of an antibiotic-free selection system. A problematic issue are revertants, which occur in a two times higher ratio of 2.8 % in comparision to the selection with antibiotics (1.5 %). This seems not to be problematic due to the higher transformation efficiency. This might be problematic when none or very few positive colonies are formed, for example in case of difficult transformations with large plasmids. In this case the ratio of false-positive transformants might be higher, which might require the addition of L-methionine or the deletion of metC to obtain an effective selection (Kang et al., 2011).
Another aspect which has to be mentioned is that the plasmid BBa_K1465401 which is used for the charaterization of the antibiotic-free selection, still contains the coding sequence for the chloramphenicol-resistance. Until now the system is not completely detached from an antibiotic-selection, but the deletion primers for the chloramphenicol resistance gene of the pSB1C3 were already designed and can be found here. Since the selection via complementation of the alanine racemase has been proven to be functional, there is no longer a hurdle to establish the first overall antibiotic-free selection system in E. coli!
Above all mentioned results this sysem might be not only limited to E. coli. A D-alanine auxotrophy could be already demonstrated for other bacteria like Listeria monocytes (Thompson et al., 1998), Corynebacterium glutamicum (Tauch et al., 2002) or Bacillus subtilis (Ferrari et al., 1985). A selection system with complementation of the alanin racemase might be feasible for all bacteria where D-alanine is responsible for the cross-linkage of the peptidoglycane layer.

References

• E. Ferrari, D. Henner und M. Yang (1985) Isolation of an alanine racemase gene from Bacillus subtilis and its use for plasmid maintenance in B.subtilis. Nature Biotechnology, vol. 3, pp. 1003 - 1007.
• Kang L, Shaw AC, Xu D, Xia W, Zhang J, Deng J, Wöldike HF, Liu Y, Su J. (2011) Upregulation of MetC is essential for D-alanine-independent growth of an alr/dadX-deficient Escherichia coli strain. Journal of bacteriology, vol. 193, pp. 1098 - 1106.
• A. Tauch, S. Götker, A. Pühler, J. Kalinowski, G. Thierbach (2002) The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains. Journal of Biotechnology, vol. 99, pp. 79 - 91.
• R. Thompson, H. Bouwer, D. Portnoy, F. Frankel (1998) Pathogenicity and and immunogenicity of a Listeria monocytogenes strain that requires D-alanine for growth. Infection and Immunity, vol. 66, pp. 3552 - 3561.