Summary

So in conclusion it could be demonstrated, that the antibiotic-free selection system by via 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. Addition the novel sysmtem requires a shorter incubation in SOC-media after the successful tranformation to reach comparable transformation effiency than in Chloramphenicol. (On top it could be demonstrated that this system can be used for molecular cloning of normal plasmid size like BBa_I13522 with a total size of 4100 bp). Additionaly the selection via the complementation of the alanine racemase is suitable also for longer time and as constant as the plasmidstability with the antibiotic Chloramphenicol.

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

Nevertheless there are resting challenges for the furture to optimize the system of an antibiotica-free selection. One problem is that there revertants, resulting in an double high ratio of 2,8 % in comparision to the selection with antibiotics (1,5 %). This seems to be not problematic due to the higher transofrmation effiency but upon difficult transformation but this might be problematic when none or very few positive colonies are formed. In this case the ratio of false-positive might be higher so that the addition of L-methionine or the deletion of metC is neccessary to obtain and effective selection (Kang et al., 2011).
Another aspect to mentioned is, that the plasmid BBa_K1465401 used for the charaterization of the antibiotic-free selection still contains the coding sequence for the Chloramphenicol-resistance, so that up to now the system is not completly detached from an antibiotic-selection, but the primer for the deletion of the Chloramphenicol-resistance of the pSB1C3 were already designed and can be found here. And since the selection via complementation of the alanine racemase has turned out to be functional there is no hurdle to establish the first overall antibiotic-free selection system in E. coli!
And on top this sysem might be not only limited to E. coli since a D-alanine auxotrophy could be demonstrated also for other bacteria like Listeria monocytes (Thompson et al., 1998), Corynebacterium glutamicum (Tauch et al., 2002) or Bacillus subtilis (Ferrari et al., 1985) as example, so that it might be feasible with any bacteria where the cross-linkage of the peptidoglycane layer is realized with D-alanine...

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.