Remaining Challenges

The E. coli strains KRX Δalr ΔdadX and DH5α Δalr ΔdadX respectively showed a strict dependence of D-alanine, but as mentioned before the ratio of false-positive transformants was slightly higher compared to the selection ratio on the antibiotic selection with chlormaphenicol. Even on the negative plate some colony forming untis were observable, while there were no colonies on the LB plate containing 30 mg/L Chloramphenicol. This effect migth be due to some revertants to the D-alanine auxotrophy and to the corresponding selection pressure.
Therefore, the revertants were analyzed by spreading an overnight culture of the strains DH5α Δalr ΔdadX and DH5α Δalr kan:dadX on normal LB and several dilution onto LB medium containing 5 mM D-alanine. The same procedure was performed with the transformation approach. In both cases nearly the same revertants rate of 3.4 10^-7 (overnight culture) and 3.11 10^-7 ± 2.29 10^-7 (transformation) was investigated. There was no significant difference between the ratio of revertants of the strain DH5α Δalr ΔdadX (3.27 10^-7 ± 2.27 10^-7) and DH5α Δalr kan:dadX (2.95 10^-7 ± 2.65 10^-7), which indicates that an effect by contamination can be excluded and additional colonies are probably revertants which are able to accumulate D-alanine in some way.
A possible explantion might be a point mutation in the coding sequence of the methionine repressor metJ, resulting in a similar mutation rate of 7 x 10^-7 (Kang et al., 2011). Under normal circumstances the MetJ represses all essential genes for the biosynthesis of L-methionin like metA, metB, metC, MetF, metE and metK as well as the genes of the metD operon by using S-adenosylmethionine (SAM) as cofactor, see Figure x below.

Figure 11: Suprresion of the Methionins biopsynthesis by MetJ. A point mutation within this repressor leads to a higher expression of MetC, which is also able to catalyze the conversion from L-alanine into D-alanine in E. coli.

It could be demonstrated that in the presence of L-methionine all genes affected are surpressed and no revertants are observable, and that the reversion could not be quantified in its absence, suggesting that there is an other methionin-repressible enzyme able to accumulate D-alanine in E. coli. The revertants formed in the absence of L-methionine showed a higher expression of the Cystathionine β-lyase and point mutations in the MetJ repressor like R42C. It could be shwon that a strict D-alanine auxotrophy can be restore by a plasmidar expression of the natural metJ repressor or the additional deletion of metC (Kang et al., 2011).
Up to now the antibiotic-free selection could be demonstrated only for relative normal plasmid like BBa_K1465401 as Backbone (3163 bp) and BBa_J04450 (RFP, 1069 bp) as insert, resulting in a plasmid-size of 4232 bp. So for greater plasmids or more complicating cloning approaches the transformation and selection 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, but for easy cloning this might be already possible without L-methionine or an additional deletion of metC.

References

• 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.