Difference between revisions of "Part:BBa K1172901"

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Alanine racemase from ''E. coli''

The alanine racemase alr (EC 5.1.1.1) from the gram-negative enteric bacteria Escherichia coli is a racemase, which catalyses the reversible reaction from L-alanine into the enantiomer D-alanine. For this reaction the cofactor pyridoxal-5'-phosphate (PLP) is typically needed. The constitutive alanine racemase (alr) is naturally responsible for the accumulation of D-Alanin, which is an essential component of the bacterial cell wall, because it is used for the crosslinkage of the peptidoglykan ([http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#References Walsh, 1989]).
The use of D-Alanine instead of a typically L-amino acids prevents the cleavage by peptdidases, but a lack of D-Alanine leeds to a bacteriolytic effect. So in the absence of D‑Alanine dividing cells will lyse rapidly. This approach is used by our Biosafety-Strain, a D-alanine auxotrophic mutant (K-12 ∆alr ∆dadX). The Safety-Strain grows only with a plasmid containing the alanine racemase (alr) for the complementation of the D-alanine auxotrophic. Because the Alanine-Racemase is therefore essential for bacterial cell division, this approach guarantees a high plasmid stability, which is extremely important when the plasmid contains a toxic gene like the Barnase. In addition this construction provides the possibility of a double kill-switch system. Because if the expression of the alanine racemase is repressed and there is no D-alanine supplementation in the media, the cells would not increase.

Figure 1: The alanine racemase (BBa_K1172901) from E. coli catalyses the reversible reaction from L-alanine to D-alanine. For this isomerisation the cofactor pyridoxal-5'-phosphate is necessary.

Usage and Biology

The alanine racemase (alr) can be used as an antibiotic free selection marker in D-alanine auxotrophic strains, to obtain a higher plasmidstability. Therefore it is ideal in a Biosafety-Plasmid like K1172909

Sequence and Features

The konstitutive Alanin-Racemase (alr) and the catabolic Alanine-Racemase (dadX) were deleted in E. coli K-12 leading to the Strain K-12 ∆alr ∆dadX.
To avoid a second recombination of the Alanine-Racemase (alr) from the plasmid with the genome, the whole coding sequence was deleted in the genome and the characterization of the Alanine-Racemase was performed with the antibiotic chlormaphenicol. For the complementation the Alanine-Racemase (alr) was brought under the control of the ptac promoter. The ptac promoter is a fusion promoter of the -35-region of the trp promoter and the -10-region the lac promoter, so that there only slight repression and the expression of the Alanine-Racemase is highly activated ([http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_Strain#References De Boer et al., 1983]). Therefore an induction with IPTG was not necessary on M9, but surprisingly it was essential on LB-agar.
The deletion of the constitutive Alanine-Racemase (alr) and the catabolic Alanine-Racemase (dadX) in E. coli leads to a strict dependance on the amino acid D-alanine, as aspected. As shown in the figure below the bacteria with this deletions are not any more able to grow on normal M9-media without D-alanine supplementation (purple curve), whereas the wild type does (red curve). The auxotrophic Safety-Strain grows only on media with D-alanine (5 mM) supplemented (blue curve) or by a complementation of the Alanine-Racemase via plasmid. Further it can be seen, that the auxotrophic mutant K-12 ∆alr ∆dadX grows slightly slower, than the wild type K-12. In contrast the bacteria containing the Alanine-Racemase (alr) on the plasmid BBa_K1172902 does hardly show a disadvantage in the cell division compared to the wild type.

Figure 2: Characterization of the D-alanine auxotrophic Biosafety-Strain. The Biosafety-Strain K-12 ∆alr ∆dadX depends strict on the presence of D-alanine in the media or a complementation via plasmid containing an intact Alanine-Racemase.

References

• Cava F, Lam H, de Pedro MA, Waldor MK (2011) Emerging knowledge of regulatory roles of d-amino acids in bacteria [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037491/pdf/18_2010_Article_571.pdf|Cell and Molecular Life Sciences 68: 817 - 831.]
• De Boer Hermann, Comstock J. Lisa and Vasser Mark (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC393301/pdf/pnas00627-0036.pdf|Proceedings of the National Academy of Science of the United States of America 80: 21 - 25.]
• Link, A.J., Phillips, D. and Church, G.M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: Application to open reading frame characterization. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC179534/pdf/1796228.pdf|Journal of Bacteriology 179: 6228-6237.]
• Walsh, Christopher (1989) Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. [http://www.jbc.org/content/264/5/2393.long|Journal of biological chemistry 264: 2393 - 2396.]