Coding

Part:BBa_K1172901

Designed by: Tore Bleckwehl   Group: iGEM13_Bielefeld-Germany   (2013-08-26)

Alanine racemase from ''E. coli''

Usage and Biology

The alanine racemase Alr (EC 5.1.1.1) from the Gram-negative enteric bacteria Escherichia coli is a racemase, which catalyses the reversible conversion of L-alanine into the enantiomer D-alanine (see Figure 1). For this reaction, the cofactor pyridoxal-5'-phosphate (PLP) is necessary. The constitutively expressed alanine racemase (alr) is naturally responsible for the accumulation of D-alanine. This compound is an essential component of the bacterial cell wall, because it is used for the cross-linkage of peptidoglycan (Walsh, 1989).

Figure 1: The alanine racemase (BBa_K1172901) from E. coli catalyses the reversible conversion from L-alanine to D-alanine. For this isomerization the cofactor pyridoxal-5'-phosphate is necessary.
The usage of D-alanine instead of a typically L-amino acid prevents cleavage by peptidases. However, a lack of D-alanine causes to a bacteriolytic characteristics. In the absence of D‑alanine dividing cells will lyse rapidly. This fact is used for our Biosafety-Strain, a D-alanine auxotrophic mutant (K-12 ∆alrdadX). The Biosafety-Strain grows only with a plasmid containing the alanine racemase (BBa_K1172901) to complement the D-alanine auxotrophy. Consequently the alanine racemase is 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 for the implementation 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 medium, cells will not grow.


In short

So in fact the alanine racemase (alr) can be used as:

  • an antibiotic free selection marker in D-alanine auxotrophic strains, like E. colialrdadX to obtain a higher plasmid stability.
  • part of a Biosafety-Plasmid like BBa_K1172909 to create powerful Biosafety-Systems.
  • ...


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 331
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 255
    Illegal BamHI site found at 957
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 373
    Illegal AgeI site found at 673
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 130



Characterizations of the alanine racemase

The konstitutive Alanin-Racemase (alr) and the catabolic Alanine-Racemase (dadX) were deleted in E. coli K-12 leading to the Strain K-12 ∆alrdadX.
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 chloramphenicol. For the complementation the alanine racemase (alr) was brought under the control of the Ptac promoter, resulting in the BioBrick BBa_K1172902. 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 (De Boer et al., 1983). Therefore an induction with IPTG was not necessary on M9 minimal medium, 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 dependence on the amino acid D-alanine, as expected. As shown in Figure 2 below the bacteria with this deletions are not any more able to grow on normal M9 medium 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. Furthermore, it could be demonstrated, that the auxotrophic mutant K-12 ∆alrdadX 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 ∆alrdadX depends strict on the presence of D-alanine in the media or a complementation via plasmid containing an intact alanine racemase.



References

[edit]
Categories
//biosafety/kill_switch
Parameters
None