Part:BBa_K1172902

alanine racemase (''alr'') under the control of the P_''tac'' promoter

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 ([http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#References 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 ∆alr ∆dadX). 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 [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#RNase_Ba_.28Barnase.29 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. coli ∆alr ∆dadX to obtain a higher plasmid stability.
• part of a Biosafety-Plasmid like BBa_K1172909 an other to create powerful Biosafety-Systems.
• ...
  

This part was used for the characterization of the complementation. The alanine racemase (alr) itself is the BioBrick BBa_K1172901.
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 385
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 309
  Illegal BamHI site found at 1011
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 427
  Illegal AgeI site found at 727
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 184
  

Characterizations of the alanine racemase

The konstitutive Alanin-Racemase (alr) and the catabolic Alanine-Racemase (dadX) were [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_Strain#Deletion_of_the_alanine_racemases 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 chloramphenicol. For the complementation the alanine racemase (alr) was brought under the control of the P_tac promoter, resulting in the BioBrick BBa_K1172902. The P_tac 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 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 ∆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.]