Part:BBa_K1172902
alanine racemase (''alr'') under the control of the P''tac'' promoter
Usage and Biology
Alanine Racemase
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 bacteriostatic characteristic. 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 (BBa_K1172901) 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 385
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 309
Illegal BamHI site found at 1011 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 427
Illegal AgeI site found at 727 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 184
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