Part:BBa_K1172902

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

Usage and Biology

The deletion of the Alanin-Racemases and araC in E. coli was not possible in the common used strains like JM109, Top10 or KRX, but in the wild type strain K-12. This is may due to the RecA1-mutations in this strains, which guarantees a better plasmid maintenance because of defect rekombinase.
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 5: 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.

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