Part:BBa_K1172903

Alanine racemase (alr) with double terminator

Usage and Biology

This is a cloning intermediate for the construction of Biosafety-Systems like BBa_K1172909 or BBa-K1172911. It contains the RBS BBa_B0034, the alanine racemase BBa_K1172901 and the terminator BBa_B0015.

This part could therefore be used as an antibiotic-free selection marker in the [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_Strain Biosafety-Strain] E. coli ∆alr ∆dadX for any other application!

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 352
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 276
  Illegal BamHI site found at 978
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 394
  Illegal AgeI site found at 694
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 151
  

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 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 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.

Terminator

Terminators are essential to terminate transcription of an operon. In procaryotes, two types of terminators exist. The rho-dependent and the rho-independent terminator. Rho-independent terminators are characterized by their stem-loop forming sequence. In general, the terminator-region can be divided into four regions. The first region is GC-rich and constitutes one half of the stem. This region is followed by the loop-region and another GC-rich region that makes up the opposite part of the stem. The terminator closes with a poly uracil region, which destabilizes the binding of the RNA-polymerase. The stem-loop of the terminator causes a distinction of the DNA and the translated RNA. Consequently the binding of the RNA-polymerase is cancelled and the transcription ends after the stem-loop ([http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#References Carafa et al., 1990]).
For our Bioafety-System araCtive the terminator is necessary to avoid that the expression of the genes under control of the rhamnose promoter P_Rha, like the Repressor AraC and the alanine racemase (alr) results in the transcription of the genes behind the arabinose promoter P_BAD, which contains the toxic Barnase BBa_K1172904 and would lead to cell death.

Figure 5: Stem-loop structure of the terminator BBa_B0015, which is used for the termination. The terminator is used to make sure that solely the alanine racemase Alr is expressed but the transcription of the genes behind is avoided and needs seperate regulations.