Difference between revisions of "Part:BBa K1172902"

Revision as of 13:21, 6 October 2013

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 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.

Figure 1: The alanine-racemase (BBa_K1172901) from E. coli catalyses the reversible reaction from L-alanine to D-alanine. For this isomerisation the cofactor pyridoxal-5'-phosphate is necessary.

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

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 ===Usage and Biology===
 <p align="justify">
-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. <br>
+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]).<br>
-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.<br>
+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 (<bbpart>BBa_K1172901</bbpart>) 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.</p>
-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 <bbpart>BBa_K1172902</bbpart> does hardly show a disadvantage in the cell division compared to the wild type.</p>
 <br>
+[[Image:IGEM Bielefeld 2013 alr isomerase bearbeitet.png|600px|thumb|center|'''Figure 1:''' The alanine-racemase (<bbpart>BBa_K1172901</bbpart>) from ''E. coli'' catalyses the reversible reaction from L-alanine to D-alanine. For this isomerisation the cofactor pyridoxal-5'-phosphate is necessary.]]
-[[File:Team-Bielefeld-Biosafety-Strain-D-Alanine-Complementation.jpg|600px|thumb|center|'''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.]]
 <span class='h3bb'>Sequence and Features</span>