Difference between revisions of "Part:BBa K1172901"

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	__NOTOC__		__NOTOC__
	<partinfo>BBa_K1172901 short</partinfo>		<partinfo>BBa_K1172901 short</partinfo>
		+	===Usage and Biology===
	<p align="justify">		<p align="justify">
−	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>	+	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]).<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>	+
−	<br>	+
−	[[Image:IGEM Bielefeld 2013 alr isomerase bearbeitet.png|600px|thumb|center|'''Figure 5:''' 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.]]	+
		+	[[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 conversion from L-alanine to D-alanine. For this isomerization the cofactor pyridoxal-5'-phosphate is necessary.]]
−	<!-- Add more about the biology of this part here	+	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 (<bbpart>BBa_K1172901</bbpart>) 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.</p>
−	===Usage and Biology===	+	<br>
−	<!-- -->	+	==In short==
−	<span class='h3bb'>Sequence and Features</span>	+	So in fact the alanine racemase (''alr'') can be used as:
−	<partinfo>BBa_K1172901 SequenceAndFeatures</partinfo>	+	*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 <bbpart>BBa_K1172909</bbpart> to create powerful Biosafety-Systems.
		+	*...
		+	<span class='h3bb'>'''Sequence and Features'''</span>
		+	<partinfo>BBa_K1172901 SequenceAndFeatures</partinfo>
		+	<br>
		+	<br>
		+	===Characterizations of the alanine racemase===
	<p align="justify">		<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 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''. <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>	+	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<sub>''tac''</sub> promoter, resulting in the BioBrick <bbpart>BBa_K1172902</bbpart>. The P<sub>''tac''</sub> 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.<br>
−	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>	+	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 <bbpart>BBa_K1172902</bbpart> does hardly show a disadvantage in the cell division compared to the wild type.</p>
	<br>		<br>
−	[[File:Team-Bielefeld-Biosafety-Strain-D-Alanine-Complementation.jpg|600px|thumb|center|'''Figure 1:''' 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.]]	+	[[File:Team-Bielefeld-Biosafety-Strain-alrdadXdeletion.jpg|600px|thumb|center|'''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.]]
		+	<br><br>
		+	==References==
		+	<p align="justify">
		+	*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.'']
	<!-- Uncomment this to enable Functional Parameter display		<!-- Uncomment this to enable Functional Parameter display
	===Functional Parameters===		===Functional Parameters===
	<partinfo>BBa_K1172901 parameters</partinfo>		<partinfo>BBa_K1172901 parameters</partinfo>
	<!-- -->		<!-- -->

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Latest revision as of 14:05, 30 October 2013

Alanine racemase from ''E. coli''

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]).

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 to create powerful Biosafety-Systems.
• ...

Sequence and Features

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.

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.]