Difference between revisions of "Part:BBa K1172903"

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	===Usage and Biology===		===Usage and Biology===
−	===Alanine Racemase===	+	This is a cloning intermediate for the construction of Biosafety-Systems like <bbpart>BBa_K1172909</bbpart> or <bbpart>BBa-K1172911</bbpart>. It contains the RBS <bbpart>BBa_B0034</bbpart>, the alanine racemase <bbpart>BBa_K1172901</bbpart> and the terminator <bbpart>BBa_B0015</bbpart>.
−	<br>	+
−	[[Image:IGEM Bielefeld 2013 biosafety alr test.png|left]]	+	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!<br>
		+
		+
		+	<span class='h3bb'>'''Sequence and Features'''</span>
		+	<partinfo>BBa_K1172903 SequenceAndFeatures</partinfo>
		+
		+
		+	===Functional parameters===
		+	==='''Alanine racemase'''===
	<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 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.]]
−	[[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.]]	+
−	<br><br>	+	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.</p>
	<br>		<br>
		+
	==='''Terminator'''===		==='''Terminator'''===
−	<br>
−	[[File:IGEM Bielefeld 2013 biosafety Terminator.png|left]]
	<p align="justify">		<p align="justify">
−	Terminator are essential for the end of an operon. In procaryot exists rho-depending and independing terminator. Rho-independing terminators are characterized by an stem-loop, which is caused by special sequence. In general the terminator-region can be divided into four regions. Starting with a GC-rich region, which performs the stem and followed by the loop-region. The third region is made up from the opposite part of the stem, so that this region concerns also GC-rich portion. After that the terminator ends by an 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, so that the binding of the RNA-polymerase is canceld and the transcription ends after the stem-loop ([http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#References Carafa ''et al.'', 1990]).<br>	+	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]).<br>
−	For our Safety-System the terminator is necessary to avoid that the expression of the genes under the control of the Rhamnose promoter pRHA, like the Repressor araC and the Alanine-Racemase (''alr''), are transcripted but not the genes of the Arabinose promoter pBAD, which contains the toxic Barnase and would lead to cell death.</p>	+	For our Bioafety-System araCtive the terminator is necessary to avoid that the expression of the genes under control of the rhamnose promoter P<sub>''Rha''</sub>, like the Repressor AraC and the alanine racemase (''alr'') results in the transcription of the genes behind the arabinose promoter P<sub>''BAD''</sub>, which contains the toxic Barnase <bbpart>BBa_K1172904</bbpart> and would lead to cell death.</p>
	<br>		<br>
−	[[File:Team Bielefeld Biosafety Terminator.png|400x600px|thumb|center| '''Figure 6:''' Stem-loop structure of the terminator <bbpart>BBa_B0015</bbpart>, which is used for the biosafety system. The terminator is used to make sure that only the repressor and the Alanine-Racemase is transcripted and avoids a transcription of the toxic Barnase.]]	+	[[File:Team Bielefeld Biosafety Terminator.png|400x600px|thumb|center| '''Figure 5:''' Stem-loop structure of the terminator <bbpart>BBa_B0015</bbpart>, 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.]]
	<br>		<br>
−
−
−	<span class='h3bb'>Sequence and Features</span>
−	<partinfo>BBa_K1172903 SequenceAndFeatures</partinfo>
−
	<!-- Uncomment this to enable Functional Parameter display		<!-- Uncomment this to enable Functional Parameter display

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Latest revision as of 15:12, 30 October 2013

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

Functional parameters

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]).

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.