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Team:Bielefeld-Germany/Biosafety/Biosafety System L
From 2013.igem.org
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| - | ===lacI=== | + | ==='''lacI'''=== |
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[[Image:IGEM Bielefeld 2013 biosafety lacI test.png|left]] | [[Image:IGEM Bielefeld 2013 biosafety lacI test.png|left]] | ||
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===Alanine Racemase=== | ===Alanine Racemase=== | ||
| - | [[Image:IGEM Bielefeld 2013 biosafety alr test.png|left]] | + | <!--[[Image:IGEM Bielefeld 2013 biosafety alr test.png|left]] |
<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. The use of D-Alanin instead of a typically L-amino acids prevents the cleavage by peptdidases, but a lack of D-Alanin leeds to a bacteristatic characteristic. So in the absence of D‑Alanine dividing cells will lyse rapidly. So if the expression of the Alanin-Racemase is repressed and there is no D-Alanine-Supplementation in the media, the cells would not increase. </p> | 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. The use of D-Alanin instead of a typically L-amino acids prevents the cleavage by peptdidases, but a lack of D-Alanin leeds to a bacteristatic characteristic. So in the absence of D‑Alanine dividing cells will lyse rapidly. So if the expression of the Alanin-Racemase is repressed and there is no D-Alanine-Supplementation in the media, the cells would not increase. </p> | ||
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[[Image:IGEM Bielefeld 2013 alr isomerase bearbeitet.png|600px|thumb|center|'''Figure x:''' The alanine-racemase from ''E. coli'' catalyses the reversible reaction from L-alanine to D-alanine.]] | [[Image:IGEM Bielefeld 2013 alr isomerase bearbeitet.png|600px|thumb|center|'''Figure x:''' The alanine-racemase from ''E. coli'' catalyses the reversible reaction from L-alanine to D-alanine.]] | ||
| - | <br><br><br><br> | + | <br><br><br><br>--> |
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| + | [https://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System_S Alanine-Racemase] | ||
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==='''Terminator'''=== | ==='''Terminator'''=== | ||
| - | [[File:IGEM Bielefeld 2013 biosafety Terminator.png|left]] | + | <!--[[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 ([https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#References Carafa ''et al.'', 1990]).<br> | 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 ([https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#References Carafa ''et al.'', 1990]).<br> | ||
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[[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 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.]] | ||
| - | <br> | + | <br>--> |
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| + | [https://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System_S Terminator] | ||
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==='''Lactose promoter (plac)'''=== | ==='''Lactose promoter (plac)'''=== | ||
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[[File:IGEM Bielefeld 2013 biosafety dlac-promoter test.png|left]] | [[File:IGEM Bielefeld 2013 biosafety dlac-promoter test.png|left]] | ||
<p align="justify"> | <p align="justify"> | ||
Naturally the lac operon regulates the catabolism of the disaccharide lactose (4-O-(β-D-Galactopyranosyl)-D-glucopyranose) in ''E. coli''. The operon consists of a CAP-binding site, the lac promoter, the lac operator and the genes lacZ, lacY and lacA downstream of the promoter. The transcription of the lactose promoter is regulated by the lacI gene, which is found upstream of the operon under the control of a weak promoter. In the absence of lactose the transcription of the genes behind the lactose promoter is blocked caused by the binding of the lacI pressor. While in the presence of Lactose the repressor is released from the operator and the genes can be transcripted. Typically the transcription is enhanced by a high intracellular level of cAMP.</p><br> | Naturally the lac operon regulates the catabolism of the disaccharide lactose (4-O-(β-D-Galactopyranosyl)-D-glucopyranose) in ''E. coli''. The operon consists of a CAP-binding site, the lac promoter, the lac operator and the genes lacZ, lacY and lacA downstream of the promoter. The transcription of the lactose promoter is regulated by the lacI gene, which is found upstream of the operon under the control of a weak promoter. In the absence of lactose the transcription of the genes behind the lactose promoter is blocked caused by the binding of the lacI pressor. While in the presence of Lactose the repressor is released from the operator and the genes can be transcripted. Typically the transcription is enhanced by a high intracellular level of cAMP.</p><br> | ||
[[File:IGEM Bielefeld 2013 Biosafety laci ohne hintergrund.png|600px|thumb|center|'''Figure 1:'''Structure of the lactose operon and regulatory units.]] | [[File:IGEM Bielefeld 2013 Biosafety laci ohne hintergrund.png|600px|thumb|center|'''Figure 1:'''Structure of the lactose operon and regulatory units.]] | ||
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| + | The lactose promoter thereby regulates the transcription of the genes lacZ, lacY and lacA. The lacZ gene encodes for the ß-Galactosidase a enzyme, who breaks down the lactose to glucose and galactose. The ß-Galactosidase catalyses additional the degradation from Lactose to Allolactose. By binding on the lacI repressor, it changes his conformation an is not any more able to bind on the operator sequnece and to block the transcription. As only one enzyme is necessary to gain a substrate of the glycolysis is becomes clear, why the degradation of Lactose is more preferable compared to L-arabinose or L-rhamnose. <br> | ||
| + | To realize a preference of lactose, the transcription of the lactose promoter is not repressed as that strong. This is caused by the fact that the lacY gene, coding for the integral membrane protein lactose permease, is necessary for the lactose uptake and has to be transcripted on a low level.<br> | ||
| + | The last gene of the lac operon, lacA, encodes for a Transacetylase, who acetylizes glycosides that can not be metabolized. The acetylated glycosides are transported outside the cell to avoid the accumulation of lactose. <br> | ||
| + | In our Biosafety-System the lac-promoter is used for the regulation of GFP or the Barnase. As the lac promoter shows a high basal transcription, its might not ideal for the regulation of a toxic gene product, but the the Biosafety-System Lac of growth is ideal for comparison with the other Systems to measure the level of basal transcription under repressed and unrepressed conditions. Besides we improved the leakiness of the lactose promoter by adding a second lacI-binding site 12 nt downstream of the excisting bining site. As this distance corresponds to about one whorl of the double helix, this should allow an additional lacI repressor to bind on the other site of the DNA and tighten the repression of the lactose promoter. Unfortunately the improvement of the so called double lac promoter could not be quantified, because lac of time.</p><br> | ||
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===Barnase=== | ===Barnase=== | ||
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[[Image:IGEM Bielefeld 2013 biosafety RNase Ba test.png|left]] | [[Image:IGEM Bielefeld 2013 biosafety RNase Ba test.png|left]] | ||
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As shown in the graphic below, the transcription of the DNA, which encodes the Barnase produces a 474 nt RNA. The translation of the RNA of this ribonuclease starts about 25 nucleotids downstream from the transcription start and can be divided into two parts. The first part (colored in orange) is translated into a signal peptide at the N-Terminus of the Barnase. This part is resoponsible for the extracellular translocation of the RNase Ba, while the peptid sequence for the active Barnase starts 142 nucleotides downstream from the transcription start (colored in red). For the Biosafety-System we used only the coding sequence of the Barnase itself to prevent the extracellular translocation of the toxic gene product. This leds to a rapid cell death if the expression of the Barnase isn't repressed by the repressor of the Biosafety-System.</p> | As shown in the graphic below, the transcription of the DNA, which encodes the Barnase produces a 474 nt RNA. The translation of the RNA of this ribonuclease starts about 25 nucleotids downstream from the transcription start and can be divided into two parts. The first part (colored in orange) is translated into a signal peptide at the N-Terminus of the Barnase. This part is resoponsible for the extracellular translocation of the RNase Ba, while the peptid sequence for the active Barnase starts 142 nucleotides downstream from the transcription start (colored in red). For the Biosafety-System we used only the coding sequence of the Barnase itself to prevent the extracellular translocation of the toxic gene product. This leds to a rapid cell death if the expression of the Barnase isn't repressed by the repressor of the Biosafety-System.</p> | ||
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| + | [https://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System_S Barnase] | ||
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Revision as of 10:54, 4 October 2013
Biosafety System Lac of Growth
Overview
comming soon... (will only take a couple of hours)
Genetic Approach
Rhamnose promoter
lacI
Naturally the lac operon regulates the catabolism of the disaccharide lactose (4-O-(β-D-Galactopyranosyl)-D-glucopyranose) in E. coli. The operon contains the lactose promoter (plac) and the genes for the catabolism of the Lactose to Glucose and Galactose. Upstream of the lac operator exists the coding sequence for the repressor lacI under the control of a weak promoter.
Compared to the catabolism of the sugars L-Rhamnose or L-arabinose Lactose, as a disaccharide has a higher energy content and is therefore used more preferable. This is a reason, why the basal transcription of this promoter is even more higher. The leakiness of the lac promoter is caused by the fact that the lacY need to be expressed for an efficient Lactose uptake, while in the arabinose system the uptake is regulated separate.
In our Safety-System the lacI (<bbpart>BBa_C0012</bbpart>) is used for the repression of the lactose promoter (plac).
Alanine Racemase
Terminator
Lactose promoter (plac)
Naturally the lac operon regulates the catabolism of the disaccharide lactose (4-O-(β-D-Galactopyranosyl)-D-glucopyranose) in E. coli. The operon consists of a CAP-binding site, the lac promoter, the lac operator and the genes lacZ, lacY and lacA downstream of the promoter. The transcription of the lactose promoter is regulated by the lacI gene, which is found upstream of the operon under the control of a weak promoter. In the absence of lactose the transcription of the genes behind the lactose promoter is blocked caused by the binding of the lacI pressor. While in the presence of Lactose the repressor is released from the operator and the genes can be transcripted. Typically the transcription is enhanced by a high intracellular level of cAMP.
The lactose promoter thereby regulates the transcription of the genes lacZ, lacY and lacA. The lacZ gene encodes for the ß-Galactosidase a enzyme, who breaks down the lactose to glucose and galactose. The ß-Galactosidase catalyses additional the degradation from Lactose to Allolactose. By binding on the lacI repressor, it changes his conformation an is not any more able to bind on the operator sequnece and to block the transcription. As only one enzyme is necessary to gain a substrate of the glycolysis is becomes clear, why the degradation of Lactose is more preferable compared to L-arabinose or L-rhamnose.
To realize a preference of lactose, the transcription of the lactose promoter is not repressed as that strong. This is caused by the fact that the lacY gene, coding for the integral membrane protein lactose permease, is necessary for the lactose uptake and has to be transcripted on a low level.
The last gene of the lac operon, lacA, encodes for a Transacetylase, who acetylizes glycosides that can not be metabolized. The acetylated glycosides are transported outside the cell to avoid the accumulation of lactose.
In our Biosafety-System the lac-promoter is used for the regulation of GFP or the Barnase. As the lac promoter shows a high basal transcription, its might not ideal for the regulation of a toxic gene product, but the the Biosafety-System Lac of growth is ideal for comparison with the other Systems to measure the level of basal transcription under repressed and unrepressed conditions. Besides we improved the leakiness of the lactose promoter by adding a second lacI-binding site 12 nt downstream of the excisting bining site. As this distance corresponds to about one whorl of the double helix, this should allow an additional lacI repressor to bind on the other site of the DNA and tighten the repression of the lactose promoter. Unfortunately the improvement of the so called double lac promoter could not be quantified, because lac of time.
Barnase
Results
References
Agnes Ullmann (2001): Escherichia coli Lactose Operon. In: Encyclopedia of Life Sciences
Stumpp et al.: Ein neues, L-Rhamnose-induzierbares Expressionssystem für Escherichia coli, In: Biospektrum 6. Jahrgang S. 33
Carsten Voss, Dennis Lindau, and Erwin Flaschel, Production of Recombinant RNase Ba and Its Application in Downstream Processing of Plasmid DNA for Pharmaceutical Use, Biotechnology Progress, 22, 2006 p. 737-44.
Danuta E. Mossakowska, Kerstin Nyberg, and Alan R. Fersht, Kinetic Characterization of the Recombinant Ribonuclease from Bacillus amyloliquefaciens (Barnase) and Investigation of Key Residues in Catalysis by Site-Directed Mutagenesis, Biochemistry, 28, 1989, p. 3843 – 3850.
C. J. Paddon, N. Vasantha, and R. W. Hartley, Translation and Processing of Bacillus amyloliquefaciens Extracellular Rnase, Journal of Bacteriology, 171, 1989, p. 1185 – 1187.
- Autoren (Jahr) Titel [Link|Paper Ausgabe: Seiten].
