Antibiotic-free selection in E. coli: new considerations for optimal design and improved production
Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Peubez, Isabelle [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2010 |
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| Schlagwörter: |
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| Anmerkung: |
© Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
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| Übergeordnetes Werk: |
Enthalten in: Microbial cell factories - London : Biomed Central, 2002, 9(2010), 1 vom: 07. Sept. |
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| Übergeordnetes Werk: |
volume:9 ; year:2010 ; number:1 ; day:07 ; month:09 |
| Links: |
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| DOI / URN: |
10.1186/1475-2859-9-65 |
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SPR028558278 |
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| 520 | |a Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. | ||
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| 700 | 1 | |a Chaudet, Nicolas |4 aut | |
| 700 | 1 | |a Mignon, Charlotte |4 aut | |
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| 700 | 1 | |a Speck, Denis |4 aut | |
| 700 | 1 | |a Sodoyer, Régis |4 aut | |
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10.1186/1475-2859-9-65 doi (DE-627)SPR028558278 (SPR)1475-2859-9-65-e DE-627 ger DE-627 rakwb eng Peubez, Isabelle verfasserin aut Antibiotic-free selection in E. coli: new considerations for optimal design and improved production 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. Fermentation (dpeaa)DE-He213 Essential Gene (dpeaa)DE-He213 Antibiotic Resistance Gene (dpeaa)DE-He213 Kanamycin Resistance Gene (dpeaa)DE-He213 Plasmid Loss (dpeaa)DE-He213 Chaudet, Nicolas aut Mignon, Charlotte aut Hild, Géraldine aut Husson, Stéphanie aut Courtois, Virginie aut De Luca, Karelle aut Speck, Denis aut Sodoyer, Régis aut Enthalten in Microbial cell factories London : Biomed Central, 2002 9(2010), 1 vom: 07. Sept. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:9 year:2010 number:1 day:07 month:09 https://dx.doi.org/10.1186/1475-2859-9-65 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2010 1 07 09 |
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10.1186/1475-2859-9-65 doi (DE-627)SPR028558278 (SPR)1475-2859-9-65-e DE-627 ger DE-627 rakwb eng Peubez, Isabelle verfasserin aut Antibiotic-free selection in E. coli: new considerations for optimal design and improved production 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. Fermentation (dpeaa)DE-He213 Essential Gene (dpeaa)DE-He213 Antibiotic Resistance Gene (dpeaa)DE-He213 Kanamycin Resistance Gene (dpeaa)DE-He213 Plasmid Loss (dpeaa)DE-He213 Chaudet, Nicolas aut Mignon, Charlotte aut Hild, Géraldine aut Husson, Stéphanie aut Courtois, Virginie aut De Luca, Karelle aut Speck, Denis aut Sodoyer, Régis aut Enthalten in Microbial cell factories London : Biomed Central, 2002 9(2010), 1 vom: 07. Sept. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:9 year:2010 number:1 day:07 month:09 https://dx.doi.org/10.1186/1475-2859-9-65 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2010 1 07 09 |
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10.1186/1475-2859-9-65 doi (DE-627)SPR028558278 (SPR)1475-2859-9-65-e DE-627 ger DE-627 rakwb eng Peubez, Isabelle verfasserin aut Antibiotic-free selection in E. coli: new considerations for optimal design and improved production 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. Fermentation (dpeaa)DE-He213 Essential Gene (dpeaa)DE-He213 Antibiotic Resistance Gene (dpeaa)DE-He213 Kanamycin Resistance Gene (dpeaa)DE-He213 Plasmid Loss (dpeaa)DE-He213 Chaudet, Nicolas aut Mignon, Charlotte aut Hild, Géraldine aut Husson, Stéphanie aut Courtois, Virginie aut De Luca, Karelle aut Speck, Denis aut Sodoyer, Régis aut Enthalten in Microbial cell factories London : Biomed Central, 2002 9(2010), 1 vom: 07. Sept. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:9 year:2010 number:1 day:07 month:09 https://dx.doi.org/10.1186/1475-2859-9-65 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2010 1 07 09 |
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10.1186/1475-2859-9-65 doi (DE-627)SPR028558278 (SPR)1475-2859-9-65-e DE-627 ger DE-627 rakwb eng Peubez, Isabelle verfasserin aut Antibiotic-free selection in E. coli: new considerations for optimal design and improved production 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. Fermentation (dpeaa)DE-He213 Essential Gene (dpeaa)DE-He213 Antibiotic Resistance Gene (dpeaa)DE-He213 Kanamycin Resistance Gene (dpeaa)DE-He213 Plasmid Loss (dpeaa)DE-He213 Chaudet, Nicolas aut Mignon, Charlotte aut Hild, Géraldine aut Husson, Stéphanie aut Courtois, Virginie aut De Luca, Karelle aut Speck, Denis aut Sodoyer, Régis aut Enthalten in Microbial cell factories London : Biomed Central, 2002 9(2010), 1 vom: 07. Sept. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:9 year:2010 number:1 day:07 month:09 https://dx.doi.org/10.1186/1475-2859-9-65 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2010 1 07 09 |
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10.1186/1475-2859-9-65 doi (DE-627)SPR028558278 (SPR)1475-2859-9-65-e DE-627 ger DE-627 rakwb eng Peubez, Isabelle verfasserin aut Antibiotic-free selection in E. coli: new considerations for optimal design and improved production 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. Fermentation (dpeaa)DE-He213 Essential Gene (dpeaa)DE-He213 Antibiotic Resistance Gene (dpeaa)DE-He213 Kanamycin Resistance Gene (dpeaa)DE-He213 Plasmid Loss (dpeaa)DE-He213 Chaudet, Nicolas aut Mignon, Charlotte aut Hild, Géraldine aut Husson, Stéphanie aut Courtois, Virginie aut De Luca, Karelle aut Speck, Denis aut Sodoyer, Régis aut Enthalten in Microbial cell factories London : Biomed Central, 2002 9(2010), 1 vom: 07. Sept. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:9 year:2010 number:1 day:07 month:09 https://dx.doi.org/10.1186/1475-2859-9-65 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 9 2010 1 07 09 |
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Antibiotic-free selection in E. coli: new considerations for optimal design and improved production Fermentation (dpeaa)DE-He213 Essential Gene (dpeaa)DE-He213 Antibiotic Resistance Gene (dpeaa)DE-He213 Kanamycin Resistance Gene (dpeaa)DE-He213 Plasmid Loss (dpeaa)DE-He213 |
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antibiotic-free selection in e. coli: new considerations for optimal design and improved production |
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Antibiotic-free selection in E. coli: new considerations for optimal design and improved production |
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Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
| abstractGer |
Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
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Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery. © Peubez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains. Results In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process. Conclusions We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Fermentation</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Essential Gene</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Antibiotic Resistance Gene</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Kanamycin Resistance Gene</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Plasmid Loss</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Chaudet, Nicolas</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Mignon, Charlotte</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Hild, Géraldine</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Husson, Stéphanie</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Courtois, Virginie</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">De Luca, Karelle</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Speck, Denis</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Sodoyer, Régis</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">Microbial cell factories</subfield><subfield code="d">London : Biomed Central, 2002</subfield><subfield code="g">9(2010), 1 vom: 07. 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