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A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli

BACKGROUND: The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have pro...

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Autores principales: Banerjee, Sampali, Kumar, Jitendra, Apte-Deshpande, Anjali, Padmanabhan, Sriram
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882348/
https://www.ncbi.nlm.nih.gov/pubmed/20459760
http://dx.doi.org/10.1186/1475-2859-9-30
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author Banerjee, Sampali
Kumar, Jitendra
Apte-Deshpande, Anjali
Padmanabhan, Sriram
author_facet Banerjee, Sampali
Kumar, Jitendra
Apte-Deshpande, Anjali
Padmanabhan, Sriram
author_sort Banerjee, Sampali
collection PubMed
description BACKGROUND: The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc. RESULTS: We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts. CONCLUSIONS: This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.
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spelling pubmed-28823482010-06-09 A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli Banerjee, Sampali Kumar, Jitendra Apte-Deshpande, Anjali Padmanabhan, Sriram Microb Cell Fact Research BACKGROUND: The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc. RESULTS: We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts. CONCLUSIONS: This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein. BioMed Central 2010-05-11 /pmc/articles/PMC2882348/ /pubmed/20459760 http://dx.doi.org/10.1186/1475-2859-9-30 Text en Copyright ©2010 Banerjee et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Banerjee, Sampali
Kumar, Jitendra
Apte-Deshpande, Anjali
Padmanabhan, Sriram
A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli
title A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli
title_full A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli
title_fullStr A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli
title_full_unstemmed A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli
title_short A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli
title_sort novel prokaryotic vector for identification and selection of recombinants: direct use of the vector for expression studies in e. coli
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882348/
https://www.ncbi.nlm.nih.gov/pubmed/20459760
http://dx.doi.org/10.1186/1475-2859-9-30
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