Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineering
<p>Abstract</p> <p>Background</p> <p>Nowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthet...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2007-06-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/7/34 |
| _version_ | 1818258815006539776 |
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| author | Soetaert Wim K Lequeux Gaspard J Maertens Jo De Mey Marjan Vandamme Erick J |
| author_facet | Soetaert Wim K Lequeux Gaspard J Maertens Jo De Mey Marjan Vandamme Erick J |
| author_sort | Soetaert Wim K |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Nowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthetic promoters of <it>Escherichia coli </it>as a useful tool for fine tuning gene expression is discussed here.</p> <p>Results</p> <p>A degenerated oligonucleotide sequence that encodes consensus sequences for <it>E. coli </it>promoters separated by spacers of random sequences has been designed and synthesized. This 57 bp long sequence contains 24 conserved, 13 semi-conserved (W, R and D) and 20 random nucleotides. This mixture of DNA fragments was cloned into a promoter probing vector (pVIK165). The ligation mixtures were transformed into competent <it>E. coli </it>MA8 and the resulting clones were screened for GFP activity by measuring the relative fluorescence units; some clones produced high fluorescence intensity, others weak fluorescence intensity. The clones cover a range of promoter activities from 21.79 RFU/OD<sub>600 </sub>ml to 7606.83 RFU/OD<sub>600 </sub>ml. 57 promoters were sequenced and used for promoter analysis. The present results conclusively show that the postulates, which link promoter strength to anomalies in the -10 box and/or -35 box, and to the length of the spacer, are not generally valid. However, by applying Partial Least Squares regression, a model describing the promoter strength was built and validated.</p> <p>Conclusion</p> <p>For <it>Escherichia coli</it>, the promoter strength can not been linked to anomalies in the -10 box and/or -35 box, and to the length of the spacer. Also a probabilistic approach to relate the promoter sequence to its strength has some drawbacks. However, by applying Partial Least Squares regression, a good correlation was found between promoter sequence and promoter strength. This PLS model can be a useful tool to rationally design a suitable promoter in order to fine tune gene expression.</p> |
| first_indexed | 2024-12-12T18:05:32Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-12T18:05:32Z |
| publishDate | 2007-06-01 |
| publisher | BMC |
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| series | BMC Biotechnology |
| spelling | doaj.art-a257ceca1868489799d15803475f186d2022-12-22T00:16:31ZengBMCBMC Biotechnology1472-67502007-06-01713410.1186/1472-6750-7-34Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineeringSoetaert Wim KLequeux Gaspard JMaertens JoDe Mey MarjanVandamme Erick J<p>Abstract</p> <p>Background</p> <p>Nowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthetic promoters of <it>Escherichia coli </it>as a useful tool for fine tuning gene expression is discussed here.</p> <p>Results</p> <p>A degenerated oligonucleotide sequence that encodes consensus sequences for <it>E. coli </it>promoters separated by spacers of random sequences has been designed and synthesized. This 57 bp long sequence contains 24 conserved, 13 semi-conserved (W, R and D) and 20 random nucleotides. This mixture of DNA fragments was cloned into a promoter probing vector (pVIK165). The ligation mixtures were transformed into competent <it>E. coli </it>MA8 and the resulting clones were screened for GFP activity by measuring the relative fluorescence units; some clones produced high fluorescence intensity, others weak fluorescence intensity. The clones cover a range of promoter activities from 21.79 RFU/OD<sub>600 </sub>ml to 7606.83 RFU/OD<sub>600 </sub>ml. 57 promoters were sequenced and used for promoter analysis. The present results conclusively show that the postulates, which link promoter strength to anomalies in the -10 box and/or -35 box, and to the length of the spacer, are not generally valid. However, by applying Partial Least Squares regression, a model describing the promoter strength was built and validated.</p> <p>Conclusion</p> <p>For <it>Escherichia coli</it>, the promoter strength can not been linked to anomalies in the -10 box and/or -35 box, and to the length of the spacer. Also a probabilistic approach to relate the promoter sequence to its strength has some drawbacks. However, by applying Partial Least Squares regression, a good correlation was found between promoter sequence and promoter strength. This PLS model can be a useful tool to rationally design a suitable promoter in order to fine tune gene expression.</p>http://www.biomedcentral.com/1472-6750/7/34 |
| spellingShingle | Soetaert Wim K Lequeux Gaspard J Maertens Jo De Mey Marjan Vandamme Erick J Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineering BMC Biotechnology |
| title | Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineering |
| title_full | Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineering |
| title_fullStr | Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineering |
| title_full_unstemmed | Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineering |
| title_short | Construction and model-based analysis of a promoter library for <it>E. coli</it>: an indispensable tool for metabolic engineering |
| title_sort | construction and model based analysis of a promoter library for it e coli it an indispensable tool for metabolic engineering |
| url | http://www.biomedcentral.com/1472-6750/7/34 |
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