Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering.
BACKGROUND
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 Escherichia coli as a useful tool for fine tuning gene expression is discussed here.


RESULTS
A degenerated oligonucleotide sequence that encodes consensus sequences for E. coli 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 E. coli 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/OD600 ml to 7606.83 RFU/OD600 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.


CONCLUSIONS
For Escherichia coli, 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.
De Mey
Marjan
M
Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, Ghent, Belgium. marjan.demey@ugent.be
Maertens
Jo
J
Lequeux
Gaspard J
GJ
Soetaert
Wim K
WK
Vandamme
Erick J
EJ
D003001:Cloning, Molecular / Q000379:methods; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015723:Gene Library; D008957:Models, Genetic; D015233:Models, Statistical; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins