Promotores que são reconhecidos por diferentes fatores sigma em Escherichia coli

Abstract

Introduction: Genetic strategies have been used for decades, improving bacterial bioprocesses aimed at simplifying and costing the production of recombinant proteins. Such strategies include the design of efficient expression vectors and the improvement of bacterial strains, in this context, the promoters are elements of an expression vector that determine the strength and duration of the transcription, and consequently, the yield of the hertoiseprotein to be produced. Bacterial promoters are recognized by sigmas factors, which are proteins that engage rna polymerase to perform transcription. Sigma s factors are connected to the promoters that respond to various environmental conditions that the cell faces, among them the best known are the sigmas factors 70 and 38, which recognize exponential and stationary phase promoters of the cell. Goal: Design, cloning and analysis of synthetic promoters recognized by sigmas factors σ70 and σ38 in Escherichia coli. Material and Methods: From the knowledge of the consensual elements of the promoters, recognized by the sigma factors 70 and 38 of Escherichia coli, an oligonucleotide was planned where the most important consensual regions of the two types of promoters were maintained and the sequences of adjacent regions that could influence the strength and specificity of the promoters were degenerated. A partially degenerated oligonucleotide of 95 nucleotides was synthesized by chemical synthesis, amplified with a pair of primers located on its edges. The resulting amplicon was digested with the restriction enzymes SpeI and NdeI, purified by electrophoresis in 2% agarose gel, eluided and cloned between the SpeI and NdeI sites of the pCDM plasmid, thus used as a hunting vector that contains the GFP gene as a reporter. The recombinant colonies were chosen according to the production intensity of PFM, initially with the naked eye, later by fluorimetry to quantify the production. The synthetic promoters of the clones chosen at this stage were sequenced and analyzed. Results and Discussion: A library with 1,741,824 sequence possibilities constructed by chemical synthesis was produced in E. coli. From a cloning, about 1200 recombinant clones containing different promoters were obtained, at an initial screening 66 gfp-producing clones were chosen, and after confirmation of containing synthetic promoters, the expression of PFM was performed in liquid medium. 16 clones were elected: 5 that express low levels of PFM, 5 medium levels and 6 high levels; some of them are capable of acting in the exponential phase of growth, others in the stationary phase and some in both phases. The new promoters of the 16 clones chosen were sequenced and analyzed to identify the consensus regions – 10, -10 extended and -35. We also analyzed the non-consensual regions that were degenerated, as well as the intensity of PFM expression. Some promoters were modified in the spacing between position -10 and -35 and in one of the clones the region -35 was modified. Clones J8, J13 and J14 produced high levels of PFM with more than 900,000 fluorescence units, and clones J1, J2 and J3 produced less PFM of all clones analyzed. Through this strategy it was possible to obtain promoters of different forces and express themselves in different phases of cell growth, and some have surprisingly high levels of expression with possible applications in gene expression for industrial purposes.