Construção de novos promotores funcionais em Escherichia coli por meio de síntese química de DNA randomizada

Abstract

The interest in genetic manipulation tools increase production of heterologous proteins for biotechnological purposes, especially therapeutic proteins, industrial enzymes and significantly increased worldwide. For the production of heterologous proteins choice of expression vector & host system is essential. For the construction of a library of adjustable and promoters functional in E. coli, two-deoxy oligonucleotides degenerate regions were produced by random chemical synthesis of DNA. To obtain the double-stranded promoters, Lac operator sequences were looped and submitted by treatment with Klenow enzyme. For cloning and analysis of the functionality of the novel promoters, the edges of the double-stranded deoxy-oligonucleotide and vector were digested with Bam HI and Hind III and following were purified. Synthetic promoters were then linked to the promoter vector pKK232-8 hunting so stay in proper position for expressing the CAT reporter gene. The recombinant plasmids were then introduced into E. coli DH5α F'Iq and transformants cells were obtained and classified according to the size of the colony on Petri plates containing different concentrations of chloramphenicol in the presence and absence of the inducer IPTG. After analysis of the colonies were chosen 42 recombinant clones, their DNA extracted plamidiais and the sequences of the promoters completely certain. 6 The following recombinant plasmids were reintroduced into E. coli DH5α F'Iq and clones selected on chloramphenicol, with or without IPTG. Of these six clones, 4 were selected to examine the growth in liquid medium with chloramphenicol in the presence and absence of the inducer IPTG. All recombinant clones in E. coli demonstrated ability to grow in liquid medium, 3 of them grew better with IPTG, indicating that their promoters are regulated. The other clone did not have their growth affected by the inductor, confirmed by deficiency following the Lac operator. The results show that the methodology used to generate and clone novel promoters running and new promoters have the potential of being used for the development of new, powerful and regulated expression vectors.