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Pesquisa do Estado do Amazonas CAPES - Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior CNPq - Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico 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 ??70 and ??38 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. Introdu????o: Estrat??gias gen??ticas t??m sido utilizadas por d??cadas, melhorando os bioprocessos bacterianos visando a simplifica????o e custo da produ????o de prote??nas recombinantes. Tais estrat??gias incluem o desenho de vetores de express??o eficientes e a melhoria das linhagens bacteriana, neste contexto, os promotores s??o elementos de um vetor de express??o que determinam for??a e dura????o da transcri????o, e consequentemente, o rendimento da prote??na heter??loga a ser produzida. Os promotores bacterianos s??o reconhecidos pelos fatores sigmas, que s??o prote??nas que se acoplam a RNA polimerase para realizar a transcri????o. Os fatores sigmas se ligam aos promotores que respondem a diversas condi????es ambientais que a c??lula enfrenta, dentre eles os mais conhecidos s??o os fatores sigmas 70 e 38, que reconhecem promotores de fase exponencial e estacion??ria da c??lula. Objetivo: Design, clonagem e an??lise de promotores sint??ticos reconhecidos pelos fatores sigmas ??70 e ??38 em Escherichia coli. Material e M??todos: A partir do conhecimento dos elementos consensuais dos promotores, reconhecidos pelos fatores sigma 70 e 38 de Escherichia coli, planejou-se um oligonucleot??deo onde mantinham-se as regi??es consensuais mais importantes dos dois tipos de promotores e foram degeneradas as sequ??ncias de regi??es adjacentes que poderiam influenciar na for??a e especificidade dos promotores. Um oligonucleot??deo parcialmente degenerado de 95 nucleot??deos foi sintetizado por s??ntese qu??mica, amplificado com um par de primers localizados em suas bordas. O amplicon resultante foi digerido com as enzimas de restri????o SpeI e NdeI, purificado por eletroforese em gel de agarose 2%, eluido e clonado entre os s??tios de SpeI e NdeI do plasm??deo pCDM, dessa forma utilizado como um vetor ca??a promotor que cont??m o gene da GFP como rep??rter. As col??nias recombinantes foram escolhidas de acordo com a intensidade de produ????o da GFP, inicialmente a olho nu, posteriormente por fluorimetria para quantificar a produ????o. Os promotores sint??ticos dos clones escolhidos nessa etapa foram sequenciados e analisados. Resultados e Discuss??o: Produziu-se em E. coli, uma biblioteca com 1.741.824 possibilidades de sequ??ncias constru??das por s??ntese qu??mica. A partir de uma clonagem, foram obtidos cerca de 1200 clones recombinantes contendo diferentes promotores, em uma triagem inicial foram escolhidos 66 clones produtores de GFP, e ap??s a confirma????o de conterem promotores sint??ticos foi feita a an??lise da express??o de GFP em meio l??quido. Foram eleitos 16 clones: 5 que expressam baixos n??veis de GFP, 5 n??veis m??dios e 6 altos n??veis; sendo alguns deles capazes de atuar na fase exponencial de crescimento, outros na fase estacion??ria e alguns em ambas as fases. Os novos promotores dos 16 clones escolhidos foram sequenciados e analisados para identificar as regi??es consenso ??? 10, -10 estendidas e -35. Analisou-se tamb??m as regi??es n??o consensuais que foram degeneradas, bem como a intensidade de express??o de GFP. Alguns promotores foram modificados no espa??amento entre a posi????o -10 e a -35 e em um dos clones foimodificada a regi??o -35. Os clones J8, J13 e J14 produziram altos n??veis de GFP com mais de 900.000 unidades de fluoresc??ncia, e os clones J1, J2 e J3 foram os que produziram menos GFP de todos os clones analisados. Por essa estrat??gia foi poss??vel obter promotores de diferentes for??as e que se expressam em diferentes fases do crescimento celular, sendo que alguns apresentam n??veis de express??o surpreendentemente altos com poss??veis aplica????es na express??o g??nica para fins industriais.