In the nature, lots of organism apply different kinds of lights such as flourscence or luminoscence for some purposes such as defence or hunting. Firefly luciferase and Bacterial luciferase are the most famous ones which have been used to design Biosensors or Bioreporters in recent decades. Their applications are so extensive from detecting pollutions in the environment to medical and treatment usages. To design Copper Bioreporter, copper resistance promoter from COP operon which plays an important role in Pseudomonas syringae and pGL3 plasmid which has luciferase gene were utilized. To achieve that target, sequences of promoter were synthesized and inserted to pCR2.1 vector, then suitable primers with considering restriction sites were designed to get high concentration of DNA. After digestion of pGL3 and interested gene by Nhe I and Sac I enzymes, ligation was performed, and then recombinat plasmids were transferred to E. coli BL-21 as a host cell. Finallay, luciferase assay of designed bioreporter was performed by Luminometer in presence of different concentration of CuSO4. The result was maginificant that confirmed design of Copper Bioreporter.

Orientador: Sergio Roberto Peres Line / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-13T02:01:37Z (GMT). No. of bitstreams: 1 Almeida_CarolinaVieirade_M.pdf: 673929 bytes, checksum: de7a7d8f324324fae24215bb7eb7ed66 (MD5) Previous issue date: 2009 / Resumo: O gene PAX9 pertence a uma família de fatores de transcrição denominada família Pax. Esse gene é expresso em tecidos embrionários como somitos, bolsa endodérmica da faringe, brotos distais dos membros e mesênquima derivado da crista neural. Alguns polimorfismos na região promotora desse gene em humanos vêm sendo associados a formas de agenesias não-sindrômicas autossômicas dominantes. No presente trabalho, nós verificamos a expressão gênica e a influência de duas seqüências da região promotora na transcrição do gene PAX9 em ensaios in vitro com células de tecido embrionário de ratos de 13.5 dias obtidas a partir de seus membros, face e regiões do mesencéfalo e romboencéfalo. Os fragmentos da região promotora foram clonados em plasmídios repórteres e transfectados nos três diferentes tecidos embrionários. Nossos resultados das análises de RT-PCR demonstraram que em culturas in vitro, as células dos membros e do SNC expressam o gene PAX9, porém, aquelas obtidas a partir da face não o expressaram. Além disso, os resultados dos ensaios de luciferase mostraram que a atividade dessa nos vetores construídos são mais fracas do que do vetor pGL3-Basic sozinho, sugerindo que essas regiões não são suficientes para guiar a transcrição gênica. / Abstract: PAX9 belongs to a transcriptional factor genes family named Pax. This gene is expressed in embryonic tissues like somites, pharyngeal pouch endoderm, distal limb buds and neural crest-derived mesenchyme. Some polymorphisms in the upstream promoter region of the human PAX9 have been associated with human non-syndromic forms of autossomal dominant agenesis. In the present study we verified the in vitro expression of this gene and the influence of two promoter sequences in the transcription of PAX9 gene, in embryo tissues obtained from digits, face and midbrain and hindbrain regions. These fragments were cloned on reporter plasmid and were transfected into the different cells cultures. Our RT-PCR results showed that in vitro E13.5 limb bud and CNS cells express PAX9, but not in derived facial region cells. Moreover, the luciferase activity of the Pax9 promoter vectors were weaker than pGL3-Basic alone, suggesting that the PAX9 sequences of promoter region are not sufficient to drive PAX9 gene transcription. / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental