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La Molécula de la Vida Ascaris megalocephala 2 Cebolla 16 Drosophila 8 cromosomas Hombre 46 Perro 78 Los Acidos Nucleícos ADN, ARN, ATP, NAD, FAD 1) Una base nitrogenada (purina o pirimidina) 2) Un azúcar (pentosa) 3) Una molécula de ácido fosfórico Purinas: Pirimidinas: Estructura del ADN NH terminal 5' N HO 2 N N Adenina A N O NH N O O P 2 O N O Citosina C O HO O N NH O O P N O NH N 2 Guanina G O HO O CH 3 NH O O P N O O Timina T O HO OH terminal 3' Estructura del ADN Watson y Crick 1953 LA INFORMACION NECESARIA PARA GENERAR LAS PROTEINAS INCUYENDO LAS ENZIMAS SON CODIFICADAS POR GENES. Expresión genética promotor Secuencia de unión al ribosoma Gene estructural (secuencia codificante) codón de inicio codón de paro ADN P RBS Transcripción ARN RBS Traducción Proteína terminador Preservación y transmisión de la información genética • • • • • • • GEN: Segmento de ADN que contienen la información necesaria para formar una proteína. Replicación. Copia del ADN para formar moléculas hijas idénticas. EXPRESION Transcripción. Proceso mediante el cual, el mensaje genético del ADN es transcrito en forma de ARN mensajero. (ARN polimerasa, ARNm). Señal: promotor Traducción. Proceso por el cual el mensaje genético es descifrado en los ribosomas, donde el RNA se utiliza como matriz dirigiendo la secuencia aminoácida específica para la síntesis proteica (ARNm, ARNt, ribosoma) Señal: RBS. Modificación post-traduccional Eliminación de exones (eucariotes) La replicación es realizada por enzimas Replicación del ADN Fenómeno mediante el cual el ADN se duplica usándose a si mismo como templado. Replicación del ADN Dogma Central Transcripción •Synthesis of RNA from a DNA Template. •Requires DNA-dependent RNA polymerase plus the four nucleotides (ATP, GTP. CTP and UTP). •Synthesis begins at a the initiation site on DNA. •The template strand is read 3' to 5' and the mRNA is synthesized 5' to 3‘. Transcripción Transcripción Código Genético Nirenberg and Khorana, Premio Nobel in Fisiologia 1968 Traducción Tecnología del ADN recombinante (Ingeniería genética) Los genes se pueden: Aislar y amplificar (obtener múltiples copias) Secuenciar Expresar con intensidad Desactivar Detectar con sensibilidad y especificidad Herramientas Enzimas de restricción (endonucleasas) Enzimas de modificación Ligasa ADN polimerasa Nucleasas kinasa, transferasa,etc,etc,etc. Vectores (plásmidos, fagos, cósmidos, etc) AISLAMIENTO DE UN GEN Kary Mullis in Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents and a source of heat. The DNA sample that one wishes to copy can be pure, or it can be a minute part of an extremely complex mixture of biological materials. The DNA may come from a hospital tissue specimen, from a single human hair, from a drop of dried blood at the scene of a crime, from the tissues of a mummified brain or from a 40,000-year-old wooly mammoth frozen in a glacier." http://nobelprize.org/chemistry/educational/pcr/ Reacción en Cadena de la Polimerasa “PCR” Taq DNA polimerasa 1993 Premio Nobel en Quimica a Kary Mullis Enzimas de Restricción Enzimas de Restricción BfuI BciVI GTATCC(N)6/5^ ER1501/2 BglI BglI GCCNNNN^NGGC ER0071/2 BglII BglII A^GATCT ER0081/2 Bme1390I ScrFI CC^NGG ER1421/2 BoxI PshAI GACNN^NNGTC ER1431/2 BpiI BbvII GAAGAC(N)2/6^ ER1011/2 BplI BplI ^8/13(N)GAG(N)5CTC(N)13/8^ ER1311/2 Bpu10I Bpu10I CCTNAGC-5/-2^ ER1181/2 Enzimas de Restricción Fermentas Specificity Prototype Enzyme 5'=>3' new new Catalog # Bpu1102I EspI GC^TNAGC ER0091/2 AarI AarI CACCTGC(N)4/8^ ER1581/2 BseDI SecI C^CNNGG ER1081 AasI DrdI GACNNNN^NNGTC ER1721/2 BseGI FokI (GGATG(N)9/13^) GGATG(N)2/0^ ER0871/2 AatII AatII GACGT^C ER0991/2 BseJI BsaBI GATNN^NNATC ER1711 BsiYI CCNNNNN^NNGG ER1201/2 Acc65I KpnI (GGTAC^C) G^GTACC ER0901/2 BseLI AdeI DraIII CACNNN^GTG ER1231/2 BseMI BsrDI GCAATG(N)2/0^ ER1261/2 AlfI AlfI ^(10/12)GCA(N)6TGC(12/10)^ ER1801 BseMII BseMII CTCAG(N)10/8^ ER1401/2 BsrI ACTGG(N)1/-1^ ER0881/2 AloI AloI ^7/12-13(N)GAAC(N)6TCC(N)12-13/7^ ER1491/2 BseNI AluI AluI AG^CT ER0011/2 BseSI BseSI GKGCM^C ER1441/2 Alw21I HgiAI GWGCW^C ER0021/2 BseXI BbvI GCAGC(N)8/12^ ER1451/2 FnuDII CG^CG ER0921/2 Alw26I BsmAI GTCTC(N)1/5^ ER0031/2 Bsh1236I Alw44I ApaLI G^TGCAC ER0041/2 Bsh1285I McrI CGRY^CG ER0891 ApaI ApaI GGGCC^C ER1411/2 BshNI HgiCI G^GYRCC ER1001/2 BamHI BamHI G^GATCC ER0051/2/3 BshTI AgeI A^CCGGT ER1461/2 BauI BsiI CACGAG(-5/-1)^ ER1841 Bsp68I NruI TCG^CGA ER0111/2 AsuII TT^CGAA ER0121 BclI BclI T^GATCA ER0721/2 Bsp119I BcnI CauII CC^SGG ER0061/2 Bsp120I ApaI (GGGCC^C) G^GGCCC ER0131/2/3 BcuI SpeI A^CTAGT ER1251/2 Bsp143I MboI ^GATC ER0781/2 HaeII RGCGC^Y ER0791/2 BfiI BfiI ACTGGG(N)5/4^ ER1591/2 Bsp143II BfmI SfeI C^TRYAG ER1161/2 Bsp1407I Bsp1407I T^GTACA ER0931/2 BfuI BciVI GTATCC(N)6/5^ ER1501/2 BspLI NlaIV GGN^NCC ER1151/2 BinI GGATC(N)4/5^ ER1321/2 BglI BglI GCCNNNN^NGGC ER0071/2 BspPI BglII BglII A^GATCT ER0081/2 BspTI AflII C^TTAAG ER0831/2 Bme1390I ScrFI CC^NGG ER1421/2 Bst1107I SnaI GTA^TAC ER0701/2 BoxI PshAI GACNN^NNGTC ER1431/2 BstXI BstXI CCANNNNN^NTGG ER1021/2 BpiI BbvII GAAGAC(N)2/6^ ER1011/2 Bsu15I ClaI AT^CGAT ER0141/2 BsuRI HaeIII GG^CC ER0151/2/3 BveI BspMI ACCTGC(N)4/8^ ER1741 BplI BplI ^8/13(N)GAG(N)5CTC(N)13/8^ Bpu10I Bpu10I CCTNAGC-5/-2^ ER1311/2 ER1181/2new Bpu1102I EspI GC^TNAGC ER0091/2 BseDI SecI C^CNNGG ER1081 BseGI FokI (GGATG(N)9/13^) GGATG(N)2/0^ ER0871/2 BseJI BsaBI ER1711 GATNN^NNATC Ligación T4 DNA ligasa VECTORES O PLASMIDOS Son moléculas de AND Con origen de replicación. Muchas portan genes de resistencia a antibióticos. Pueden transferirse de una célula a otra. VECTOR DE CLONACION Vectores 4363 pb 2686 pb Vectores de expresión PROMOTORES Inducidos por diversos factores. Inductores: •Calor •Fuente de carbono •Presencia de algun compuesto inductor SELECCION Operón lac CLONACION DE UN GEN TRANSFORMACION DEL PLASMIDO ANALISIS DE LAS CLONAS DIVERSA Aisla enzimas de fuentes naturales Obtiene muestras de diferentes regiones del mundo. Aisla el ADN (metagenoma) Construye bibliotecas genomicas Caracteriza con equipos roboticos miles de clonas a la vez, tanto sus capacidades cataliticas como sus propiedades cineticas Secuenciación del ADN •Maxam-Gilbert •Sanger Expresión heteróloga ENZIMAS PRODUCCION DE ENZIMAS RECOMBINANTES A NIVEL INDUSTRIAL (FERMENTACIONES) INGENIERIA DE PROTEINAS MUTAGENESIS SITIO DIRIGIDA MUTAGENESIS POR PCR COMBINATORIO Recombinant Chymosin PROTEINAS DE LA LECHE Chymosin Reaction Recombinant Chymosin Production The problems outlined above for chymosin and its microbial substitutes can be alleviated by cloning the bovine gene into a suitable production strain and producing the enzyme by fermentation. The most important decisions to be made were the choice of host/vector system. There are several possibilities for aproduction host: Eschrichia coli. E. coli is the favourite organism of molecular biologists and is most frequently used in gene cloning experiments. The problem with E. coli, however, is that recombinant proteins are frequently synthesised as intracellular inclusion bodies, increasing process costs cosiderably. Another issue with E. coli is that it is not generally recognised as safe for human consumption. Bacillus sp. Bacillus species are non-pathogenic and are used industrially to produce several enzymes used in food processing such as amylases. B. licheniformis could produce the chymosin, but the signal sequences did not allow the secretion of the enzyme into the medium. by addition of benzoic acid and the chymosin is isolated by filtration. Recombinant Chymosin Production Lactococcus lactis. This host was chosen as it is already used in starter cultures, but production levels were found to be very low. Saccharomyces cerevisiae. Difficulties were experienced in achieving high secretion rates in yeast. Kluveromyces lactis. K. lactis is used for the production of dairy grade beta-galactosidase and its fermentation properties are well understood. It was found that the chtmosin could be produced in this host and good levels of secretion into the medium were achieved. The chymosin gene was inserted into the K. lactis chromosome and the yeast is grown by fed-batch fermentation. After fermentation, the yeast is killed by addition of benzoic acid and the chymosin is isolated by filtration.
