You are on page 1of 56

La Molcula de la Vida

Ascaris megalocephala 2 Cebolla 16 Drosophila 8 cromosomas Hombre 46 Perro 78

Los Acidos Nuclecos


ADN, ARN, ATP, NAD, FAD 1) Una base nitrogenada (purina o pirimidina) 2) Un azcar (pentosa) 3) Una molcula de cido fosfrico

Purinas:

Pirimidinas:

Estructura del ADN


NH
2

terminal 5'

HO O

Adenina A
NH
2

O O P HO O O N

N
O

Citosina C
O N

O P HO O O N N

NH

NH

Guanina G

O CH O P HO O O N
3

NH O

Timina T

OH

terminal 3'

Estructura del ADN


Watson y Crick 1953

LA INFORMACION NECESARIA PARA GENERAR LAS PROTEINAS INCUYENDO LAS ENZIMAS SON CODIFICADAS POR GENES.

Expresin gentica
promotor Secuencia de unin al ribosoma Gene estructural (secuencia codificante) codn de inicio codn de paro terminador

ADN
P RBS

Transcripcin

ARN
RBS Traduccin

Protena

Preservacin y transmisin de la informacin gentica


GEN: Segmento de ADN que contienen la informacin necesaria para formar una protena. Replicacin. Copia del ADN para formar molculas hijas idnticas. EXPRESION Transcripcin. Proceso mediante el cual, el mensaje gentico del ADN es transcrito en forma de ARN mensajero. (ARN polimerasa, ARNm). Seal: promotor Traduccin. Proceso por el cual el mensaje gentico es descifrado en los ribosomas, donde el RNA se utiliza como matriz dirigiendo la secuencia aminocida especfica para la sntesis proteica (ARNm, ARNt, ribosoma) Seal: RBS. Modificacin post-traduccional Eliminacin de exones (eucariotes)

La replicacin es realizada por enzimas

Replicacin del ADN


Fenmeno mediante el cual el ADN se duplica usndose a si mismo como templado.

Replicacin del ADN

Dogma Central

Transcripcin
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.

Transcripcin

Transcripcin

Cdigo Gentico

Nirenberg and Khorana, Premio Nobel in Fisiologia 1968

Traduccin

Tecnologa del ADN recombinante (Ingeniera gentica)

Los genes se pueden:


Aislar y amplificar (obtener mltiples copias) Secuenciar Expresar con intensidad Desactivar Detectar con sensibilidad y especificidad

Herramientas
Enzimas de restriccin (endonucleasas)

Enzimas de modificacin Ligasa ADN polimerasa Nucleasas kinasa, transferasa,etc,etc,etc. Vectores (plsmidos, fagos, csmidos, 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/

Reaccin en Cadena de la Polimerasa PCR


Taq DNA polimerasa

1993 Premio Nobel en Quimica a Kary Mullis

Enzimas de Restriccin

Enzimas de Restriccin

BfuI

BciVI

GTATCC(N)6/5^ GCCNNNN^NGGC A^GATCT CC^NGG GACNN^NNGTC GAAGAC(N)2/6^ ^8/13(N)GAG(N)5CTC(N)13/8^ CCTNAGC-5/-2^ GC^TNAGC C^CNNGG

ER1501/2 ER0071/2 ER0081/2 ER1421/2 ER1431/2 ER1011/2 ER1311/2 ER1181/2 ER0091/2 ER1081 ER0871/2 ER1711 ER1201/2 ER1261/2 ER1401/2 ER0881/2 ER1441/2 ER1451/2 ER0921/2 ER0891 ER1001/2 ER1461/2 ER0111/2 ER0121 ER0131/2/3 ER0781/2 ER0791/2 ER0931/2 ER1151/2 ER1321/2 ER0831/2 ER0701/2 ER1021/2 ER0141/2 ER0151/2/3 ER1741

Enzimas de Restriccin
BoxI BpiI PshAI BbvII

BglI BglI BglII BglII Bme1390I ScrFI

Fermentas Specificity Prototype Enzyme 5'=>3' AarI AarI AasI DrdI AatII Acc65I AdeI AlfI AloI AluI Alw21I Alw26I Alw44I ApaI new BamHI BauI BclI BcnI BcuI BfiI BfmI BfuI AatII KpnI (GGTAC^C) DraIII AlfI AloI AluI HgiAI BsmAI ApaLI ApaI BamHI BsiI BclI CauII SpeI BfiI SfeI BciVI

Catalog # CACCTGC(N)4/8^ GACNNNN^NNGTC GACGT^C G^GTACC CACNNN^GTG ^(10/12)GCA(N)6TGC(12/10)^ AG^CT GWGCW^C GTCTC(N)1/5^ G^TGCAC GGGCC^C G^GATCC CACGAG(-5/-1)^ T^GATCA CC^SGG A^CTAGT ACTGGG(N)5/4^ C^TRYAG GTATCC(N)6/5^ GCCNNNN^NGGC A^GATCT CC^NGG GACNN^NNGTC GAAGAC(N)2/6^ ^8/13(N)GAG(N)5CTC(N)13/8^ CCTNAGC-5/-2^ GC^TNAGC C^CNNGG ER1581/2 ER1721/2 ER0991/2 ER0901/2 ER1231/2 ER1801 ER0011/2 ER0021/2 ER0031/2 ER0041/2 ER1411/2 ER0051/2/3 ER1841 ER0721/2 ER0061/2 ER1251/2 ER1591/2 ER1161/2 ER1501/2 ER0071/2 ER0081/2 ER1421/2 ER1431/2 ER1011/2 ER1311/2 ER1181/2new ER0091/2 ER1081 ER0871/2 ER1711

BplI Bpu10I

BplI Bpu10I EspI SecI

Bpu1102I BseDI BseGI BseJI BseLI BseMI BseMII BseNI BseSI BseXI Bsh1236I Bsh1285I BshNI BshTI Bsp68I Bsp119I Bsp120I Bsp143I Bsp143II Bsp1407I BspLI BspPI BspTI Bst1107I BstXI Bsu15I BsuRI BveI

FokI (GGATG(N)9/13^) GGATG(N)2/0^ BsaBI GATNN^NNATC BsiYI CCNNNNN^NNGG BsrDI BseMII BsrI BseSI BbvI FnuDII McrI HgiCI AgeI NruI AsuII ApaI (GGGCC^C) MboI HaeII Bsp1407I NlaIV BinI AflII SnaI BstXI ClaI HaeIII BspMI GCAATG(N)2/0^ CTCAG(N)10/8^ ACTGG(N)1/-1^ GKGCM^C GCAGC(N)8/12^ CG^CG CGRY^CG G^GYRCC A^CCGGT TCG^CGA TT^CGAA G^GGCCC ^GATC RGCGC^Y T^GTACA GGN^NCC GGATC(N)4/5^ C^TTAAG GTA^TAC CCANNNNN^NTGG AT^CGAT GG^CC ACCTGC(N)4/8^

new

^7/12-13(N)GAAC(N)6TCC(N)12-13/7^ ER1491/2

BglI BglI BglII BglII Bme1390I ScrFI BoxI BpiI BplI Bpu10I Bpu1102I BseDI BseGI BseJI PshAI BbvII BplI Bpu10I EspI SecI

FokI (GGATG(N)9/13^) GGATG(N)2/0^ BsaBI GATNN^NNATC

Ligacin
T4 DNA ligasa

VECTORES O PLASMIDOS Son molculas de AND Con origen de replicacin. Muchas portan genes de resistencia a antibiticos. Pueden transferirse de una clula a otra.

VECTOR DE CLONACION

Vectores

4363 pb

2686 pb

Vectores de expresin
PROMOTORES Inducidos por diversos factores.
Inductores: Calor Fuente de carbono Presencia de algun compuesto inductor

SELECCION

Opern 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

Secuenciacin del ADN


Maxam-Gilbert Sanger

Expresin heterloga

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 betagalactosidase 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.

You might also like