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La Mol cula de la Vida

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La Mol cula de la Vida

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    1. La Molécula de la Vida

    5. Estructura del ADN

    9. Estructura del ADN

    15. Dogma Central

    22. 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

    24. AISLAMIENTO DE UN GEN

    36. SELECCION

    41. DIVERSA

    44. ENZIMAS

    46. INGENIERIA DE PROTEINAS

    47. MUTAGENESIS SITIO DIRIGIDA

    51. Recombinant Chymosin

    52. PROTEINAS DE LA LECHE

    53. Chymosin Reaction

    54. 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.

    55. 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.

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