DEEP WORK

RECOMBINEERING INCREASES THE SPEED OF GENE CLONINGAssembling new DNA vectors with different genes of interest can become difficult when the gene is long, since it can be hard to identify unique restriction enzymes compatible with a polylinker that do not cut within the gene. Large recombinant DNA vectors can be created using homologous recombination, a process called recombineering (Fig. 3.25). ..

FEATURES OF EXPRESSION VECTORSBecause foreign protein can be toxic to E. coli, especially if made in large amounts, the promoter used to express the foreign gene is critical.  critical 대단히 중요한 If too much foreign protein is made, the host cell may die. To control protein production, expression vectors have promoters with on/off switches; therefore, the host cell is allowed to grow and then after..

EUKARYOTIC EXPRESSION LIBRARIESIn expression libraries, the vector has sequences required for transcription and translation of the insert. This means that the insert DNA is expressed as RNA and then translated into a protein. An expression library, in essence, generates a protein from every cloned insert, whether it is a real gene or not.  When eukaryotic DNA is studied, expression libraries are..

SCREENING THE LIBRARY OF GENES BY HYBRIDIZATIONOnce the library is assembled, researchers often want to identify a particular gene or segment of DNA within the library. Sometimes the gene of interest is similar to one from another organism. Sometimes the gene of interest contains a particular sequence. For example, many enzymes use ATP to provide energy. Enzymes that bind ATP share a common sign..

GETTING CLONED GENES INTO BACTERIA BY TRANSFORMATIONOnce the gene of interest is cloned into a vector, the construct can be put back into a bacterial cell through a process called transformation (Fig. 3.18) (see Box 3.2).FIGURE 3.18 Transformation Bacterial cells are able to take up DNA such as recombinant plasmids by incubation with metal ions such as Ca++ on ice. This destabilizes the bacteria..