细菌的基因表达课件.ppt

Gene expression of Bacteria Contents Introduction Gene expression and regulation of bacteria Gene cloning and protein expression Types of life Procaryotes Eukaryotes Viruses Three kindom of organisms Two types of bacteria Bacterial shapes Bacterial cell structure Capsules Flagella and Motility Fimbriae and Sex pili Gram-Positive Cell Walls Gram-Negative Cell Walls The Cytoplasmic Matrix 1. DNA, genes and chromosomes Gene clone and expression 1. Gene Cloning This describes the process of copying fragments of DNA which can then be used for many different purposes, such as creating GM crops, or finding a cure for disease. There are two types of gene cloning: in vivo, which involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cells. The other type is in vitro which is using the polymerase chain reaction (PCR) method to create copies of fragments of DNA. 1) Gene cloning: in vivo DNA fragments isolation (flanking sequence) Gene Libraries construction (genomic library, cDNA library) Screen libraries Clone and subclone DNA fragments isolation Mapping Reverse PCR Tail PCR Gene Libraries construction A gene library is a large collection of cloned DNA sequences from a single genome. A genomic library, in theory, would contain at least one copy of every sequence in an organism’s genome. These are used to investigate the structure of a given chromosome, or to clone specific genes. These types of libraries may be prepared from a subset of the entire genome (for example, a single chromosome). The first step in creating a genomic library is to break up, or ‘fractionate’, the genome using physical methods or restriction enzymes. The fragments are then linked to appropriate vectors and cloned in a suitable host cell population. A cDNA library (complementary DNA) contains DNA present in a given cell population which is prepared from the mRNA (messenger RNA) using the enzyme reverse transcriptase. The resulting cDNA re