挑菌（Pick the bacteria）.docVIP

挑菌（Pick the bacteria） Blue spot screening experiment principle: blue spot screening, blue spot screening is a genetically engineered commonly used recombinant bacteria screening methods. The beta galactosidase produced by wild-type Escherichia coli can cut the colorless compounds X-gal (5-, bromide, -4-, chloride, -3-, indole, beta, -D-, galactose) into galactose and dark blue, 5-, bromine, -4-, indigo. Colored substances can cause color changes in cultures of colonies, and color change is the most intuitive and effective way to identify and screen colonies. White spot screening is a method of recombinant screening: the selection of recombinant plasmids according to the genetic characteristics of vectors, such as alpha complementation, antibiotic genes, etc.. Many vectors currently in use contain a short segment of Escherichia coli DNA, which has regulatory sequences for the beta galactosidase gene (lacZ) and coding information for the first 146 amino acids. In the encoding region into a multiple cloning site (MCS), it does not disrupt the reading frame, but can make a few amino acids into the beta galactosidase N-terminal and does not affect the function of the host cell carrier for encoding beta galactosidase C flanking sequence. Therefore, host and plasmid encoded fragments do not have enzyme activity, but when they exist together, proteins with enzymatic activity can be formed. Thus, the lacZ gene is complementary to a plasmid that lacks a host cell near the manipulated gene segment and a plasmid with a complete proximal control gene segment, known as alpha complementation. LacZ+ bacteria, produced by alpha complementation, are susceptible to recognition by the presence of the inducer IPTG, which produces blue colonies when the chromogenic substrate X-Gal is present. However, when exogenous DNA is inserted into the polyclonal site of the plasmid, it almost inevitably leads to an amino - terminal fragment with no alpha complementary ability, allowing bacteria wit