Phosphatase

this is very good notes for r-DNA technology.

1. Phosphatases  catalyze the hydrolysis of a phosphomonoester , removing a  phosphatemoiety  from the substrate. Water is split in the reaction, with the -OH group attaching to the phosphate ion, and the H+ protonating the hydroxyl group of the other product This reaction is not reversible This shows totally opposite activity from enzyme like kinases & phaspharylase that add a phosphate grope to their substrate. PHOSPHATASE

2. REACTION

3. TYPES OF PHASPHATASE On the basis of their activity there are two types of phosphatase ACID PHOSPHATASE ALKALINE PHOSPHATASE In both forms the alkaline phosphate are most common special class of phophatase that remove a phosphate group from protein called phasphoprotein phosphatase

4. ACID PHOSPHATASE It shows is optimal activity at Ph between 3 to 6 range Acid phosphatase is a ubiquitous  lysosomal  enzyme that  hydrolyses  organic phosphates at an acid pH. Although the postpuberteral prostatic  epithelial  cell contains a uniquely high concentration of acid phosphatase , cellular components of bone, spleen, kidney, liver, intestine, and blood also contain this enzyme.

5. ALKALINE PHOSPHATASE Alkaline phosphatase or basic phosphatase is a homodimeric protein enzyme of 86 kilodaltons . Each monomer contains five cysteine residues, two zinc atoms, and one magnesium atom crucial to its catalytic function, and it is optimally active at alkaline pH environments. They shows their optimal activity at pH of about 10 In humans,  alkaline phosphatase  is  present  in all tissues throughout the entire body, but is particularly concentrated in the liver, bile duct, kidney, bone, intestinal mucosa and placenta .

6. Calf-intestinal alkaline phosphatase  ( CIAP / CIP ) Calf-intestinal alkaline phosphatase  ( CIAP / CIP ) is a type of alkaline phosphatase that catalyzes the removal of phosphate groups from the 5' end of DNA strands. This enzyme is frequently used in DNA sub-cloning , as DNA fragments that lack the 5' phosphate groups cannot  ligate .  This prevents recircularization of linearized  vector DNA and improves the yield of vector containing the appropriate insert.

7. Calf-intestinal alkaline phosphatase  ( CIAP / CIP )

8. Transformation  results in the  genetic  alteration of the recipient cell. Exogenous DNA is taken up into the recipient cell from its surroundings through the cell membrane (s).  Transformation occurs  naturally in some species of bacteria, but it can also be affected by artificial means in other cells GENETIC TRANSFORMATION

9. Transformation is the process by which an organism acquires exogenous DNA. Transformation can occur in two ways: natural transformation and artificial transformation. Natural transformation describes the uptake and incorporation of naked DNA from the cell’s natural environment. Artificial transformation encompasses a wide array of methods for inducing uptake of exogenous DNA. In cloning protocols, artificial transformation is used to introduce recombinant DNA into host bacteria ( E. coli ). The most common method of artificial transformation of bacteria involves use of divalent cations (e.g., calcium chloride) to increase the permeability of the bacterium’s membrane, making them chemically competent, thereby increasing the likelihood of DNA acquisition. Another artificial method of transformation is electroporation , in which cells are shocked with an electric current, to create holes in the bacterial membrane. With a newly-compromised cell membrane, the transforming DNA is free to pass into the cytosol of the bacterium. Regardless of which method of tranformation is used, outgrowth of bacteria following transformation allows repair of the bacterial surface and selection of recombinant cells if the newly acquired DNA conveys antibiotic resistance to the transformed cells .

10. ELECTROPORATION Electroporation , or  electropermeabilization , is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane allowing chemicals, drugs, or DNA to be introduced into the cell (also called  electrotransfer )

11. HEAT SHOCK METHOD Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. A short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42°C for 45 seconds (heat shock) and then placed back in ice.