Aulani "GE" Presentation 8 Recombinant DNA Aulanni’am Biochemistry Laboratory Chemistry Laboratory Brawijaya University

Aulani "GE" Presentation 8 Definition  To put DNA from one organism into the DNA of bacteria Methods Transformation Transduction Conjugation

Aulani "GE" Presentation 8 Method: Transformation  The modification of the genotype of a cell (usually prokaryotic) by introducing DNA from another source  The uptake of DNA from an organism’s environment  The uptake and expression of DNA in a bacterium

Aulani "GE" Presentation 8 Method: Transformation  3 elements required 1. Suitable host 2. Self-replicating vector 3. Means of selecting of host cell that have taken up the gene  Plasmids  Small, circular, extra-chromosomal DNA molecules found in bacteria which can replicate on their own outside of a host cell  Rapid growth rate  Cloning limit kb

Aulani "GE" Presentation 8 Method: Transformation  Plasmid (cont.)  Fertility-(F-)plasmids, which contain only tra-genes. Their only function is to initiate conjugation.conjugation  Resistance-(R-)plasmids, which contain genes that can build a resistance against antibiotics or poisons.antibioticspoisons  Col-plasmids, which contain genes that code for (determine the production of) colicines, proteins that can kill other bacteria.colicinesproteins  Degrative plasmids, which enable the digestion of unusual substances, e.g., toluole or salicylic acid.toluolesalicylic acid  Virulence plasmids, which turn the bacterium into a pathogen. pathogen

Aulani "GE" Presentation 8 Method: Transformation  The modification of the genotype of a cell (usually prokaryotic) by introducing DNA from another source  The uptake of DNA from an organism’s environment  Cosmids  Extrachromosomal circular DNA molecules that combines features of plasmids and phage  Cloning limit kb

Aulani "GE" Presentation 8 Method: Transduction  Process of transfection with phage introduction(equivalent to transformation)  Phages  Derivatives of bacteriophage lambda Virus which infects E. coli  Linear molecules  Transformation efficiency 100X greater than plasmid vector  Cloning limit 8-25 kb

Aulani "GE" Presentation 8 Method: Conjugation  Bacterial Artificial Chromosomes (BAC)  Based on bacterial mini-F plasmids  Small pieces of episomal bacterial DNA that give the bacteria the ability to initiation conjugation with adjacent bacteria  Cloning limit kb

Aulani "GE" Presentation 8 Definitions for rDNA process  Restriction endonuclease  Natural function  to protect bacteria by cutting and inactivating the DNA of infecting viruses  rDNA  Recognize specific base sequences and cleave the DNA molecule EcoR1 cuts at GAATTC BamHI cuts CCTAGG HindIII cuts TTCGAA

Aulani "GE" Presentation 8 Definitions for rDNA process  “Sticky ends”  Single stranded extensions of DNA created from a restriction enzyme  Cloning Vector  A DNA molecule that carries foreign DNA into a host cell, where it replicated  Phages  Plasmid  Cosmid

Aulani "GE" Presentation 8 Definitions for rDNA process  Donor cells  Cell supplying the snippet of DNA  Recipient cells  The cell supplying the cell as well as the rest of the bacterial chromosome  DNA ligase  Enzyme used to bind sticky ends

Aulani "GE" Presentation 8 rDNA Steps (generalized) 1. Plasmid isolated and cut with enzyme 2. DNA isolated from donor cell and cut with same enzyme 3. Donor and plasmid DNA mixed and sealed with DNA ligase 4. rDNA introduced into a bacterium 5. Replication of plasmids

Aulani "GE" Presentation 8 Results  3 results  Cells that lack plasmids (picked up human DNA)  Cells that contain plasmid that do not contain a foreign gene (picked up plasmid DNA)  Cells that contain plasmid that have picked up a foreign gene (picked up rDNA)

Aulani "GE" Presentation 8 Distinguishing rDNA  Antibiotic resistance marker(s) added to plasmid (ex. Ampicillin and tetracycline genes)  Human DNA only – sensitive to both  Plasmid DNA only – resistant to both  rDNA –resistant to ampicillin but sensitive to tetracycline (the human DNA is inserted into the tetracycline coding sequence, thus disrupting the gene)

Aulani "GE" Presentation 8 Other info  Gene isolation  Genomic library  collection of recombinant bacteria that harbor pieces of a genome  DNA probe (gene sequence in question)  A labeled short sequence of DNA that corresponds to a specific gene.  Emits a signal when it binds to its complement in a bacterial cell that contains a recombinant plasmid

Aulani "GE" Presentation 8 Applications  Epidermal growth factor  Accelerates healing of wounds and burns  treats gastric ulcers  Insulin  Allows cells to take up glucose in treatment of type I diabetes mellitus  Renin inhibitor  Lowers blood pressure  Interleukin-2  Treats kidney cancer