11.6 Hosts for Cloning Vectors Ideal hosts should be Capable of rapid growth in inexpensive medium ·Nonpathogenic Capable of incorporating DNA Genetically stable in culture Equipped with appropriate enzymes to allow replication of the vector Escherichia coli,Bacillus subtilis,Saccharomyces cerevisiae Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology 11.6 Hosts for Cloning Vectors Ideal hosts should be • Capable of rapid growth in inexpensive medium • Nonpathogenic • Capable of incorporating DNA • Genetically stable in culture • Equipped with appropriate enzymes to allow replication of the vector Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae
Figure 11.13 Bacteria Eukaryote- Escherichia coli Bacillus subtilis Saccharomyces cerevisiae Well-developed Easily transformed Well-developed genetics Nonpathogenic genetics Many strains Naturally secretes Nonpathogenic available proteins Can process mRNA Best known Endospore formation and proteins bacterium simplifies culture Easy to grow Potentially Genetically unstable Plasmids unstable pathogenic Genetics less Will not replicate Periplasm traps developed than most bacterial proteins in E.coli plasmids Advantages Disadvantages 2012 Pearson Education,Inc
Figure 11.13 Well-developed genetics Many strains available Best known bacterium Easily transformed Nonpathogenic Naturally secretes proteins Endospore formation simplifies culture Well-developed genetics Nonpathogenic Can process mRNA and proteins Easy to grow Potentially pathogenic Periplasm traps proteins Genetically unstable Genetics less developed than in E. coli Plasmids unstable Will not replicate most bacterial plasmids Advantages Disadvantages Escherichia coli Bacillus subtilis Saccharomyces cerevisiae Bacteria Eukaryote © 2012 Pearson Education, Inc
Transfection:转染 Introduction of dNA into mammalian cells is called transfection Originally done through phagocytosis of DNA by host cell ·Can also be done using ·Microinjection ·Electroporation ·Gene gun Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology Transfection转染 Introduction of DNA into mammalian cells is called transfection • Originally done through phagocytosis of DNA by host cell • Can also be done using • Microinjection • Electroporation • Gene gun
Figure 11.14 Before gas release After gas release Plunger Helium gas Gas vent Disc ●。9●●●●0●0。工 Microprojectiles with transfecting nucleic acid Fine screen Rough screen Target tissue (a) 2012 Pearson Education,Inc DNA gun for transfection of eukaryotic cells
Figure 11.14 Plunger Helium gas Gas vent Disc Microprojectiles with transfecting nucleic acid Fine screen Rough screen Target tissue Before gas release After gas release © 2012 Pearson Education, Inc. DNA gun for transfection of eukaryotic cells
11.7 Shuttle Vectors and Expression Vectors Shuttle vecfors:vectors that are stably maintained in two or more unrelated host organisms (e.g.,E.coli and B. subtilis or E.coli and yeast)穿梭载体 Bacterial plasmid engineered to function in eukaryotes Add a eukaryotic origin of replication Add a centromere recognition sequence Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology 11.7 Shuttle Vectors and Expression Vectors Shuttle vectors: vectors that are stably maintained in two or more unrelated host organisms (e.g., E. coli and B. subtilis or E. coli and yeast) 穿梭载体 • Bacterial plasmid engineered to function in eukaryotes • Add a eukaryotic origin of replication • Add a centromere recognition sequence