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TORTORA • FUNKE • CASE Microbiology AN INTRODUCTION EIGHTH EDITION B.E Pruitt & Jane J. Stein Chapter 9, part A Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA • Biotechnology: – The use of microorganisms, cells, or cell components to make a product – Foods, antibiotics, vitamins, enzymes – Old science - cheese, beer, wine and bread • Recombinant DNA Technology: – Insertion or modification of genes to produce desired proteins Figure 9.1.1 Figure 9.1.2 Table 9.1.1 Table 9.1.2 Tools of Biotechnology • Enzymes that do the work: – – – – Restriction enzymes DNA polymerase DNA ligase Reverse Transscriptase • Vectors to carry DNA – Plasmids – Viruses • DNA sources – Synthetic – Gene libraries • Important procedures – PCR – Blotting Southern / northern / western Tools: Restriction Enzymes • Cut specific sequences of DNA – Palindromes: Madam I’m Adam; Race car; wow and mom • In wild cells they at as a type of disease defense by destroying bacteriophage DNA in bacterial cells • Cannot digest (host) DNA with methylated cytosines • May cut DNA blunt or with “sticky ends” Figure 9.2 Vectors • Carry new DNA to desired cell • Shuttle vectors can exist in several different species • Plasmids and viruses can be used as vectors Vectors Figure 9.3 (PCR) • To make multiple copies of a piece of DNA enzymatically • Used to – Clone DNA for recombination – Amplify DNA to detectable levels – Sequence DNA – Diagnose genetic disease – Detect pathogens PCR Figure 9.4.1 PCR Figure 9.4.2 DNA can be inserted into a cell by: • Transformation • Electroporation • Protoplast fusion • Microinjection • transduction Figure 9.5b DNA can be inserted into a cell by: • Microinjection • Gene gun Figure 9.6 & 7 Obtaining DNA • Gene libraries are made of pieces of an entire genome stored in plasmids or phages • cDNA is made from mRNA by reverse transcriptase – Why??? • Synthetic DNA is made by a DNA synthesis machine Selection & Mutation • Selection: Culture a naturally-occurring microbe that produces desired product • Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait • Site-directed mutagenesis: Change a specific DNA code to change a protein • Select and culture microbe with the desired mutation