Identify and explain the three types of classifications

Monday Notes  Fred Griffith 1928 o Smooth (capsulegives virulence, protects from body killing it) step bacteria (causes pneumonia) and rough (no capsule) strep  Heat killed S doesn’t kill mouse  Mix of heat killed S and live R kills mouse  R was transformed into S  Transformation-cell takes up naked DNA and incorporates it into genome  Transduction-DNA transferred by bacteriophage o General-phage accidentally takes up random bacterial DNA piece o Specialized-happens during lysogenic, part if bacterial DNA excised with prophage Wednesday Notes  Genetic engineering-modifying organisms genetic info by changing the nucleotide sequence  Recombinant DNA technology-carry out genetic engineering o Some developments are restriction enzymes, genetic cloning, cDNA synthesis, and gel electrophoresis  Cloning-used to generate large number of identical DNA molecules  Biotechnology-uses organism to make products  Industrial microbiology-uses microbes to make compounds or the microbes are the products themselves  Cloning steps o Isolate DNA o Restriction enzymes or PCR to cut up DNA o Insert fragments into a cloning vector to make recombinant molecule o Put recombinant into new host to express host  Restriction enzymes bind to specific sequences in DNA, recognition sites  Cuts DNA at site or specific distance from it  Produce sticky or blunt ends in DNA o Sticky ends are more discriminatory  Commercially available  Jackson, Symons and Bergs made the first recombinant molecules using plasmid as vector (carries foreign DNA)  Reverse transcriptase makes double stranded DNA from mRNA o The mRNA makes complementary DNA (cDNA)  Gel electrophoresis separates molecules by charge and molecular  Agarose or acrylamide used for DNA o Migrates to positive end o Small fragments travel faster and farther  The first recombinant molecule was cDNA for human insulin into E. coli vector in 1982 o The E. coli could produce insulin  Polymerase chain reaction (PCR) amplifies the gene, rapidly synthesizing many copies of the fragment o Oligonucleotides (DNA or RNA) serve as the DNA primers and can be from 2-30 or 50-100 nucleotides o The mix has primers, target DNA, thermostable DNA polymerase, and nucleotides Friday Notes  PCR has three 3 steps: denaturation, annealing, elongation o Denaturation from heat at 90 degrees Celsius or above  Need thermo stable PCR enzyme (DNA polymerase taq)  To anneal temp is reduced to 65 degrees  To elongate temp is brought back up to 70-75 degrees o Known as thermo cycle  PCR is used on DNA, RT-PCR also has reverse transcriptase when starting with mRNA  Cloning uses bacteria because they replicate asexually, gene stays pure  Both bacterial chromosome and plasmid have own origin of replication  Bacteria also has restriction enzymes to cut up foreign DNA  Restriction enzymes create the same sticky ends on everything they cut, so use restriction enzyme on DNA you want to clone and on plasmid  Transformation o For bacteria to pick up naked DNA from environment you have to make it competent, open pores in membrane  Heat shock it or use CaCl2treatment  Then mix with plasmid solution  Ones that take in plasmids will express plasmid genes  Transduction o Uses phage o Generalizedassembled phage takes up bacterial chromosome piece, leaves host and take this piece to ne bacteria  Any piece can be transferred, random o Specializedwhen prophage leaves bacterial chromosome it takes piece of chromosome with it, piece goes into all assembled phages that leave host  Only chromosome pieces that are connected to prophage can be transduced this way  Easier to figure out the transduced sequence when specialized transduction occured