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Lecture notes for Week 9 of BISC 300

by: Jj Lynch

Lecture notes for Week 9 of BISC 300 BISC300

Marketplace > University of Delaware > Biological Sciences > BISC300 > Lecture notes for Week 9 of BISC 300
Jj Lynch

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notes from Monday, Wednesday and Friday
Carlton Cooper
Class Notes
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This 3 page Class Notes was uploaded by Jj Lynch on Saturday April 9, 2016. The Class Notes belongs to BISC300 at University of Delaware taught by Carlton Cooper in Summer 2015. Since its upload, it has received 11 views. For similar materials see Microbiology in Biological Sciences at University of Delaware.

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Date Created: 04/09/16
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


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