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Exam 2 M 10/29 at 7-8:30pm in UTC 2.102A Review Th 10/25 at 5-7pm in WRW 102 Bonus #1 due now Last day to drop with a ‘Q’…10/24 Genetic Engineering: Direct manipulation of DNA Bacteria can be modified or serve as intermediates a typical bacteria Bacterial DNA plasmid DNA A typical bacterial plasmid used for genetic engineering Moving a gene into bacteria via a plasmid What problems exist for expressing eukaryotic gene in bacteria? Bacterial DNA plasmid DNA Reverse transcriptase can be used to obtain coding regions without introns. After RT, PCR will amplify the gene or DNA Fig 20.14 Moving a gene into bacteria via a plasmid RT and PCR Restriction Enzymes cut DNA at specific sequences Restriction enzymes cut DNA at a specific sequence Cutting the plasmid and insert with the same restriction enzyme makes matching sticky ends Fig 20.2 A typical bacterial plasmid used for genetic engineering Using sticky ends to add DNA to a bacterial plasmid Fig 20.2 If the same restriction enzyme is used for both sides, the plasmid is likely to religate to itself. Fig 20.2 The plasmid is treated with phosphatase to remove the 5’-P, preventing selfligation Fig 20.2 Transformation of bacteria can happen via several different methods. Fig 20.8 Bacteria can take up DNA from the environment Fig 7.2 Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane: •Electroporation •Heat shock •Osmotic Stress Fig 20.8 How can you know which bacteria have been transformed, and whether they have the insert? Fig 20.5 Resistance genes allow Figurebacteria 20-5 with the plasmid to be selected. Bacteria with the resistance gene will survive when grown in the presence of antibiotic Fig 20.5 Is the insert present? FigurePlasmids 20-5 with the MCS in the lacZ gene can be used for blue/white screening… A typical bacterial plasmid used for genetic engineering Fig 20.5 Intact lacZ makes a Figureblue 20-5 color when expressed and provided X-galactose Fig 20.5 When the lacZ gene is Figuredisrupted, 20-5 the bacteria appear white Blue/white screening: Transformed bacteria plated on antibiotic and Xgal plates. Each colony represents millions of clones of one transformed cell. Successful transformation will grow a colony of genetically modified bacteria Fig 20.4 RT and/or PCR Inserting a gene into a bacterial plasmid