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Molecular Biology Lecture 4 Chapter 4 Molecular Cloning Methods Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. pUC and b-galactosidase pUC plasmid 4-2 pUC multiple cloning site 4-3 pUC and b-galactosidase pUC plasmid lac promoter O MCS lacZ (a-peptide) -Induced by lactose or IPTG -Under the control of the lac repressor -The MCS is a cluster of sequences recognized by restriction endonucleases 4-4 pUC and b-galactosidase a-complementation Plasmid contains part of the lacZ gene coding for the Nterminal extremity of the b-galactosidase enzyme. When expressed in E. coli lacZ strain = no activity Host bacterial strain contains a truncated lacZ gene encoding a polypeptide missing the N-terminal extremity When expressed in E. coli = no activity To use the pUC plasmid for selection, the host strain used for transformation must be lacZ- and lacZ M15 4-5 pUC and b-galactosidase a-complementation The two partial gene products can cooperate to form an active enzyme Reconstituted tetramer E. coli lacZ M15 pUC a-peptide + inactive inactive Active tetramer 4-6 pUC selection procedure Plate transformants on a petri dish containing IPTG (synthetic inducer of the lac operon) and X-gal (synthetic substrate of b-galactosidase) a-peptide is synthesized and complementation will take place Blue colony lacZ gene is disrupted a-peptide is not synthesized and complementation will not take place white colony 4-7 Non-directional cloning EcoR1 EcoR1 1) 2) 3) 4) 5) EcoR1 Cut plasmid DNA and insert with EcoR1 De-phosphorylate the cut vector (alkaline phosphatase) Mix de-phosphorylated vector and insert + DNA ligase and ATP Transformation Selection De-phosphorylation of cut vector will reduce the number of false positive 4-8 Directional cloning EcoR1 1) 2) 3) 4) BamH1 EcoR1 BamH1 Cut plasmid DNA and insert with EcoR1 and BamH1 Mix cut vector and insert + DNA ligase and ATP Transformation Selection Digestion of the vector with two restriction endonucleases prevents religation 4-9 Summary 4-10 cDNA Cloning • cDNA is the abbreviation for complementary DNA or copy DNA • A cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time – Such a library can contain tens of thousands of different clones 4-11 Making a cDNA Library 4-12 Making a cDNA Library 4-13 Making a cDNA Library 4-14 Making a cDNA Library 4-15 Making a cDNA Library cDNA plasmid Recombinant plasmid 4-16 Methods of Expressing Cloned Genes Cloning a gene permits • Production of large quantities of a particular DNA sequence for detailed study • Large quantities of the gene’s product (protein or RNA) can also be obtained for further use 4-17 Expression Vectors • Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen • Expression vectors are those that can yield protein products of the cloned genes 4-18 Fusion Proteins pUC plasmid • If inserted DNA is in the same reading frame as interrupted gene, a fusion protein results – These have a partial bgalactosidase sequence at amino end – Inserted cDNA protein sequence at carboxyl end 4-19 Oligohistidine Expression Vector • Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His – Oligohistidine has a high affinity for divalent metal ions like Ni2+ – Permits purification by nickel affinity chromatography – His tag can be removed using enzyme enterokinase without damage to the protein product 4-20 Summary • Expression vectors frequently produce fusion proteins – One part of the protein comes from coding sequences in the vector – Other part from sequences in the cloned gene 4-21 Inducible Expression Vectors • Main function of expression vector is to yield the product of a gene – usually more is better • For this reason, expression vectors have very strong promoters • Prefer keep a cloned gene repressed until time to express – Large quantities of eukaryotic protein in bacteria are usually toxic – Can accumulate to levels that interfere with bacterial growth – Expressed protein may form insoluble aggregates, inclusion bodies 4-22 Controlling the lac Promoter • lac promoter is somewhat inducible – Stays off until stimulated – Actually repression is incomplete or leaky – Some expression will still occur 4-23 Summary • Expression vectors are designed to yield the protein product of a cloned gene • When a lac inducer is added, cell begins to transcribe the gene of interest 4-24 Using the Ti Plasmid to Transfer Genes to Plants • Genes can be introduced into plants with specialized vectors • Common bacterial vector promoters and replication origins are not recognized by plant cells • Plasmids are used containing T-DNA – T-DNA is derived from a plasmid known as tumor-inducing (Ti) – Ti plasmid comes from bacteria that cause plant tumors called crown galls 4-25 Ti Plasmid Infection • Bacterium infects plant, transfers Ti plasmid to host cells • T-DNA integrates into the plant DNA causing abnormal proliferation of plant cells • T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant 4-26 Ti Plasmid Transfers Crown Gall 4-27 Use of the T-DNA Plasmid 4-28