Biotechnology and Recombinant DNA Chapter 9 Biotechnology and Recombinant DNA

SLOs Compare and contrast biotechnology, recombinant DNA technology, and genetic engineering. Identify the roles of a clone and a vector in making recombined DNA. Compare selection and mutation. Define REs, and outline their use to make recombinant DNA. List some properties of vectors and describe their use. Outline the steps in PCR and provide an examples of its use. Describe various different ways of getting DNA into a cell. Differentiate cDNA from synthetic DNA. Explain how each of the following are used to locate a clone: antibiotic-resistance genes, DNA probes, gene products. Outline advantages of engineering with either E. coli, Saccharomyces cerevisiae, mammalian cells, or plant cells. List some advantages of, and problems associated with, the use of genetic modification techniques.

Terminology and Definitions Biotechnology: Manipulation (as through genetic engineering) of living organisms or their components to produce useful commercial products Recombinant DNA (rDNA) technology: Insertion or modification of genes to produce desired proteins Clones and cloning: Cell clones “to clone a gene” - question: Interest in DNA or in gene product? Clone: population of cells arising from one cell; each carries the new gene

Biotechnology Toolkit Natural vs. artificial selection Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait. Now: Site-directed mutagenesis Restriction Enzymes (RE): Molecular scissors Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells Methylases protect own DNA by methylating cytosines Bunt ends vs. sticky ends Review Microbiology Animations with Quizzes in Mastering Microbiology

Restriction Enzymes = REs (= Restriction Endonucleases) Site of cleavage EcoRI Recognition sequence is always a palindrome Fig 8-25 Review Table 9.1

Origin and Naming of Restriction Enzymes

Role of Restriction Enzymes in Making Recombinant DNA Molecules Fig 9.2

Vectors Also known as cloning vectors. Must be Small and easy to manipulate. ________ & _________ serve as vectors. self-replicating  large quantities When they carry “insert”: = Recombinant DNA molecules Introduce foreign DNA (desired gene) into host cells Shuttle vectors can exist in several different species.

... One of most commonly used vectors: pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers.[1] The designation "puce" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early work on the plasmid series had been conducted.[2] It is a circular double stranded DNA and has 2686 base pairs.[3] pUC19 is one of the most widely used vector molecules as the recombinants

Polymerase Chain Reaction (PCR) Makes 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 For details see Lab Fig 9.4 Review Microbiology Animations with Quizzes in Mastering Microbiology

Inserting Foreign DNA into Cells Techniques of Genetic Modification Inserting Foreign DNA into Cells DNA can be inserted into a cell by Transformation Protoplast fusion Electroporation Microinjection Fig 9.7

Blue and White Screening Method for Selecting a Clone (or Recombinant DNA Molecule) Direct selection of engineered vector via antibiotic-resistance markers (ampR) on plasmid vectors. Vector also contains-galactosidase gene for blue-white screening Desired gene is inserted into the -galactosidase gene site  gene inactivated Three possible outcomes: Bacteria lack vector  _______________ Bacterial clones contain vector without the new gene  colony type? _______________ Bacterial clones contain recombinant vector  resistant to Ampicillin and unable to hydrolyze X-gal  colony type? _______________ Three possible outcomes: Bacterial clones contain recombinant vector  resistant to Ampicillin and unable to hydrolyze X-gal (white colonies). Bacterial clones contain vector without the new gene  blue colonies. Bacteria lack vector  will not grow.

Possible Method to detect recombinant bacteria: Blue–White Screening 1) Plasmid cloning Fig 9.11

2) Selecting Recombinant Bacteria

Which type of colonies do you want? White Blue I don’t want any

Making a Gene Product E. coli: prokaryotic workhorse of biotech. Easily grown and genomics well understood. Disadvantage: Cells must be lysed to get product  release of ______ Yeast: Saccharomyces cerevisiae is eukaryotic workhorse of biotechnology. Advantage: Continuous secretion of gene product. Mammalian cells: May express eukaryotic genes easily. Disadvantage: Harder to grow. Plant cells: Easy to grow. May express eukaryotic genes easily.

Some Applications of DNA Technology Forensic Microbiology & Diagnostics: PCR and DNA probes can be used to quickly identify a pathogen in body tissue or food. Therapeutic Applications: Insulin production Subunit vaccines DNA vaccines Gene therapy to replace defective or missing genes Pharmaceutical applications Hormone and Antibiotics production See Table 9.2

Safety Issues and Ethics of Using rDNA Strict safety standards avoid accidental release of genetically modified microorganisms. Some microbes used in cloning have been altered so that they cannot survive outside the laboratory. Microorganisms intended for use in the environment may be modified to contain suicide genes  organisms do not persist in the environment. Safety and ethical concerns beyond microbiology: Who will have access to an individual's genetic information? Are genetically modified crops safe for release to environment?

A Typical Genetic Modification Procedure Foundation Figure Fig 9.1 The End