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?

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

Biotechnology Toolkit

Review Microbiology Animations with Quizzes in Mastering Microbiology

Fig 8-25

Site of cleavage

Recognition sequence is always a palindrome

EcoRI

Review Table 9.1

Restriction Enzymes = REs(= Restriction Endonucleases)

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:

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

Review Microbiology Animations with Quizzes in Mastering Microbiology

Fig 9.4

Inserting Foreign DNA into Cells

DNA can be inserted into a cell by Transformation Protoplast fusion Electroporation Microinjection

Fig 9.7

Techniques of Genetic Modification

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:

1. Bacteria lack vector _______________

2. Bacterial clones contain vector without the new gene colony type? _______________

3. Bacterial clones contain recombinant vector resistant to Ampicillin and unable to hydrolyze X-gal colony type? _______________

Possible Method to detect

recombinant bacteria:

Blue–White Screening

Fig 9.11

1) Plasmid cloning

2) Selecting Recombinant Bacteria

Which type of colonies do you want?

a)White

b)Blue

c)I don’t want any

Making a Gene ProductE. 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:1. Insulin production

2. Subunit vaccines

3. DNA vaccines

4. Gene therapy to replace defective or missing genes

5. Pharmaceutical applications Hormone and Antibiotics production

See Table 9.2

http://www.dnalc.org/view/15087-Genentech-s-approach-to-making-insulin-David-Goeddel.html

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 FigureFig 9.1