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1 Biotechnology • What is Biotechnology? – Use of organisms, especially microbes, to produce useful products? •Beer, wine, bread, organic solvents, antibiotics •By this definition, very very old. – Use of recombinant DNA techniques to harness the power of organisms to make useful products. •Very new technology •Includes herbicide-resistant plants, human proteins produced in yeasts, new vaccines.

1 Biotechnology What is Biotechnology? –Use of organisms, especially microbes, to produce useful products? Beer, wine, bread, organic solvents, antibiotics

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1Biotechnology

• What is Biotechnology?– Use of organisms, especially microbes, to produce

useful products?• Beer, wine, bread, organic solvents, antibiotics• By this definition, very very old.

– Use of recombinant DNA techniques to harness the power of organisms to make useful products.• Very new technology• Includes herbicide-resistant plants, human

proteins produced in yeasts, new vaccines.

2Biotechnology has several applications:overview

• Agriculture– Herbicide resistant plants– Improved nutritional qualities

• Pharmaceuticals– Production of human proteins as drugs– Production of vaccines

• Medical, legal, biological– Screening for, treatment of genetic disease– DNA fingerprinting, biological conservation

3Herbicide resistance

• Example: glyphosate resistant plants– More than 2/3 of US soybeans and cotton– Glyphosate inhibits EPSP synthase

• Engineered plants have extra copies of gene, make more enzyme, so are more resistant.

• Steps in engineering:– Gene from E. coli. Put next to strong promoter – Cloned into Ti plasmid, plasmid put back into

A. tumefaciens which carries plasmid to plant cell.– Grow whole plant from engineered plant cell

4Why and why not?

• Use of herbicide-resistant plants means less herbicide use, no-till farming. – less erosion and less non-point source pollution.

• Safe to eat? Why not?– Proteins not automatically destroyed during

digestion; allergies possible. Otherwise, what’s the problem?

• Environmental concerns– Toxic pollen? Herbicide resistant weeds?

• Biotech: same only more targeted and quicker.

5Ag-2: improved nutrition

• Not every food product has complete nutrition– Corn very low in the amino acid lysine– Countries relying on rice have low intake of beta-

carotene– Some plants have health-improving chemicals

• Transgenic plants can provide relief– Daffodil gene inserted into rice to make beta-

carotene, precursor to Vitamin A = golden rice• Critics say: not enough to make a difference.

6Pharmaceuticals

• Dwarfism, diabetes, cancer can be treated using human proteins– Obtained with difficulty– Insulin from slaughterhouse animals

• Recombinant insulin first from E. coli– Required combination of cloning, chemical

treatment– Starting point: mRNA, reverse transcriptase, then

insertion into plasmid vector– E. coli or yeast cells used.

7Future directions

• “pharming”: growing of protein drugs in farm plants and animals– Cloning into sheep (etc.) with mammary specific

promoter, only expressed in that tissue.• Released in, collected from milk.

– Using tobacco plants, especially for vaccines• Tobacco easy to grow, easy to engineer, easy to

harvest• Years of agricultural experience

– Use of hairy roots• Cloned genes inserted w/ Ti plasmid

8Vaccines

• Exposing host to antigens found on pathogen– Whole, live, weakened pathogen

• Strong immunity, but risk of live pathogen– Whole, dead pathogen

• Nucleic acid not “dead”; • cancer or toxic reaction

– Subunit vaccine: using a molecule from pathogen• Host reacts, then protects against later exposure

to entire pathogen

9Vaccines-2

• Recombinant vaccines– Clone gene for surface antigen of pathogen– Express gene i.e. get antigenic proteins made

• Collect proteins, process into vaccine• Get proteins into harmless virus

– Express proteins in food• Because there are food allergies, proteins taken

orally can result in immune reactions• Eliminates worries about sterilization, storage,

needle-phobia

10Transgenic vaccine

11Medical diagnosis

• Sickle cell anemia– Fetal cell samples– CVS or amniocentesis– Gene obtained from fetal DNA

• Sickle cell anemia caused by a single nucleotide base substitution that removes a MstII site.

– Different banding pattern on gel indicates whether fetus will be a carrier or have disease (homozygous)

12Medical diagnosis -2

• Cystic fibrosis– Most cases causes by a specific deletion of DNA– PCR used to make allele-specific oligonucleotides

• This DNA hybridizes to region in normal gene that is deleted in faulty allele

• Absence of hybridization means deletion is present, person has the Cf allele.

• Huntington disease– Because of variable number of trinucleotide

repeats, probably PCR or VNTR-type test looking for varying lengths of DNA fragments.

13Ethics!

• Genetic engineering, medical tests opens up wide range of issues and questions– Environmental and global economic issues– Stem cell research and cloning– Who owns the data? Can someone else patent your

genes? Privacy issues.• Should your boss, insurance company,

government have access to your data?– We can tell you that you have the disease, but

• We can’t do anything about it!

14Gene Therapy

• Gene therapy: the attempt to cure an underlying genetic problem by insertion of a correct copy of a gene.– Tantalizingly simple and profound in theory,

maddeningly difficult to actually achieve.– Easiest targets: access to or retrieval of cells

• Respiratory and blood• Engineered virus, infects cells, carries in good

gene.• Engineer cell in tube, return to body.

15Recent successes

• Adenosine Deaminase deficiency– Defect in nucleoside metabolism especially affects

white blood cells

• X-linked chronic granulomatous disease– Neutrophils fail to make superoxide

• General scheme: Retrovirus used to replace gene in bone marrow cells, return cells to patient

16Failures

• Genes don’t always make into genome– “cure” is short-lived as DNA disappears

• Viruses carrying genes insert in bad places– Cause over-expression of genes or DNA deletions

• Cancer

• Immune system becomes sensitized to vector

• Death of patient in clinical trial in 1999.

17New development

• Sleeping Beauty transposon system– First transposon available in vertebrates?– Originally from inactive fish transposon

• Engineer with gene of interest– Transposon inserts with gene, getting it into the

chromosome– Inserts in different places from viral vector– Much higher rate of gene insertion

18Sleeping Beauty Transposon system

Active transposon engineered from an inactive fish transposon.

Transposon jumps into chromosome bringing good gene with it.

http://www.discoverygenomics.net/sbts.html