Genetic Engineering and Biotechnology

Ways to insert DNA into a Cell

- Transformation - Electroporation - Protoplast fusion - Microinjection

Advantages and Disadvantages of Genetic Engineering

-Allows controlled expression of only the product of interest -Traditional Methods may have led to the production of unintended byproducts like toxins -It can be difficult to predict the effect of an engineered product on the population -How do we keep genetic engineered organism traits separate from the non-engineered organisms.

Biotech Animal Applications

-Animals can be bioreactors -scientists can create transgenic animals to express proteins in milk in high numbers. -Goats, cattle, sheep, and chickens are a source of antibodies.

Molecular Pharming

-Use of genetically modified plants or animals as a source of pharma products -Usually recombinant proteins with therapeutic value. -emerging but challenging field -uses manipulation of protein glycosylation (polysaccharide chain addition) -sub-cellular protein targeting in plant cells

Other biotech products

-home pregnancy tests (monoclonal antibodies) -frost resistant strawberries (GMOs) -mostly medical and agricultural -fading jeans with engineered enzymes like amylase and cellulase.

Biotech laboratory

-most widely used biotech products are recombinant proteins (gene cloning in cell culture) -Cell cultures- growing cells in lab under control settings (in vitro) -in vitro- controlled environment outside of a living organism. -Bioreactors- used industrially, large culturing containers where DNA of interest can be mass produced.

Ethics of rDNA

-preventing accidental release of engineered microbes -suicide genes so microbes can't survive outside lab. -who has access to modifications -implications for modifying higher organisms

Basics of Genetic Engineering

1. Identify and clone the gene of interest. May have to remove eukaryotic introns so bacterial cells can express or modify codons for efficient expression. 2. Insert into appropriate expression vector. Need an inducible/repressible promoter to express protein in bacteria. Need a strong viral promoter or tissue specific promoter for expression in animals. 3. Transform into cells and select for recombinants.

Steps of DNA Recombination

1. Restriction enzymes cut double stranded DNA at its particular recognition sites. 2. These cuts produce DNA fragments with sticky ends 3. Where two such fragments of DNA cut by the same restriction enzyme come together they can join through base pairing. (remove or add something in-between) 4. The joining of fragments will usually form either a linear or circular plasmid. 5. The enzyme DNA ligase is used to unite the backbones of the two DNA fragments producing a molecule of recombinant DNA.

Genetic Modification Procedure

1. Vector such as a plasmid is isolated 2. DNA is cleaved by an enzyme into fragments 3. Gene is inserted into plasmid (through recombination procedure) 4. Plasmid is taken up by a cell such as a bacterium 5. Cells with genes of interests are cloned. 6.A. Copies of genes are harvested 6.B. Cells make product proteins 7. Proteins are harvested

Top Fermenting Yeast

Ales Yeast remains uniformly throughout fermenting pear and is carried to the top by the CO2 during fermentation. Higher Temperatures (14-25C) 5-7 day shorter cycle

Biotech Aquatic Applications

Aquaculture- Process of raising fish or shellfish in controlled settings as a food source. -transgenic salmon (increased growth rate) -disease resistant oysters -vaccines against viruses that infect aquatic life - rich new source for genes, proteins, and metabolic processes.

Knockouts

Basic research tool for IDing function of a gene. Active gene is replaced by a non functioning one and can observe the effects

Biotech Environmental Applications

Bioremediation to degrade a variety of natural and manmade products that contribute to pollution. Helps clean up environment and helps explain microbial biotech. Clean up oil spills with GMO pseudomonas species that degraded the crude oil quickly

Cloning Human Insulin

Cloned before reverse transcriptase and PCR Very small size allowed the synthesis and clone of both the A and B subunits separately in E. coli under the control of the lac promoter. Induction of Lac was sufficient for the expression of A and B chain. They were purified, incubated and then mature insulin was formed.

pUC19

Commonly used cloning vector with many cut sites and ampicillin resistance and an oriC.

Strepyomyces

Complex life cycle that is linked to molecule production- iron scavenging, surfactant, quorum sensing, and germination inhibitor. Use as an antibiotic Ex- used as a de-worming agent and won a nobel prize for helping river blindness

Biotech Forensic Applications

DNA fingerprinting used by crime scene investigation- first used to convict a rapist in 1987. Can also ID human remains, paternity tests, endangered species, and epidemiology

Complementary DNA (cDNA)

DNA made from mRNA by reverse transcriptase in eukaryotic cells. Complement does not have introns- you can put it into prokaryotes

Applications of Biotechnology

Diagnostics (PCR and DNA probes can be used to quickly identify a pathogen in body tissue or food) Gene therapy (to replace a defective or missing gene) Pharmaceutical (hormone and antibiotics production, vaccines (subunit vaccines, DNA vaccines, nonpathogenic viruses carrying genes for pathogen's antigens as vaccines))

Insulin

Discovered in 1922 as a pancreatic enzyme extract. Originally gotten from cow and pig pancreas'. Posed a risk for allergic reactions and disease transfers.

tPA

Early genetically engineered product- tissue plasminogen activator Blood clot dissolving enzyme used immediately after heart attacks or strokes to hep clear blood vessels

Plant Cells

Easy to grow and may express eukaryotic genes easily

Biotech Agricultural Application

Estimated worth of 6 billion -pest resistant plants -higher protein and vitamin contents in foods -drugs developed and grown in plants -drought resistant and cold tolerant and higher-yielding crops. (use other plant genes)

Yeast (saccharomyces cerevisiae)

Eukaryotic workhorse of biotechnology -continuous secretion of gene product by manipulating the various promoters.

Stanley Cohen and Herbert Boyer

Fathers of recombinant DNA. Had the idea at a convention in Hawaii in 1972. Boyer's lab recently isolated a restriction enzyme used to selectively cut DNA. Cohen developed a method to introduce antibiotic carrying plasmid into bacteria Developed a way to isolate and clone genes in bacteria.

Humalin

First FDA approved biotech drug, made by genentech to treat diabetes with human insulin grown in E. coli.

Dolly

First cloned animal (1996) cloned from a cell from a 6 year old sheep A lot of controversy about ethics of doing so

Biotech Treatments

Gene Therapies- replace a defective gene with a normal one Tissue Engineering- designing and growing tissues for regenerative reasons E. Coli was the first GMO making somatostatin (HGH) and humalin was the first approved by the FDA.

Flanders Institute of Biotechnology

Genetically engineered yeast to make a superior beer. Currently no beers are made with genetically modified yeasts, but some wines are and so it should soon happen according to Verstrepen. Engineer so no head aches?

Obtaining DNA

Genomic Libraries- gene stores in plasmids or phages (natural copies of genes, made from mRNA by reverse transcriptase (cDNA), synthetic DNA from machines)

Bottom Fermenting Yeast

Lager Yeast settles to the bottom Lower temperatures (6-12C) 8-14 days longer cycle

PCR

Makes multiple copies of a piece of DNA enzymatically (makes a lot out of a little) Used to: clone DNA for recombination, amplify DNA to detectable levels, sequence DNA, diagnose genetic diseases, detect pathogens. Process: 30-40 cycles 1. Denaturing double stranded to single stranded DNA at 94C. 2. Annealing assembles the primers to the strands at 54C. Forward and reverse primers 3. Extension by adding the DNTPs to the primers to re-synthesize the DNA at 72C

Biotechnology

Manipulation (genetic engineering) of living organisms or their components to produce useful commercial products. The exploitation of biological processes for industrial purposes especially the genetic manipulation of microorganisms for the production of antibiotics and hormones.

Mammalian Cells

May express eukaryotic genes easily but harder to grow

Recombination DNA (rDNA) Technology

Most Common; insertion or modification of genes to produce desired proteins.

Biotech Microbial Applications

Often Yeast and bacteria Used to better enzymes, making food, simplifying manufacturing, better decontamination processes for waste Drugs Batch clone important proteins

Drug Discovery today

Post-genomic- genome mining- allows us to sequence the whole genome and see what molecules and variants are all there and then use those. A lot more molecules. Must also consider feedback and regulation impact within cell. 1. isolation 2. 16s rDNA sequencing 3. phylogeny 4. Database blast 5. Molecule 6. bioassay- must predict chemistry Can use homologous expression of non producing host (activating expression translation) or heterozygous clean host (cloning gene synthesis) to find new peaks

Biotech Medical Applications

Prevention, diagnostic, and treatment Human genome project Gene therapy and stem cell technology to create specialized targeted cells to treat disease

E. Coli

Prokaryotic workhorse of biotechnology -easily understood and grown. -need to eliminate endotoxin from products -cells must be lysed to get the products

Plant advantage

Promotes agricultural biotech Cost of producing plant material with recombinant proteins in plants is often lower than bacteria Can also be combined with medical biotech for molecular pharming.

Restriction Enzymes (Restriction endonuclease)

Recognize different specific sequences to cleave them. Recognition is usually palindromic. Creases sticky ends to be rejoined. A lot of different ones for bacteria.

Cloning Vectors

Requires a significant amount of DNA. They are recombinant DNA molecules to introduce foreign DNA into the host cells to self replicate in large quantities. Plasmids and viruses are common vectors and shuttle vecots can exist in several difference species. pUC19 is commonly used.

Blue White Screening Method

Selects for the Transformed clone -directed selection for engineered vector via antibiotic-resistance markers (ampR) on plasmid vectors -Vector also contains beta-galactosidase gene for blue-white screening. -Desired gene is inserted into the beta-galactosidase gene site-> gene inactivated - Disrupt lacZ gene by cutting with restriction enzymes and new gene is added in altering the color Outcomes: 1. Bacterial clones contain recombinant vector with resistance to amp. and is unable hydrolyze gal- white colonies. 2. Bacterial clones contain vector without new gene- blue colonies 3. Bacteria lack vector and will not grow

Genetic Engineering

Techniques used to cut up and join together genetic material (from different spices) and to introduce the result into an organism in order to change one or more of its characteristics

Forensic Microbiology

Uses PCR with a primer for a specific organism that allows for detection if the organism is present. Real time PCR- newly made DNA tagged with a fluorescence that can be measured after every PCR cycle. Reverse-transcription (RT-PCR): reverse transcriptase makes DNA from viral RNA or mRNA. Combined allows for easy strand identification

Insulin recombination

Very small in size- ideal for rDNA. Was the first protein to have its primary sequence determined. Restriction enzyme cut a plasmid and human insulin gene was inserted and linked. Then transformed into a bacteria (E. coli) to be cloned and expressed

Stem Cells

We use chemicals to make them develop a certain way. Newest and most promising area but also controversial.

Kirin Beer Research Laboratory

Yeast development Select and develop a superior yeast to enhance beer quality through research to select, breed, and employ yeasts to make beer flavor and quality characteristics

Transgenic

containing genes from another source

Traditional drug discovery

find and grind- pregenomic; requires bioactivity or abundance of isolatable stuff 1. isolation 2. cultivation 3. Liquid chromatography and mass spectrometry 4. Molecule identification 5. bioassay

Actinomycete

gifted bacterial drug makers 2/3 of all antibiotics today Secondary metabolites are very important- extreme diversity of molecules

Microbial enzymes at work

recombinant rennet in cheese transglutaminase meat glue clothing conditioning and detergent paper-making/biofuels/bioremediation microbiology digestion of things and waste management

Chitin

shellfish waste management- chitosan is one of the strongest blood coagulants in clinical use. Used to help stop bleeding in massive cuts. Chitosan can also be used to deliver drugs by docking them to the chitosan which then get endocytosed into cells and released inside. tricks cells into taking up things like insulin and released in cell pH change.

Bioprospecting

systematic search for new sources of chemical compounds, genes, micro-organisms, macro-organisms, and other valuable products from nature. Economically valuable genetic and biochemistry resources from nature. GET MORE MICRBES- get from extreme heat, animals that degrade hard lignocellulose, desserts, deep sea vents, or just anywhere.