If your eyes follow the movement of the rotating pink dot, you will only see one color, pink. If you stare at the black + in the center, the moving dot turns to green. Now, concentrate on the black + in the center of the picture. After a short period of time, all the pink dots will slowly disappear, and you will only see a green dot rotating if you're lucky! It's amazing how our brain works. There really is no green dot, and the pink ones really don't disappear. This should be proof enough, we don't always

see what we think we see.

Recombinant DNARecombinant DNAMicrobiology 2314

California 2003California 2003

• In Sacramento, a group of scientists have genetically altered fish. Basically they took ordinary zebra fish, added genetic jellyfish stuff, and made pet fish that glow in the dark, when placed under a blacklight. They hit the shelves in 2004 to be sold as pets.

• They will be on sale, just about everywhere except California (which bans lab-engineered species.)

So…We can genetically engineer all the animals we want, just can’t sell em.

Note:

• The genetic material of each living organism-plant or animal, bacterium or virus-possesses sequences of its nucleotide building blocks (usually DNA, sometimes RNA) that are uniquely and specifically present only in its own species.

What is Genetic Engineering?What is Genetic Engineering?

• Genetic engineering (GE) is the transfer of genes from one organism to another through means that do not occur in nature, but through human intervention. This involves isolating and then moving genes within and without different species by recombinant DNA techniques and other manipulation of the genetic construct outside the traditional practices such as sexual and asexual breeding, hybridization, fermentation, in-vitro fertilization and tissue culture.

IntroductionIntroduction

• Biotechnology

- The use of microorganism cells or cell components to make a new product.

• Idea More than 60 Years Old

• Delay Due to Technology

1. Cut

2. Combine

3. Reinsert

Advent of Recombinant DNAAdvent of Recombinant DNA

• Closely Related Organisms Can Exchange Genes

• Labs Can Facilitate Transfer Between Unrelated Species

• DNA Transfers Genes

• Recombinant DNA Technology is a term used to refer to experimental protocols leading to the transfer of genetic information (DNA) from one organism to another.

Tools and TechniquesTools and Techniques

• Restriction Enzyme Makes Cuts

• Cuts at Specific DNA Sequences

• Accurate

• 75 Known Restriction Enzymes

• Restriction enzymes take apart the DNA in a certain area and allow for a plasmid to be inserted within the gap that is created.

Sticky Ends/Blunt EndsSticky Ends/Blunt Ends

DNA LigaseDNA Ligase

• Vectors

- Called Chimeras

• Shuttle Vectors

- Plasmids that can exist in several different species.

• Transformation

- Process by which a new gene is inserted into a cell

ProcessProcess

• Isolate the Source and Vector DNA

• Use Restrictive Enzymes to Make Cuts in Both

• Mix Plasmid and Vector DNA Together and Bond Via Ligase

• Clone

Overview of ProcessOverview of Process

Properties of a Good Vector

1. It should be able to replicate autonomously. When the objective of cloning is to obtain a large number of copies of the DNA insert, the vector replication must be under relaxed control so that it can generate multiple copies of itself in a single host cell.

2. It should be easy to isolate and purify.

3. It should be easily introduced into the host cells. In other words transformation of the host with the vector should be easy.

4. The vector should have suitable marker genes that allow easy detection and/or selection of the transformed host cells.

5. When the objective is gene transfer, it should have the ability to integrate either itself or the DNA insert it carries into the genome of the host cell.

6. The cells transformed with the vector containing the DNA insert (recombinant DNA) should be identifiable be selectable from those transformed by the unaltered vector.

7. A vector should contain unique target sites for as may restriction enzymes as possible into which the DNA insert can be integrated.

8. When expression of the DNA insert is desired, the vector should contain at least suitable control elements, e.g., promoter, operator and ribosome binding sites.

Plasmids Serve As Vectors

• Plasmids are considered "replicons", capable of autonomous replication within a suitable host.

• Plasmids can be found in all three major domains: Archea, Bacteria and Eukarya.

• Similar to viruses, plasmids are not considered by some to be a form of "life".

Methods for Inserting Foreign DNA Into Cells

• Cells can take up naked DNA by transformation. Chemical treatments are used to make cells that are not naturally competent take up DNA.

• Pores (holes) can be made in protoplasts and animal cells by electric current during the process of electroporation to provide an entrance for DNA.

• Foreign DNA can be introduced into plant cells by shooting DNA-coated articles into the cells.

• Foreign DNA can be injected into animal cells by using a fine glass micropipette.

Why Genetic Engineering?Why Genetic Engineering?

• 3000 Known Genetic Diseases

Crop ImprovementCrop Improvement

• The improvement of crops with the use of genetics has been occurring for years. Traditionally, crop improvement was accomplished by selecting the best looking plants/seeds and saving them to plant for the next year’s crop.

DNA ExtractionDNA Extraction

• DNA extraction is the first step in the genetic engineering process. In order to work with DNA, scientists must extract it from the desired organism. A sample of an organism containing the gene of interest is taken through a series of steps to remove the DNA.

Gene CloningGene Cloning

• The second step of the genetic engineering process is gene cloning. During DNA extraction, all of the DNA from the organism is extracted at once. Scientists use gene cloning to separate the single gene of interest from the rest of the genes extracted and make thousands of copies of it.

Gene DesignGene Design

• Once a gene has been cloned, genetic engineers begin the third step, designing the gene to work once inside a different organism. This is done in a test tube by cutting the gene apart with enzymes and replacing gene regions that have been separated.

TransformationTransformation

• The modified gene is now ready for the fourth step in the process, transformation or gene insertion.

• Since plants have millions of cells, it would be impossible to insert a copy of the transgene into every cell. Therefore, tissue culture is used to propagate masses of undifferentiated plant cells called callus. These are the cells to which the new transgene will be added.

Backcross BreedingBackcross Breeding

• The fifth and final part of producing a genetically engineered crop is backcross breeding. Transgenic plants are crossed with elite breeding lines using traditional plant breeding methods to combine the desired traits of elite parents and the transgene into a single line. The offspring are repeatedly crossed back to the elite line to obtain a high yielding transgenic line. The result will be a plant with a yield potential close to current hybrids that expresses the trait encoded by the new transgene.

Common Terms (FYI Only)Common Terms (FYI Only)

• Agbiotech = the agricultural arm of the biotechnology industry• Biotech = the biotechnology industry• GE = genetic engineering/genetically engineered• GM = genetically modified• GMO = genetically modified organism• Pharm crop = a GE crop that creates its own pharmaceutical byproducts in virtually all parts of the plant• Transgenic = another name for GE

GMO / Genetically Modified GMO / Genetically Modified OrganismOrganism

• A GMO is a plant, animal or microorganism (e.g., bacteria) that is created by means that overcome natural boundaries.

• Genetic engineering involves crossing species which could not cross in nature.

• For example, fish genes have been inserted into strawberries.

• The most widely grown GE crops are soybeans, corn, canola (rapeseed) and cotton. Nearly all GE crops grown today are one of two varieties: "insect resistant" and "herbicide tolerant" crops.

Good Reasons Do ExistGood Reasons Do Exist

• For example, the splicing of a specific flounder gene for producing a unique blood "antifreeze" protein into tomatoes, to render them frost resistant; splicing insect proteins into zucchinis to create a taste and fragrance that is repugnant to other insect pests; or growing potatoes endowed with built-in pesticides.

According to the FDAAccording to the FDA

• While the Food and Drug Administration insists that foods produced by genetic engineering are the same as foods from traditional breeding, their own scientists reported that, "the processes of genetic engineering and traditional breeding are different and... they lead to different risks."

GENETIC ENGINEERING: A GENETIC ENGINEERING: A CAUTIONARY APPROACHCAUTIONARY APPROACH

• The Institute of Science, Technology and Public Policy has taken a strong precautionary stand on genetic engineering. In collaboration with leading scientists and other public service organizations, it has launched a nation-wide public awareness campaign to alert the public about the dangers of genetically engineered foods, and is calling for rigorous safety testing and mandatory labeling of such foods.

Humans?Humans?

• The biotech industry's rationale for the genetic engineering of humans is the predisposition of human beings to certain diseases. If such human frailties could be fortified by genetically transplanting traits of other animals, insects, bacteria or viruses, then it might be possible for biotechnologists to improve upon our species.

The ArgumentThe Argument

• Genetic surgery performed on fetuses would, with high probability, infect the germ line (egg or sperm) cells.

• As a consequence, any such genetic defects would be passed on to future generations, causing irreversible gene pollution and the potential for new genetic diseases.

• In addition to the immediate and long-term gross health risks posed by irreversible gene pollution, we have no idea what the subtle effects of incomplete or mutated human DNA will be on the human race.

The Fact Exists That There Are Some Diseases The Fact Exists That There Are Some Diseases Out There We Cannot Cure Without Genetic Out There We Cannot Cure Without Genetic

Engineering. Engineering.

Take a Stand. Have a View. Take a Stand. Have a View.

But Ask Yourself…Would That View Change If It Were But Ask Yourself…Would That View Change If It Were Your Child?Your Child?

Real Life Real Life ExampleExample

SCIDSCID

Babies born without immune systems quickly become ill during weaning when the protection of their mother's milk – which contains maternal antibodies – begins to wear off. Withouttransplant medicine of some kind, the only way of keeping these children alive is to cocoon them in a sterile environment free of potentially lethal microbes.In the Sixties and Seventies, hospitals put babies with severe combined immune deficiency (SCID) in plastic "bubbles" where the the air is filtered and direct contact with the outsideworld is minimized.

Paul Simon

Boy in the Bubble

Rhys Evans (April 2002 UK)Rhys Evans (April 2002 UK)

• Gene Therapy used to cure a toddler of SCID

• Took Bone Marrow from the child, then used a virus to carry a new version of the gene into the immune cells from the marrow.

PCR• What is a polymerase? A polymerase is a naturally

occurring enzyme, a biological macromolecule that catalyzes the formation and repair of DNA (and RNA).

• The accurate replication of all living matter depends on this activity -- an activity scientists have learned to manipulate.

• In the 1980s, Kary Mullis at Cetus Corporation conceived of a way to start and stop a polymerase's action at specific points along a single strand of DNA.

PCR

• The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

A strip of eight PCR tubes, each containing a 100 μl reaction mixture

• The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.

• There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

3 Major Steps to PCR

1. Denaturation at 94°C :

2. Annealing at 54°C :

3. Extension at 72°C :

1. Denaturation at 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).

2. Annealing at 54°C :The primers are jiggling around, caused by Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.

3. Extension at 72°C :This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions.

Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment.

The bases (complementary to the template) are coupled to the primer

Why PCR?

• PCR, the quick, easy method for generating unlimited copies of any fragment of DNA, is one of those scientific developments that actually deserves timeworn superlatives like "revolutionary" and "breakthrough."

To Sum It All Up

• PCR makes it possible to have enough DNA to trace genetic material back to its origin, identifying with precision at least what species of organism it came from, and often which particular member of that species.

Gene Cloning• Gene cloning is the process in which a gene of

interest is located and copied (cloned) out of DNA extracted from an organism.

• When DNA is extracted from an organism, all of its genes are extracted at one time. This DNA, which contains thousands of different genes. The genetic engineer must find the one specific gene that encodes the specific protein of interest.

• Since there is no way to locate a gene by visibly looking at all of the DNA, scientists must use other methods such as …

1. DNA Probes

2. Antibiotic-Resistance Genes

3. Gene Products

1. A DNA probe is a labeled segment of DNA or RNA used to find a specific sequence of nucleotides in a DNA molecule. Probes may be synthesized in the laboratory, with a sequence complementary to the target DNA sequence.

If the DNA probe finds a match, it will adhere to the target gene.

2. A very common method for identifying a particular protein is by using antibodies, or immunoglobulins, that bind specifically to that protein. This is often done to select antibiotic resistant genes.

3. A gene product is the biochemical material, either RNA or protein, resulting from expression of a gene. A measurement of the amount of gene product is sometimes used to infer how active a gene is.