BIOTECHNOLOGY : NUCLEIC ACID ISOLATION and GENETIC MANIPULATION MICROBIOLOGY

* DNA fingerprinting - What type of biotechnology is it used in? - Does DNA fingerprinting use Gel Electrophoresis - WHat is DNA fingerprinting based on?

-DNA fingerprinting used in Forensic Biotechnology ( to be able to find our defendant) > Yes, DNA+ fingerprinting use Gel Electrophoresis - DNA fingerprinting Based on DNA results from gel

* Transformed Cell identification -How are transformed cell identified by? (2) - 2 Types of markers ( Define each)

-Transformed cells identified by: > the plasmid "marker" >And by disruption of plasmid metabolic identifier -Two types of markers >Selectable : only allows cells that have the plasmid to grow eg. antibiotic resistance Ex) Plasmids that have antibiotic resistance, only grows in certain media that grow antibiotic resistant >Screenable : shows a visual difference between those cells that have inserted gene vs. those that only have plasmid.

*Restriction Endonucleases: Vectors - Define Vectors >What must they be able to do? ( give 2 examples) - What are 4 Useful properties of vectors - 3 different types of vectors ^What do viruses and transposons carry?

-Vectors: Nucleic acid molecules that deliver a gene into a cell > Must be able to self replicate such as plasmids or viral genome - 4 Useful properties of vectors: > Small enough to manipulate in a lab >Survive inside cells >Contain recognizable genetic marker ( Distinguish between cells that have plasmids and cells that dont) >Ensure genetic expression of gene ( Certain gene cannot produce another product and places that they are able to work in properly) - 3 different types of vectors: >viral genomes >transposons ( jumping genes) >plasmids ^ Viruses and transposons can carry larger genes than plasmids

-Define Agricultural Biotechnology - What do the United Nations Food and Agricultural Org. predicts by 2050 will happen? -5 things that Agricultural Biotechnology does

- Agricultural Biotechnology: Increase ability for world to feeds itself - United Nations Food and Agricultural Org. predicts by 2050, we will need to feed a world population of 9.1 billion! This requires raising food production by approximately 70%! -5 things it does: >Plants more environmentally friendly that yield more per acre (genetically engineered) >Resistance to diseases and insects >Foods with higher protein or vitamin content >Drugs developed and grown as plant products >These better plants ultimately reduce production costs to help feed the growing world population

-Define Animal Biotechnology - 3 things it does

- Animal Biotechnology: animals used to produce proteins and medicines and experiments human disease( Animals used to grow and doing experiment on them to see human disease to help cure diseases) - 3 things it does > Animals as a source of medically valuable proteins *Antibodies *Transgenic animals >Animals as important models in basic research *Gene "knockout" experiments *Design and testing of drugs and genetic therapies >Animal cloning *Source of transplant organs

* Clone Selection - Define Clone -2 essential components

- Clone: Formed by selective cells containing recombinant vector is grown in culture ( Many copies of gene of interest) - 2 essential components: > X-gal substances for Beta Galactosidase ( Breaks down lactose) > antibiotic ampicillin ( prevents growth of bacteria that has not recieve ampicillin gene from plasmid)

*Genetic manipulation to produce a specific biological chemical - What does current biotechnology do with natural occurring substances? - 5 natural occurring substances that are manipulated ( short description of where they take it from) >Insulin >Penicillin >Spider web protein >Human growth factor >Erythropoetin

- Current biotechnology takes natural occurring substances and making it occur faster that it normally would ->Insulin: taking Genes out of humans to grow in bacteria to help people with diabetes >Penicillin: penicillin gene taken out of mold and put in bacteria cheaper to make >Spider web protein: spider thread resist breaking "tension strength" , taking gene from protein and put it in goats to make spider proteins in milk and spin out webs to isolate protein >Human growth factor—->Used to treat disorders in humans produced from bacteria >Erythropoetin——————> used to treat disorders in humans (produced from bacteria)

*Separation of DNA Molecules: Gel Electrophoresis - How does the Gel Electrophoresis relate to PCR -Define Electrophoresis -WHat does Gel electrophoresis allows scientists to do? > How is DNA seperated? > If the size of the DNA is known what can be pinpointed? WHat can be used?

- Gel Electrophoresis relates to PCR because it it used to see the Amplificatiion of DNA from the PCR -Electrophoresis: involves separating molecules based on electrical charge, size, and shape -Gel electrophoresis allows scientists to isolate DNA of interest >DNA is seperated by size > If the size of the DNA is known we can pinpoint the piece of DNA based on where it is on the cell and use Gel electrophoresis to isolate it

- Define Gene therapy technology - Does it Still have barriers to overcome before this technology becomes safe and effective? - what 3 Obstacles does it include?

- Gene therapy technology: Replacing or augmenting defective genes with normal copies of the gene - yes, it Still have barriers to overcome before this technology becomes safe and effective -3 Obstacles include: >How can normal genes be delivered to virtually all cells in the body? >What are the long-term effects of introducing extra genes in humans? > What must be done to ensure the proper protein is made after the genes are delivered to the body?

* DNA in Cells -In Eukaryotic cells all the DNA is found where and what does it form? > some cells, what type of DNA is there? (Define general term) - >Are plasmids used more than Genomic DNA ? > When used as a vector what does it do? > Because of the Plasmid size how does it Isolate genomic DNA ? - Define Recombinant DNA

- In the nucleus and forms a necessary part of the genetic make-up of the cell > in some cells, there is Plasmids: there may be other, small piece of DNA -> Yes, plasmids are more used than Genomic DNA > When used as vectors, it moves desired piece of DNA around > it isolated genomic DNA from Plasmid DNA -Recombinant DNA: circular pieces of DNA in cytoplasm In cell can be extracted from the cells, changed and replaced giving new properties to the cell

*Improvements in Nutritional Value and Yield - WHat improvements have been made to Tomatoes? > WHy is this? - WHat does BGH allow cattles to do? - Where has β-carotene (precursor to vitamin A) been inserted? *Scientists considering transplanting genes coding for entire metabolic pathways*

-Tomatoes allowed to ripen on vine and shelf life increased > Because Gene for enzyme that breaks down pectin are suppressed -BGH allows cattle to gain weight more rapidly, have meat with lower fat content, and produce 10% more milk -Gene for β-carotene (precursor to vitamin A) inserted into rice

* DNA Technology -what tools are used? - 5 parts to Manipulate genes involved in producing biotechnological products? (Define each) > DNA extraction and purification >Enzymes that interact with DNA and RNA >Plasmid DNA >Host organisms >Electrophoresis

-Tools :Using natural processes of replication, transcription, translation, etc. to manipulate genes. - 5 parts to Manipulate genes involved in producing biotechnological products: > DNA extraction and purification: Precise Gene we are interested in >Enzymes that interact with DNA and RNA: Be able to manipulate enzyme >Plasmid DNA: be able to construct & manipulate plasmids DNA >Host organisms: Have a set of organisms to have DNA—> RNA, and RNA—> Protein >Electrophoresis: separates pieces of DNA and RNA based on size

*Restriction Endonucleases: Gene cloning and recombinant DNA -Give an example of Gene cloning and recombinant DNA of Insuline with E.coli

-With insuline, it is identifies which chromosome the insuline gene is on -The insuline gene is then cut out with Endonucleases - Plasmid is pulled out of E.coli - E.coli is cut with the same Endonucleases - Insuline gene and E.coli are combined together using enzyme DNA ligase, and now a brand new piece of Cellular DNA ( recombinate DNA) is made - If new piece is sticked into cell, cell should be able to produce the product of insuline chain - Insuline is now produced by bacteria, so it no longer needs to be extracted out

*Regulatory Biotechnology - 2 types of Regulatory Biotechnology (Define) - Together what does QA and QC do together?

>Quality Assurance (QA): making sure processes and tests companies's preform on product is actually telling companies information it wants (All activities involved in regulating the final quality of a product) > Quality Control (QC): Monitors process and products itself ( Part of QA process that involves lab testing and monitoring of processes and applications to ensure consistent product standards) -Together QA and QC ensure that biotechnology products meet strict standards for purity and performance

- Define Medical Biotechnology > 3 things it does - What does the new information from the Human Genome Project allow? - What type of technology does it involve > Define stem cell, and what is it currently used for?

- Medical Biotechnology: Involves treatment of disease, involves prevention of disease (Involved with whole spectrum of human medicine ->Preventive medicine) >Diagnosis of health and illness >Treatment of human diseases -Human Genome Project allows for Gene therapy - involves Stem cell technologies >Stem cells - grown in lab and then treated with different chemicals to allow them to develop into specific kinds of tissues needed for transplant >Current use: stem cells are used for diabetes; spinal cord injuries ( Helps cure genetic defects)

* Modern Biotechnology > what type of activities does Modern Biotechnology involve doing with genes? - 3 Modern Examples ( Short descriptions for each ) >Gene cloning >Genetic engineering >Recombinant DNA ( rDNA) technology -Human Genome Project did what 4 things

- Modern biotechnology involves isolation, identification, and manipulation of specific genes to alter living organisms and the way they grow - Modern Examples >Gene cloning: repetitive of production of a single gene >Genetic engineering: Inserting gene to change the way organisms function, can also creates medicines and vaccines using genes of organisms >Recombinant DNA technology: bring together genetic materials from multiple sources -Example: Human Genome Project -development of disease-resistant plants -food crops that produce greater yields -"golden rice" engineered to be more nutritious -genetically engineered bacteria that can degrade environmental pollutants

* Ethics and Safety of Recombinant DNA Technology (Conti.) - Have Studies shown any risks to human health or environment so far? - What are imposed in labs involved with recombinant DNA technology - What type of wepon can be used with this same technology?

- No, Studies have not shown any risks to human health or environment so far - Standards imposed on labs involved in recombinant DNA technology -Can create biological weapons using same technology

* Process of how the Restriction Endonucleases works >What is the Restriction site called on the DNA? (Define) ^ are all sides called this? —————————————- - State the 3 steps of the process of how the Restriction Endonucleases works with "Sticky ends " ————————————- - How does this process works with blunt ends? (3 steps) ------- * Which are more important for rDNA, sticky ends or blunt ends? And Why?

- Palindrome: Sequence is same on one side and the other side ( 5'->3' & 3'->5' are same sequence) ^ Not all site called this but most are —————————————— 1. With Palindrome , Restrictive enzyme breaks 2 DNA into "sticky sides"( sequence base from 2 different DNA that are complementary base pairs that allow the different stands to bind to one another) 2. Restrictive fragments from 2 different organisms are cut by the same restrictive enzyme( enzyme breaks hydrogen bonds) ( They are staggered cuts in the 2 strands ( Not directly opposite from each other)) ( If the 2 pieces of different DNA are digested, by the same restrictive enzyme, as long as 2 molecules have same ends they are able to bind to each other, regardless if the DNA comes from 2 different species) 3. These 2 Restrictive fragments use the enzyme DNA Ligase to one combine with one another from the sticky ends creating recombinant DNA molecules ( repairing hydrogen bonds) —————— 1. 2 different strands of DNA is split by Restrictive enzyme creating blunt ends ( DNA split down the middle in same place) 2. These 2 different strands of DNA do not have complementary bases, so the blunt ends of the 2 different pieces of DNA bind together with one another ( look as if they are shoved together) 3. After blunt ends use enzyme DNA Ligase to bind together , Recombination DNA molecules are made ---------------- * Sticky ends are most important for rDNA because they can join 2 different pieces that were cut by same restricitve enzyme ( "Sticky ends" stick to stretches of single stranded DNA by complementary base pairing)

* Plasmids in detail - Define plasmids > Is there many copies of plasmids in the cell? - with plasmids occurring naturally in cells what properties are they responsible for? (3) - What do plasmids do with material? - Do some plasmids capable of exisitng in several other species? > Is Plasmids a primary vector? > What type of vectors are they called and why - How do plasmids modify cells?

- Plasmids : self-replicating, pieces of DNA, not tied to the replication cycles of the nuclear DNA > yes, there May be many copies of a plasmid inside a cell -Plasmids occurring naturally in cells may be responsible for unique properties like drug resistance , toxin production or other such non-essential traits. - Important part in manipulating material - Yes, some plasmids capable of exisitng in several other species -Yes, plasmids are primary vectors used - Called " shuttle vectors" because they are capable of moving cloned DNA sequences amaong organisms - Plasmids modify cells by being inserted into cells by transformation ( Procedures during which cells can take up DNA from the surround environment)

*Polymerase Chain Reaction (PCR) - Define Polymerase Chain Rection -Is PCR an enzyme? - where does PCR occur? - WHat does it target and is it specific? How is it specific? - What is PCR used for as a test? --------------------------- - Describe its process (7)

- Polymerase Chain Rection: Small sample of DNA can be amplified, not ENTIRE genome -Yes, PCR is an enzyme -PCR occur in test tubes - Targets the way of replicating DNA done outside of cell . Yes it is specific becuase it only amplifies small sequence of DNA as determined by choice primer -Used for a diagnostic test for the presencre of infectious agents that may be undectable ---------------------------------------------------------- - >Starts with single cell, singe gene ( area of interest) > DNA is heated, DNA is denatured, heat breaks H-Bonds holding strands together > Once strands broken, all bases on strands are free to attach to other things > 4 Nucelotides, DNA primer (Nucleic Acids that are complemetary to the ends of target DNA and will cross breed for fragments to be amplified) , and DNA polymerase ( Allows for the synthesis of New DNA strands) are mixed with bases > Solution cooled down, and DNA pieces bind to each other > Primer bind to end part of DNA , DNA polymerase will run along DNA adding nucleotides > After time passes New DNA is made > After each cycle of synthesises, DNA is heated to convert all new DNA into single strands ( As long as there are a lot of sites and nucleotide cycles can be run again and again, hundreds and millions of copies of desired pieces of DNA made)

- What does Forensic Biotechnology use? > What 5 things does it do?

- Uses DNA fingerprinting - 5 things : >Inclusion or exclusion of a person from suspicion >Paternity cases >Identification of human remains >Endangered species >Tracking and confirmation of the spread of disease done with corona virus

*Pest Resistance -Do Bt- toxin naturally occur and where is it extracted from ? Is it harmful to insects, is it biodegrabable? > Where are Bt-toxins inserted into? - What is the purpose of the pest resistance?

- Yes, Bt-toxin ( protein) naturally occurring and extracted from bacillus) harmful to insects only, and biodegradable > Gene for Bt-toxin inserted into potatoes, cotton, and corn - Organic farmers have used to reduce insect damage to crops so when insects eat plants they will be killed

*Restriction Endonucleases ( cont) -when DNA is spliced into a plasmid it forms what? - Give an example of how a Recombination vector is made ? * Are ampft and lacZ Gene, marker genes

- when DNA is spliced into a plasmid it forms a vector -Example: 1. There is a Plasmid vector with restrictive site within lacZ Gene( ecodes for Beta galactosidase which breaks down lactose ) and ampft ( ecodes for antibiotic ampicillin resistance) , the restrictive site within lacZ Gene vector is split open by a restrictive enzyme 2. A Foreign piece of DNA has a restrictive site with the gene of interest in it that is broken apart ( from rest of DNA strand) with same restrictive enzyme that split plasmid vector. 3. The vector that was split open, and the piece of foreign DNA fragment is mixed together( by being inserted into the LacZ Gene) under conditions that favor the base pairing 4. DNA Ligase join DNA pieces together 5. A Recombination Vector is created carrying the gene of interest > Summary of Above: some plasmids( which are small pieces of DNA) , that come from bacteria have restriction sites cut with specific Restriction Endonuclease . DNA is cut Is cut with same Restriction Endonuclease that works on plasmid. Then DNA piece is Carved out that has sticky ends that match each other and bind to each other. These two have created recombination vector * Yes, they are marker genes

* Biotechnology : Food modifications Example ( know at least 4 and where it comes from )

-Beer—> comes from sugar water -Cheese-> comes from milk -Yogurt->comes from milk -Tempeh-> comes from soil beans -Tofu-> Comes from soil beans -Sauerkraut-> comes from cabbage -Soy sauce->comes from extract of soil plants -Wine-> comes from grapes

- Define Bioremediation > What is an example of this?

-Bioremediation: The use of biotechnology to process and degrade a variety of natural and manmade substances ( things that contribute to environmental pollution) >Example - stimulated growth of bacteria that degrade components in crude oil -1989 Exxon Valdez oil spill in Alaska -2010 Deep Water Horizon spill promoted research into natural oil-degrading organisms and enzymes

*What is biotechnology? -Define Biotechnology - Define rDNA ( Recombinant DNA) - What are 3 Historical examples ( Give short description for for each) - Define artifical breeding > Is corn and dog artificial breeding? > why do we artificially breed?

-Biotechnology: using living organisms, or the products of living organisms, for human benefit to make a product or solve a problem -Recombinant DNA (rDNA): circular pieces of DNA in cytoplasm In cell can be extracted from the cells, changed and replaced giving new properties to the cell (Inserting, deleting or modifying genes) -3 Historical Examples >Fermentation: making wine and beer using yeast >Selective breeding: making bigger animals and crops >Use of antibiotics: improve health of animals -Artificial breeding: Select desirable breeds of animal or strains of plants to cultivate >Yes > using artificial breeding to improve crops and animals and to make them grow bigger, better and faster

-Define DNA extraction > What size of molecules are DNA, where are they found and how are they extracted? > Where is DNA released? > Where is DNA isolated from ? ^ How can DNA isolation occur?

-DNA extraction: Has and identifies DNA molecule, gets it out of cells and isolates it from cell > DNA : large molecule inside of cells Cell membrane (cell wall degraded) has to lysed >DNA released into solution > DNA isolated from other cell constituents: proteins, and lipids ^ DNA isolation can be done with individual steps or "quick-prep" filters: by lysing cell and adding cell to buffer and filter it out to get pure DNA product

* Inserting DNA into cell: Gene Therapy -Define Gene Therapy -How do some patients immune systems react to gene therapy? And why does it respond in that way? -Has Gene Therapy been sucessesful with trating patients with severe combined immunodeficiency disease? How ? - WHat are 4 diseases that respond well to Gene therapy? * Can it possibly provide a cure for genetic diseases?

-Gene Therapy: Missing or defective genes replaced with normal copies -Some patients' immune systems reacted negatively because it created an immune response against things that stayed fixed in the body (introduced genes) - Yes, gene therapy has successfully treated patients with severe combined immunodeficiency disease by increasing immune defense - 4 diseases that respond well to egen therapy: > Cystic fibrosis >sickle cell anemia > some types of hemophilia >Some types of diabetes * Yes, it can possibly provide a cure for genetic diseases

*Applications of Recombinant DNA Technology - Genome Mapping of many species includes what processes ? (2) - Why do we use DNA Fingerprinting ? - Does it involve Gene therapy? -4 Things Agricultural Applications include * Define Transgenic organisms

-Genome Mapping includes >Locating genes > Nucleotide sequencing ( We have to limit ability of PCR based on given nucleiotides) -DNA fingerprinting used for DNA analysis - Yes, it involves Gene therapy -4 things that Agricultural Applications include: >Herbicide resistance >Pest resistance >Improvements in nutritional value and yield >Transgenic organisms - recombinant plants and animals altered by addition of genes from other organisms

* Genome Mapping -Define - WHat useful facts does it provide? (3) - WHat does DNA sequencing determine?

-Genome Mapping: Locating genes on a nucleic acid -Provides useful facts concerning metabolism, growth characteristics, and relatedness to others -DNA sequencing determines the precise sequence of bases (A,T G, C)

*Herbicide Resistance (Plant crop manipulation) - Define Glyphosate (Roundup) > What is it considered? and how is it absorbed? -What does the Gene from the bacteria Salmonella create resistance towards ? >How are farmers able to kill weeds without killing crops? > What does this help reduce

-Glyphosate (Roundup): kills all plants, more specifically kills weeds > Considered a contact Herbicide, which is absorbed through the leaves -Gene from the bacteria Salmonella conveys resistance to glyphosate >Farmers can kill weeds without killing crops because a gene from Salmonella is transfered to the desired crops, which cause the crops to also become resistant to Glyphosate > Modifying crops also helps to reduce tilling and soil erosion

-Define Microbial Biotechnology > What 3 things does it do?

-Microbial Biotechnology - manipulation of microorganisms such as yeast and bacteria >Create better enzymes >More efficient decontamination processes for industrial waste product removal > Used to clone and produce large amounts of important proteins used in human medicine

* Types of Biotechnology ( short description for each) -Microbial Biotechnology -Agricultural Biotechnology -Animal Biotechnology -Forensic Biotechnology -Bioremediation -Aquatic Biotechnology -Medical Biotechnology *What type of biotechnology does all biotechnology have and why is this?

-Microbial Biotechnology: only looks at microbes ( Manipulation Microorganisms) - Agricultural Biotechnology: Altering agricultural plants -Animal Biotechnology: change genetic make up of animals -Forensic Biotechnology: using tools of biotechnology to survive crimes -Bioremediation: clean up toxic spills and contamination in environment -Aquatic Biotechnology: things live in water, increase ability of growing fish -Medical Biotechnology:Looking at producing substances can be therapeutic, used to correct genetic defects in humans *All types of biotechnology have Regulatory Biotechnology which have certain types of regulatory systems, and all areas make sure genetic changes that does not do any harm

*Techniques of Recombinant DNA Technology - What 4 repeating processes are there?

-Polymerase Chain Reaction (PCR) -Clone Selection -Separation of DNA Molecules -Inserting DNA into Cells

-Define Restriction Endonucleases >Does it matter the source of DNA?What needs to be present for the Restriction Endonucleases to do its job?

-Restriction Endonucleases: enzymes that could cut DNA) by breaking hydrogen bonds) in the middle of the strand and leave ragged ends that could be specifically bound to other pieces of DNA, allowed the ability to cut and paste different pieces of DNA together > No, it doesn't matter the source of DNA, as long as the " sticky ends" or blunt ends exist, then a particular Restriction Endonucleases can cut it it in a certain way that the "sticky ends"/ blunt ends can create a hybrid piece of DNA

*Ethics and Safety of Recombinant DNA Technology ( Describe what each question means ) -Routine screenings? -Who should pay? -Genetic privacy rights? -Profits from genetically altered organisms? -Required genetic screening? -Forced correction of "genetic abnormalities?"

-Routine screenings? --> Making sure product does not mutate -Who should pay?--> Who should pay for research either private interprizes which can cause companies to charge a lot of somthing that has the potential to be benifical globally -Genetic privacy rights?--> Right to know/ have genetic information -Profits from genetically altered organisms?--> Patenting gene -Required genetic screening?--> Ability to know if employee is sick or not -Forced correction of "genetic abnormalities?"---> Fix any genetic abonomalities that may arise

*Ethics and Safety of Recombinant DNA Technology (Cont.) -Define Supremacist view - Are the Long-term effects of transgenic manipulations known? - Is it possible for Unforeseen problems arise from every new technology and procedure -What could Natural genetics do? - What negative side affects can Transgenic organisms do?

-Supremacist view - humans are of greater value than animals - No, Long-term effects of transgenic manipulations are unknown - Yes, it is possible for Unforeseen problems arise from every new technology and procedure -Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms -Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

* Aquatic Biotechnology - >Define Aquaculture >Define Genetic engineering (3 examples of this in Aquatic biotechnology) > Define Bioprospecting (1 example)

> Aquaculture - raising finfish or shellfish in controlled conditions for use as food sources 50% of all fish consumed by humans worldwide > Genetic engineering- Inserting gene to change the way organisms function, can also creates medicines and vaccines using genes of organisms ( in this case using marine life ) *Disease-resistant strains of oysters *Vaccines against viruses that infect salmon and other finfish, * Transgenic salmon that overproduce growth hormone > Bioprospecting: rich and valuable sources of new genes, proteins and metabolic processes with important applications for human benefits * Marine plankton and snails found to be rich sources of antitumor and anticancer molecules

* Overview of Genetic Modification Procedures (5 steps)

1. Vector such as plasmid is isolated 2. DNA containing the gene of interest from different species is cut out by an enzyme into fragments 3. Desired gene is selected and inserted into a plasmid 4. Plasmid is taken up by a cell such as bacterium 5. Cells with gene of intrest are cloned to either: A) create and harvest copies of genes OR B) create and harvest protein products of a gene