Ch. 9 Biotechnology PDF

Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria: overview - transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-wh...

Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria: overview - transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  Biotechnology – what is it? Bacterial transformation of pGLO gene for florescent green protein being expressed in bacteria use of technology to control biological processes make products (foods, antibiotics, vitamins, enzymes) treat disease – discovering drugs or replacing faulty genes with functional genes  Bio-Technology Terms: o Genome: the complete genetic makeup of an organism o Clone: genetically identical cells or organism o Genetic Engineering: manipulating the genes of an organism o Gene Cloning: production of many exact copies of a single gene o Transgenic Organism: organism with functional foreign genes inserted o Recombinant DNA (rDNA) technology: insertion or modification of genes to produce desired proteins  Tools of Biotechnology: o Selection: selecting for a naturally occurring microbe that displays a certain characteristic or produces a desired product o Mutation: change in an organisms DNA sequence; mutagens cause mutations; some can result in microbes with a desirable trait o Site-directed mutagenesis: a targeted and specific change in a gene o Restriction enzymes: enzymes (from bacteria) that cut DNA at specific sights cut DNA at specific sequences evolved to destroy bacteriophage DNA in bacterial cells methylated cytosine's (C base) in bacterial DNA protect their own DNA from digestion create staggered cuts with complementary ends known as sticky ends allows donor and recipient DNA to have COMPLIMENTARY SEQUENCES for DNA to anneal!!! o Vectors: “vehicle” used to transfer DNA from source to target cell common vectors: plasmid and “modified virus”  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing Typical bacterial genetic modification procedure: DNA insertion via plasmid examples of products produced by genetically modified bacteria inserting genes into bacteria via a plasmid  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  Restriction Enzymes - cutting out / splicing in DNA enzymes (from bacteria) that cut DNA at specific spots source DNA contains gene to transfer Restriction Enzymes DNA to transfer o same restriction enzyme gene to transfer used to cut DNA out and used to insert DNA target DNA gene inserted into target DNA transferring a gene between organisms using restriction enzymes  Restriction Enzymes: cut DNA at specific sequences called “recognition sites” DNA with restriction enzyme recognition sites (G/GATCC) 1. DNA contains “recognition site” for restriction enzyme 2. restriction enzyme “cuts” DNA at specific sequence of recognition site cuts BOTH DNA strands “sticky ends” creates “sticky ends” over-hanging ends are complimentary to each other restriction enzymes – create “sticky ends” *NOTE* source and vector DNA both cut with same restriction enzyme; creates complimentary ends Restriction Enzymes role in creating Recombinant DNA using restriction enzymes to create “recombinant plasmids”  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  Vectors “vehicle” used to transfer DNA from source to target cell a vector must be able to self-replicate shuttle vectors exist in many different species and can move cloned sequences among various organisms plasmid viral vector natural “free-floating” circular DNA “disabled virus” able to infect but not reproduce  Plasmids o circular “rings” of DNA that exist in nature double stranded DNA naturally taken in by bacteria through transformation or conjugation once in bacteria, plasmids are replicated, transcribed and translated plasmid resistance plasmid  Plasmids: “circular rings” of double stranded DNA “inserted” gene of interest Recombinant DNA Plasmids: inserting gene from one organism into another recombinant DNA organism via a plasmid plasmid plasmids can MOVE INTO (and out of) bacterial cells replicate, transcribe and translate INDEPENDTLY of bacterial chromosome bacterial transformation  Viral Vector “disabled” virus used to infect cell and transfer DNA (gene) bacteriophage binding bacterial cell What is a Virus? DNA (or RNA) wrapped in a protein coat CAN NOT reproduce (replicate) on its own virus MUST INFECT host cell to be reproduced virus hijacks cells machinery to incorporate its DNA or RNA and have cell produce more virus  Viral Vector – a partially disabled virus used to transfer DNA into cells virus can infect cell and incorporate its DNA, but it CAN NOT make new virus gene to transfer is inserted into viral vector. gene transferred to cell when infected by viral vector DNA in protein “capsule” DNA of interest “spliced” into viral DNA cell membrane viral DNA inserted into target cell DNA incorporates into host genome transcribes and translated by host cell viral vector inserting DNA into host cell  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  5 “additional” ways DNA can be inserted into a cell: 1. Transformation: o cells take up DNA from the surrounding environment 2. Protoplast Fusion: o removing cell walls from two bacteria allows them to fuse Protoplast – cell that has had its cell wall removed 3. Electroporation: o introducing DNA into bacteria or other cells using electrical pulse to briefly open the pores in the cell membranes protoplast fusion to transfer bacterial DNA 4. Gene gun: device for delivering exogenous DNA to cells uses an elemental particle of a heavy metal coated with DNA (typically plasmid DNA) 5. Microinjection: use of a glass micropipette to inject foreign DNA into a cell Gene gun: used to insert DNA-coated "bullets" into a cell. Microinjection: the microinjection of foreign DNA into an egg cell  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  PCR – “Polymerase Chain Reaction” a way to rapidly copy a DNA sample result: millions….even billions of DNA copies in ~1hr! DNA of interest DNA quantity doubles with each round of copying PCR: polymerase chain reaction o used for creating millions of gene copies for transgenic or forensic biology o used for diagnostic tests for genetic diseases and detecting pathogens o to study gene function reverse-transcription PCR uses mRNA as template and reverse transcriptase enzyme called cDNA (complimentary DNA) – a complimentary DNA strand from an mRNA sequence  How are we able to do PCR? …because of bacteria! Thermus aquaticus thermal hotspring in Yellowstone National Park Thermus aquaticus thermophilic bacteria that lives in thermal hot springs of Yellowstone it’s DNA polymerase can withstand high temperature PCR requires high temperature to denature (separate) DNA double helix its DNA polymerase can survive the 100⁰C temp required to separate strands!!!  PCR: 4 essential items 1. DNA – DNA to be amplified 2. Primer – short DNA sequence complimentary to beginning of DNA sequence of interest 3. DNA Polymerase – enzyme that adds ducleotides 4. Nucleotides – dtp’s: dtpA, dtpT, dtpC and dtpG (dtp = deoxyribose tri-phosphate) * salt and buffers  PCR: polymerase chain reaction PCR steps: STEP 1: DENATURE DNA heat DNA to 95⁰C to separate double stranded DNA STEP 2: ANNEAL primers primer is small piece of DNA complementary to beg. of DNA seq. of interest cool DNA to 60 ⁰C and primers will “naturally anneal” by base pairing primers are designed and manufactured (cheap!) STEP 3: EXTENSION of DNA heat DNA to 72 ⁰C – functioning temperature for DNA polymerase DNA polymerase EXTENDS primer creating a copy of the DNA REPEAT these steps a million times in 1 hr. DNA quantity increases EXPONENTIALLY - doubles each time PCR steps:  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  Synthetic DNA: o builds genes using a DNA synthesis machine o nucleotides, primers, genes easily synthesized o synthetic labeled nucleotides used in Next Generation Sequencing – fast, cheap sequencing they made a synthetic bacteria!!! DNA Synthesis Machine how scary is that?  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  Selecting a clone: Blue-white screening used to identify if gene has been successfully inserted into plasmid uses plasmid vector containing ampicillin resistance gene (ampR) and β-galactosidase gene (lacZ) bacteria grown in media containing ampicillin and X-gal, a substrate for β-galactosidase (lactase) ampicillin function in agar: selects for successful transformation bacteria species normally sensitive to ampicillin only transformed bacteria that have successfully taken-in plasmid can grow blue-white screening: selecting colonies for recombinant plasmids  X-GAL function: selects for recombinant plasmids o substrate that mimics lactose o metabolism creates blue precipitate on colonies o non-recombinant transformed cells can metabolize X-gal o recombinant transformed cells can not metabolize X-gal o successful plasmid recombination disables lacZ gene White Colonies transformed cells - NOT metabolizing X-gal therefor no blue PPT gene successfully inserted into plasmid – disabling lacZ gene positive for recombinant DNA colonies - plasmid with inserted gene Blue colonies blue-white screening: transformed cells – ARE metabolizing X-gal, blue PPT selecting colonies for recombinant plasmids gene insertion into plasmid unsuccessful – functional lacZ gene these are non-recombinant cells – unsuccessful gene insertion plasmid without inserted gene blue-white screening procedure Blue-White Clone Screening Procedure: 1. Insert gene into ampR and lacZ plasmid gene inserts into lacZ of plasmid – disabling lacZ gene in plasmid can not metabolize X-gal 2. Grow on Ampicillin and X-gal media 3. Only “transformed” bacteria that received plasmid can grow on ampicillin plate 4. Bacteria with “recombinant” plasmid can not hydrolyze X-gal and produce white colonies – these are colonies we want 5. Bacteria without “recombinant” plasmid can hydrolyze X-gal and produce blue colonies  Blue – white screening: selecting recombinant plasmids White colonies recombinant DNA colonies plasmid with inserted gene what we want! Blue colonies non-recombinant cells plasmid with/out inserted gene what we don’t want blue-white screening: selecting colonies for recombinant plasmids  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  Human uses for Transgenic Biology: Gene therapy: to replace defective or missing genes Transgenic bacteria: growing human enzymes and other proteins in bacteria; such as insulin  Transgenic use of bacteria for human medicine Type-I Diabetes: pancreas can not make enough or any functional insulin hormone Insulin: insulin is a protein hormone hormone - signaling molecule signals cells to bring in glucose from blood Type-I Diabetes: body’s pancreas can not make insulin thus can not bring glucose into cells insulin protein cells starve and die  Producing human insulin in bacteria Restriction enzymes, plasmids and transformation human DNA plasmid human insulin gene Restriction Enzyme – used to cut DNA and plasmid cut DNA gene out of donor cell cut plasmid gene “anneals” into plasmid  Producing human insulin in bacteria Restriction enzymes, plasmids and transformation Transgenic Insulin Procedure: 1. Human insulin gene cut out with restriction enzyme 2 2. Plasmid cut with same restriction enzyme 1 3. Human insulin gene inserted into plasmid 3 4. Transformation – transfer plasmid INTO bacteria 4 5. Bacteria cells replicate transcribe / translate insulin gene 5 and 6 6. Harvest human insulin protein made from “inserted” human insulin gene steps to creating transgenic bacteria for producing human insulin Growing insulin hormone molecules for Type-I Diabetics: inserting DNA gene for “insulin” into bacteria 1. restriction enzymes / vector 2. vector / transformation 3. replication / harvest insulin 4. purify / bottle insulin  Ch.9 – Biotechnology o Biotechnology – what’s that? Terms, tools, and objectives o Genetically modifying bacteria – overview; transferring genes into bacteria o Restriction enzymes o Vectors – Plasmids and Viral Vectors o 5 “additional” ways to transfer DNA into cells o PCR o Synthetic DNA o Blue-white Screening – “selecting” a recombinant clone o Human uses of Transgenic Biology o Gene Silencing  Therapeutic Applications: Gene Silencing Small interfering RNAs (siRNAs): small RNA’s complementary to short sequence of a specific mRNA siRNA’s complex with “slicer protein” to form… RNA-induced silencing complex (RISC) RISC binds the mRNA and slicer protein “splices” and disables the mRNA RNA interference (RNAi): o regulating protein synthesis using siRNA’s insert plasmid containing DNA encoding siRNA for specific mRNA RNAi into cell disabling mRNA’s to regulate siRNA’s suppress mRNA and thus rate of protein synthesis for protein synthesis particular gene  Ch.9 Learning Objectives Compare and contrast biotechnology, genetic modification, and recombinant DNA technology. Describe the following terms: genome, clone, genetic engineering, gene cloning, transgenic organism, recombinant DNA technology. Describe the following biotechnology tools: selection, mutation, site-directed mutagenesis, restriction enzymes and vectors. Identify the roles of a clone and a vector in making recombinant DNA. Define restriction enzymes, and outline how they are a valuable tool used to make rDNA. Describe the terms recognition site and sticky ends relative to restriction enzymes. Describe the use of recombinant plasmids and viral vectors in transferring genes. Describe five additional ways genes can be transferred to cells. Describe PCR and its uses. Describe how/why reverse-transcription PCR is used. Discuss the four key ingredients of PCR What ingredient allows for PCR to work? What’s it called and where does it come from? Name and describe the PCR steps. What is the role of the different temperature used in each step. Differentiate cDNA from synthetic DNA.  Ch.9 Learning Objectives Discuss the process of Blue-white screening – what is it used for and how is it done? Be sure to include the role of each of the following: Ampicillin resistance gene and lacZ gene in the plasmid Ampicillin and X-gal in the agar Why and how do colonies become blue or white? What does that mean? Discuss three applications of DNA technology or transgenic biology and how they can be used to diagnose, treat or prevent disease. Describe the process of how bacteria are used to produce the human hormone insulin for used by Type-I Diabetics. Describe gene silencing, siRNA’s and RNAi. What are they and what do they do?