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pGLO lab The importance of DNA The DNA double helix is the code of life • DNA is the code to make proteins • The blueprint for all structures in your body which are made of protein • DNA is comprised of nucleotides Nulceotides are the monomers of nucleic acid polymers • Consist of a sugar, a phosphate, and a nitrogen-containing base • Sugar can be deoxygenated • Bases contain the genetic information There are 4 kinds of DNA bases Adenine always matches with Thymine, Cytosine always matches with GuanineHydrogen bonds hold bases together Living things are extremely complex • Cellular machinery is sophisticated and required for life • Cellular machinery is made largely of proteins • Blueprints for all cellular machinery are contained in genes • Genes are inherited from parents • Humans have ~30,000 genes Proteins give living things the variety of their structures Protein variety is generated by 1o structure- the sequence of amino acids which make the protein Amino Acids • Proteins consist of subunits called amino acids Figure 2.12 How DNA works • Replication • Transcription • Translation The sequence of DNA bases is the code for the primary structure of proteins All cells require a copy of the genome • • • • Genome- all the genes of the cell Human genome is made of DNA DNA is similar in all cells Gene- 1 DNA Molecule (+ proteins the genetic information to produce a single product (protein) • DNA replication copies all cellular DNA Replication of DNA Figure 21.2 The DNA code Computers use binary digital code 01000011 01101000 01100001 = A 01100101 01100101 01100010 =B 01110011 01100101 01000011 =c 01100010 01110101 01110010 01100111 00100111 = apostrophe 01100101 01110010 Etc. 00100000 01000100 01100101 01101100 • http://www.geek01110101 01111000 notes.com/tools/17/tex 01100101 = t-to-binary-translator/ cheeseburger deluxe • • • • • How does the DNA code work? • atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaa gttcgtatggaaggttccgttaacggtcacgagttcgaaatcga aggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgct aaactgaaagttaccaaaggtggtccgctgccgttcgcttggga catcctgtccccgcagttccagtacggttccaaagcttacgttaa acacccggctgacatcccggactacctgaaactgtccttcccgg aaggtttcaaatgggaacgtgttatgaacttcgaagacggtggt gttgttaccgttacccaggactcctccctgcaagacggtgagttc =GFP The DNA code is (nearly) universal It uses groups of 3 bases (codon) 3 bases = 1 codon = 1 amino acid The Central Dogma of Molecular Biology • DNA RNA Protein • DNARNA : Transcription • RNA Protein: Translation DNA RNA Protein Trait The Universality of the DNA code makes this possible Firefly gene (Luciferase) in a tobacco plant tRNA’s carry an amino acid at one end, and have an anticodon at the other Amino acid attachment site: Binds to a specific amino acid. Amino acid (phenylalanine) Anticodon: Binds to codon on mRNA, following complementary base-pairing rules. Anticodon mRNA Figure 21.6 The ribosome matches tRNA’s to the mRNA, thereby linking amino acids in sequence In this way, the proteins in nature are virtually limitless Proteins are incredibly diverse at the molecular level A few examples Insulin Rubisco ATP synthase Fibrin Nitrogenase Protein function depends greatly on shape Because the DNA code is universal, genes can be moved from one living thing to another Cell with gene of interest Bacterium Step 1: Isolate DNA from two sources. Step 2: Cut both DNAs with the same restriction enzyme. Plasmid Source (donor) DNA Fragments of source DNA Step 3: When mixed, the DNAs recombine by base pairing. Figure 21.14 (1 of 2) When one DNA molecule is copied to make two DNA molecules, the new DNA contains 1. 2. 3. 4. 5. A) 25% of the parent DNA. B) 50% of the parent DNA. C) 75% of the parent DNA. D) 100% of the parent DNA. E) none of the parent DNA. DNA in action • HIV and AIDS • Genetic engineering • pGLO Genetic Engineering allows DNA to be moved from one organism to another Figure 21.14 Green Fluorescent Protein • discovered in 1960s by Dr. Frank Johnson and colleagues • closely related to jellyfish aequorin • absorption max = 470nm • emission max = 508nm • 238 amino acids, 27kDa • “beta can” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore • amino acid substitutions result in several variants, including YFP, BFP, and CFP 40 Å 30 Å The pGLO plasmid has genes which can be turned on and off • ori- origin of replication • GFP- green fluorescent protein • bla- Beta-lactamase • araC- Arabinose • What are all the other marks? Why are they there? 2008 Nobel Prize- GFP • GFP mice On pGLO, the regulatory regions of the Arabinose operon have been glued to the structural sequences for GFP ara GFP Operon ara Operon B ara C A D araC GFP Gene Effector (Arabinose) Effector (Arabinose) Gene Regulation B A D araC araC GFP Gene RNA Polymerase RNA Polymerase araC B A D araC GFP Gene What will happen on the Ara (+) plates? What will happen on the Ara (-) plates? Grow? Glow? Which colonies will glow? Follow protocol On which plates will colonies grow? Which colonies will glow? 5/23/2017 34