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Lecture1-October 24.docx

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Queen's University
BIOL 205
Kenton Ko

Oct 24/ 2011 – Lecture 1 1. Introduction to Synthetic Biology Central Dogma - DNA can replicate itself and RNA is produced from DNA. - Epigenetics plays a role through modification of histones when DNA is transcribed into RNA - RNA can replicate itself and produce proteins or turn back into DNA through reverse transcriptase (found in viruses) - Some RNA does not produce protein and these are miRNAs Synthetic Biology - Construction of DNA to build organisms that can perform tasks - Develop new biofuels - Drug production - Environmental cleanup Micro RNAs - It was discovered that RNA can catalyze the splicing of themselves - Micro RNAs are short RNAs that have emerged as a new means of diagnosing and treating disease - miRNA blocks translation of mRNA into protein and initiates breakdown of normal mRNA. - Some up or down regulate a single gene or regulate multiple gene networks. o Scientists found that when miR-21 overexpression caused tumors that regressed with expression was switched off. Implantable - By directly manipulating the activity of individual neurons, scientists have given sensors/Optogenetics flies memories of a bad experience they never had - Other researchers used light to induce normal patterns of muscle contraction - This new approach allows scientists to more accurately reproduce muscle firing order - Channel rhodopsin 2 is activated by blue light and causes action potentials - This depolarization activates release of neurotransmitter at synapse between neurons (turn on a nerve) - Halo-rhodopsin generates a chloride flux when activated by yellow light which causes hyperpolarization which prevents action potentials (shuts off nerve) Nanotechnology - McGill produced DNA nanotubes that encapsulate and load cargo and then release it rapidly and completely when a specific external DNA strand is added - Nanohealing works by self-assembling peptides that forms a transparent, nanofiber-rich meshwork and allows wounds to begin healing quickly Oct /26/2011 – Lecture 2 1. Risk and Biotechnology - Genetically modified food, pharming, stem cell research, human cloning, xenotransplanation , genetic screening, alternative fuels, bioremediation, biopharmaceuticals, bioterrorism all lead to public distrust of biotechnology - In the case of biotechnology, the perceived purpose of the product is the positive driver and the process appears to be the negative one - Scientists fail in communicating the public about biotechnology in language people can understand - The general public perceives industry as not credible - promoting over consumption; drugs for profit. 2. Discovery of DNA DNA’s role - To fulfill it’s role, genetic material must meet several criteria: 1. Information: it must contain the information necessary to make an entire organism 2. Transmission: it must be passed from parent to offspring 3. Replication: it must be copied 4. Variation: it must be capable of changes - The identification of DNA as the genetic material involved a series of experiments. S/R strain Mouse experiment F. Griffith - Studied Streplococcus pneumonia which causes pneumonia in humans and is lethal in mice - This bacterium comes in 2 strains: S (smooth) and R (Rough) o Smooth strain secretes a polysaccharide capsule that protects bacterium from the immune system of animals (lethal) and produces smooth colonies on solid media o Rough doesn’t secrete a carbohydrate capsule and produces colonies with a rough appearance – not lethal - S strain live cells – mouse dies - R strain – mouse lives - Heat killed S. strain – mouse lives - R strain and Heat killed S strain – mouse dies and S strain live cells are found - Griffith thought that something from the dead type S was transforming type R Destroying components to find into type S and called this the transformation principle genetic material Avery, MacLeod and McCarty - Prepared cell extracts from type S cells containing each of these macromolecules - By destroying the components such as lipids, polysaccharides, RNA, or protein of the S strain and simultaneously adding the R strain, the mouse died - When DNA was destroyed and R strain was added, the mouse lived. Bacteriophage Radioactivity - Thus, they discovered that DNA is the transforming agent Hershey & Chase - The bacteriophage T2 is relatively simple, containing only 2 macromolecules: DNA and protein - They used 2 kinds of radioactive labels on phage heads 1. 35S labels proteins because amino acid contains Sulfyl groups 2. 32P labels phosphate backbone of DNA and there’s limited to no P in proteins. - They then allowed the phages to infect the E.coli, then blended and centrifuged to shea35phages off the cells - In the S sample, most of the radioactivity was recovered in phage ghosts - In the P sample, most of the radioactivity was recovered in bacteria - This shows that it was DNA that the phages injected into the bacteria. 3. DNA structure - DNA is a large macromolecule with several levels of complexity: 1. Nucleotides form the repeating units and is composed of phosphate group, deoxyribose sugar and any one of four bases A, G, C,T 2. Nucleotides are linked to form a strand 3. 2 strands = double helix 4. Double helix folds, bends, and interacts with proteins resulting in 3D structures in the form of chromosomes. Purine vs. Pyrimidine - Purine nucleotides: A & G - Pyrimidine nucleotides: C & T **Pyramids are never found in the CiTy - The term deoxyribose is used because compared to the RNA, there is an oxygen missing on the 2’ position - The Phosphate is always at 5’ position and is connected to OH at 3’ - Chargaff’s rules 1. T+C = A+G 2. T=A; C=G 3. A+T doesn’t always equal G+C - These rules helped solve the structure of DNA because if you have a pyrimidine + pyrimidine together, the DNA is too thin and 2 purines make the DNA too thick - Thus, having a purine and pyrimidine gives a thickness compatible with X-ray data. DNA strand - Nucleotides are covalently linked together by phosphodiester bonds to form a Phosphodiester bonds strand of DNA, which gives the strand directionality Directionality - Phosphates and sugar molecules form the backbone of the nucleic acid strand. - 2 strands are twisted together in a right-handed helix that spirals away from you in a clockwise fashion - There are 10 bases per complete twist - The 2 strands are antiparallel which means one strand runs in the 5’3’ Antiparallel direction and the other in the 3’ 5’ DNA stability - The double-bonded structure is stabilized by: 1. Hydrogen bonding between complementary bases - A+T with 2 H-bonds - G+C with 3 H-bonds 2. Base stacking - Within the DNA the bases are oriented so that the flattened regions are facing each other- Van der Waals interaction - There are hydrophobic interactions: bases on the interior and sugar and phosphate residues are hydrophilic so on the exterior interacting with water. - Cations (Mg+) and proteins can neutralize phosphate backbone - DNA also has a major and a minor groove, with most proteins fitting into the major groove. Oct 28/2011 – Lecture 3 1. Models of DNA replication Meselson and Stahl 15 Semiconservative model - M&S grew E.coli in medium containing heavy isotope of nitrogen ( N) and the heavy nitrogen was incorporated into new DNA - Then, the cells were switched to medium containing normal light nitrogen (14N) and grown for 2 generations - The DNA was isolated and separated in cesium chloride by density 1. Parental: test tube contained heavy DNA st 2. 1 ndneration: a hybrid of heavy and light nitrogen was found 3. 2 generation: a hybrid of heavy and light as well as only light was found Conservative model - Predicted that an entirely new replicated strand is produced so you should be expecting to see: 1. Parental: heavy DNA st 2. 1 ndn: half heavy and half light 3. 2 gen: mostly light and some heavy Dispersive model - Suggests that parental DNA will result in a dispersed manner througout the first and second generations 1. Parental: heavy DNA st 2. 1 ndneration: hybrid 3. 2 gen: hybrid - We ended up seeing the pattern for semiconservative model 2. How does Semiconservative DNA replication work? Polymerase 1 - Kornberg purified DNA polymerase 1 but it’s not the major polymerase Arthur Kornberg - It moves at 20 nucleotides / s (too slow for major polymerase) - There are 400 molecules/ cell – too abundant for uniqueness - Dissociates from DNA after incorparating 20-50 nucleotides – falls off too quickly - He found that DNA replication requires deoxyribonucleotides (dNTPs) which are the building blocks of DNA and are used in PCR reactions. 5 DNA polymerases in E.coli 1. Poly 1: Major repair enzyme Q: What is the difference between 2. Poly 2: Restarts replication- reinitiate syn downstream of gaps Poly 4 and 5? 3. Poly 3: Replicase – extends DNA Poly 5 is used as a bypass for 4. Poly 4: Translesion replication: roadblocks 5. Poly 5: Translesion replication - DNA polymerases can extend a chain but cannot start a chain Synthesizing the Lagging strand 1. Primase synthesizes a short RNA oligonucleotides (primer) copied from DNA which lays down a 3’ OH end to be used by DNA polymerase 3. - A Primosome protein complex contains a ‘primase enzyme’, a RNA polymerase that synthesizes RNA complementary to specific DNA region. 2. DNA polymerase 3 elongates RNA primers with new DNA which forms an Okazaki fragment Q: Why are RNA primers used but 3. The RNA primer is degraded by 5’-3’ exonuclease and DNA polymerase 1 in PCR, DNA primers are used? removes RNA at 5’ end of neighbouring fragment and fills gap 4. DNA ligase connects adjacent fragments using ATP to catalyze a phosphodiester bond. DNA synthesis in bacteria - DNA synthesis is fast and accurate. At 2000 nucleotides/s, E.coli has 2 forks moving at 1000 nucleotides/s - In 40 minutes, the 5 million base pairs of the E.coli genome will be replicated - DNA synthesis begins at an origin of replication where replication begins bidirectionally in opposite directions - When it arrives at the site where replication ends on the opposite side of origin of replication, the chromosomes sp10ts. - There is 1 nucleotide error in 10 inserted due to Pol 1 and 3 which can recognize an incorrect base pair and reverse its direction by a base pair, excise, and correct it before continuing. Oct 31/2011 – Lecture 4 1. Initiation of Replication - The origin of replication in E.coli is termed oriC (origin of Chromosomal replication) - There are 3 kinds of DNA sequences in oriC that are significant: 1. AT rich region - conserved Q: Why do we have methylation? 2. DnaA boxes-conserved A: methylation of DNA (not to be 3. GATC methylation sites- changes confused with histone methylation) is a common epigenetic signaling tool that cells use to lock genes in the "off" position Unwinding double helix - enzymes open helix and prevent overwinding: Helicase 1. Helicase clamps to DNA and breaks H bonds ahead of DNA synthesis Topoisomerase 2. Topoisomerase: bind to either single-stranded or double-stranded DNA and cut the phosphate backbone of the DNA. This intermediate break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed again Single strand binding protein 3. Single strand DNA binding proteins: bind to unwound DNA and prevent it from re-forming. Initiation of replication 1. DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences 2. This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex 3. DnaB protein (helicase), a 6 subunit enzyme binds to the origin and travels along DNA in the 5’ to 3’ direction bidirectionally, using energy from ATP o DnaC assists in the process but is released afterwards 4. SSBPs bind to unwound DNA 5. As DNA unwinds, there are some over wound regions which have coils removed by DNA gyrase/ Topoisomerase that cuts the strands, rotates the DNa to remove coils and rejoins the DNA strands. 2. DNA replication complexes 1. Primosome:
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