BIOL4380 Lecture 1 on E1 - Students - Tagged-merged PDF

BIOL4380 Cell & Molecular Biology Laboratory Dr. Sadat and Dr. Gomez Lecture 1: E1 Basic Bacterial Techniques  Lab Safety Always wear closed toed shoes No drinking or eating allowed in the lab Tie long hair back Always wear lab coat and gloves M...

BIOL4380 Cell & Molecular Biology Laboratory Dr. Sadat and Dr. Gomez Lecture 1: E1 Basic Bacterial Techniques  Lab Safety Always wear closed toed shoes No drinking or eating allowed in the lab Tie long hair back Always wear lab coat and gloves Make sure to clean bench top before and after working in the lab Wash hands before leaving lab  When you enter the lab: Sign-in Clean table-top with ethanol Get coat card in first lab and then get your coat. From next lab always get your lab coat before the beginning of lab  Syllabus Reconnaisance Talk to one another and find points to discuss from the syllabus. After discussing for 3 minutes you can ask questions  Considerations when working with bacteria Growing bacteria in the laboratory – Liquid medium – Solid medium – Growth conditions Cultivation techniques Laboratory sterilization techniques Isolation of bacterial clones Identification of genotypes of bacterial strains  Nutritional Requirements of Cells Every organism must find in Nutritional requirements its environment all – Carbon and energy source substances required for – Growth factors energy generation and – Growth media cellular biosynthesis - Temperature nutritional requirements Oxygen pH Osmolarity Waste products Segmented filamentous bacterium (SFB) in mouse intestinal epithelium. SFB were discovered in the 1970s but wasn’t successfully grown in labs until 2012. https://www.the-scientist.com/features/lost-colonies-34760 Major Factors That Influence Microbial Growth Major elements Energy Source – Phototrophs: Carbon – Hydrogen – Use light as an energy source. – Oxygen – Use CO2 as a sole source of – Sulfur carbon – Potassium Heterotrophs: – Magnesium – Iron – Use an organic form of carbon – Trace elements Lithotrophs: Zinc – oxidize inorganic compounds Cobalt Copper (e.g., NH4) Molybdenum  Culture media Require small amounts of certain organic compounds – Purines, pyrimidines, amino acids, vitamins Some bacteria called prototrophs do not require any growth factors or additional nutrients. – __________: Synthesize all essential factors themselves – ___________: mutant strains of bacteria that require some growth factors, in contrast to normal prototroph strains which do not  Culture media Depends on special needs of a particular microorganism What kind of culture will you grow if you needed a large amount of pure bacterial culture? a. Solid culture b. Liquid culture What kind of culture will you grow if you needed to isolate a single bacterial stain from a mixture of bacteria? a. Solid culture b. Liquid culture – Agar contains unique physical properties –melts at 100oC and therefore can be sterilized and poured into plates –remains liquid until cooled to 40oC –cannot be metabolized by bacteria  Types of Culture Media Minimal medium: Selective medium: – chemically defined – components added to inhibit growth of certain types or (synthetic) medium, species of bacteria; exact composition is – promote growth of desired known species or strains or mutants Complex medium: Differential medium: – full range of growth – promotes growth of a factors for unknown desired microorganism bacteria with unknown which will have different properties compared to nutritional requirements others  Culture Media Minimal medium for Neiserria 1 L minimal medium for E. - pathogen that causes coli gonorrhea – 5 g glucose – NaCl, uracil, K2SO4, hypoxanthine, – 7 g dipotassium phosphate MgCl2, spermine, NH4Cl, hemin K2HPO4 – K2HPO4, nitrilotriethanol, CaCl2, – 2 g monopotassium phosphate polyvinyl alcohol, Glycerine KH2PO4 – sodium lactate – 0.08g magnesium sulfate – Sodium acetate MgSO4 – oxaloacetate – 20 amino acids – 1 g ammonium sulfate – glucose (NH4)2SO4 – 7 vitamins 15 g agar for solid medium  Culture Media 1 liter rich (complex) medium E. coli LB (Luria-Bertani, 1951) – 10 g tryptone (pancreatic digest of casein, a nitrogen source) – 5 g yeast extract (autolysed yeast, a carbon source) – 5 g NaCl – 1 ml 1N NaOH (optional) 15 g agar for solid medium Nutrient broth – 5 g peptone – nitrogen source – 3 g meat extract – carbon source  Temperature Requirements Psychrophiles: -10⁰C – 20⁰C Mesophiles: 20 – 50⁰C Thermophiles: 40 – 90⁰C Extreme thermophiles: 80 – 110⁰C Oxygen Requirements http://diverge.hunter.cuny.edu/~weigang/Images/06-t01_effectoxygen.jpg  Enzyme content of bacteria with different requirements for oxygen Name Enzyme content for O2 detoxification Strict aerobe Catalase H2O2- (radical) H2O + O2 Superoxide dismutase (SOD) 2O2- (radicals) + 2H+ O2+H2O2- H2O + O2 Facultative anaerobe Catalase & SOD Strict anaerobe Neither catalase nor SOD Microaerophile Small amounts of catalase and SOD Aerotolerant SOD  Optimal pH Most bacteria: pH ~7 Yeast and mold: pH 5 Helicobacter pylori – acidic environment of the stomach – Urease converts urea to CO2 and ammonia – Results in neutral pH around bacteria Thiobacillus lives in low pH by quickly pumping H+ ions out of cytosol  Questions for review If a yeast strain requires tryptophan added to minimal media for growth, is it an auxotroph or a prototroph? If you add Tetracycline to LB agar, is the medium selective or differential? What is the optimal temperature and pH for the growth of pathogenic bacteria?  Four Phases of Bacterial Growth During the lag phase, there is little or no change in the number of cells, but metabolic activity is high. – DNA and enzyme synthesis occurs; may last from 1 hour to several days. During the log phase, the bacteria multiply at the fastest rate possible under the conditions provided. – Maintained by use of a chemostat – constant supply of fresh media http://jan.ucc.nau.edu/%7Efpm/bio205/grthcrv.gif  Four Phases of Bacterial Growth During the stationary phase, there is an equilibrium between cell division and death. – Nutrients are exhausted and waste products build up; pH increases. During the death phase, the number of cell deaths exceeds the number of newly formed cells. http://jan.ucc.nau.edu/%7Efpm/bio205/grthcrv.gif Bacterial growth: The results of doubling https://www.jove.com/science-education/10100/bacterial-growth-curve-analysis-and-its-environmental-applications Exponential expansion of bacterial cells  Sterilization techniques Dry heat sterilization: baking – Slow, glass or metal Flame sterilization: Bunsen burner – Fast, glass or metal Filter sterilization – Fast, expensive, liquid Radiation sterilization – Commercial, plastics, especially tissue culture http://diverge.hunter.cuny.edu/~weigang/Images/07-02_autoclave_1.jpg  Sterilization techniques Steam under pressure – Relatively fast, 121⁰C, 15psi – Glass, metal, liquid, some plastics http://www.hydroclave.com/images/graph.jpg  Sterile (aseptic) techniques Maintaining sterility in the laboratory Flaming: Bunsen burners Cleaning after the experiment – Ethanol – Washing hands  Bacterial growth Streaking for single colonies Bacterial growth technique Liquid medium – Bacterial culture Solid medium – Bacterial lawn – Single colonies https://eurekabrewing.wordpress.com/2013/03  1 ase Ph Ph sea 2 Pha s e3  Bacterial genetics Bacteria Additional small circular DNA plasmids – Prokaryotic, single cell – Plasmids are much smaller and have – Single bacterial chromosome, fewer genes circular Bacterial chromosome contains all the – Haploid essential information for living The genome of E. Plasmids often carry genes that may coli (sequenced in 1997) is benefit the survival of the organism, about 4 million base pairs (e.g., antibiotic resistance) with about 3000 genes Bacterial genomes are only about 0.1% as big as the human genome and have ~10% as many genes. http://www.lehigh.edu/~jas0/G16.html  Bacterial genetics Replication – Bacterial chromosome and plasmids replicate – Have their own origins of replication Bacterial cells reproduce by binary fission  Bacterial genetics: exchange of genetic material Forms of Horizontal Gene Transfer: _____________: Uptake of genetic material from the environment – Not all bacteria are naturally competent to take up DNA. Can be chemically manipulated (e.g., with CaCl2) to permeabilize cell membrane and allow transformation https://blog.addgene.org/plasmids-101-transformation-transduction-bacterial-conjugation-and-transfection  Bacterial genetics: exchange of genetic material Forms of Horizontal Gene Transfer: Transduction: Foreign DNA or RNA introduced by a virus or viral vector – E.g. bacteriophages can attach to bacterial membrane and inject genetic material into cell – During bacteriophage life cycle, bacterial DNA can be inadvertently packaged into the newly created phage’s genetic material. Transfer of this material into another bacterial cell = transduction – This method also used to introduce foreign DNA into eukaryotic cells E.g. mammalian cell lines can be manipulated with lentiviruses or Adeno-Associated Viruses  Bacterial genetics: exchange of genetic material Forms of Horizontal Gene Transfer: Bacterial conjugation: direct contact – Common occurrence allowing transfer of plasmids between bacteria – e.g., plasmids conferring antibiotic resistance  Bacterial genetics: exchange of genetic material Forms of Horizontal Gene Transfer: ____________: performed in eukaryotic cells – Foreign DNA deliberately introduced into eukaryotic cells via non-viral methods Physical manipulation: Electroporation, microinjection Chemical manipulation: calcium phosphate or other chemicals can neutralize DNA molecules’ charge so that DNA can cross the cell membrane  Genetic Markers To study bacterial genetics, Selective media are often used mutant bacteria with distinct to observe the mutant phenotypes are often used phenotypes – color – form Prototroph: – shape of the colonies – A wild type strain that does not need anything extra added to Mutants of the genes producing media enzymes participating in Auxotroph: metabolic processes can be – a mutant strain such as his- that distinguished by the ability to has lost the ability to synthesize produce certain metabolites its own supply of a particular nutrient, in this case histidine.  Phenotype and genotype: Nomenclature Auxotroph of E. coli for tryptophan: Trp- – May be caused by a loss-of-function mutation in any of five different enzymes that are involved in the biosynthesis of tryptophan – TrpA-, TrpB-, TrpC-, TrpD-, TrpE- Genotype: italics Trp Phenotype: regular Trp Proteins: All caps such as TRP Antibiotic resistance is designated with a superscript - Ampicillin resistant: AmpR - Ampicillin sensitive: AmpS  Determining Phenotypes Bacterial Phenotypes: – Antibiotic resistance – Activity of LacZ gene Yeast mutants: – Prototrophs – Auxotrophs: uracil, tryptophan, leucine  Replica-plating technique to screen for mutant strains of a colony-forming Determining Phenotypes microorganism Auxotrophs – Trp-: cannot produce tryptophan, require addition of tryptophan to the medium for growth Selective medium – No tryptophan – With tryptophan Methods: – Patching – Replica printing/plating Selective media  Bacteria: Basics of Drug Resistance  Modes of bacterial resistance:  Drug never reaches target  Drug is altered  Drug target is altered Bacteria: Basics of Drug Resistance Antibiotic Mechanism Antibiotic classes Resistance mechanisms Inhibit cell-wall synthesis Penicillins Altered cell wall composition Cephalosporins Altered binding protein Altered Carbapenems efflux pumps Drug destruction by beta- lactamase Penicillins are bactericidal antibiotics: They kill the infectious agent. Battling evolution to fight antibiotic resistance, M. Baker, The Scientist, Oct 2005 Bacteria: Basics of Drug Resistance Antibiotic Mechanism Antibiotic classes Resistance mechanisms Inhibit cell-wall synthesis Penicillins Altered cell wall composition Cephalosporins Altered binding protein Altered Carbapenems efflux pumps Drug destruction by beta- lactamase Inhibit protein synthesis Tetracyclines Efflux pumps Reduced cell wall permeability Mutations in bacterial ribosome Molecular degradation by lyase Tetracyclines are bacteriostatic antibiotics: They inhibit bacterial growth and are mainly effective against multiplying (actively growing) bacteria. Battling evolution to fight antibiotic resistance, M. Baker, The Scientist, Oct 2005 Beta-lactam antibiotics: Penicillin, ampicillin Beta-lactam antibiotics are bactericidal – inhibit synthesis of the peptidoglycan layer of bacterial cell walls. Irreversibily bind transpeptidases or penicillin binding proteins (PBPs). Transpeptidases facilitate the final transpeptidation step in the synthesis of peptidoglycan layer in bacterial membrane – Therefore mainly functional against Gram-positive bacteria Miyachiro et al. (2020) Macromolecular Protein Complexes II: Structure and Function  Beta-lactam antibiotics Normal peptidoglycan synthesis: How ampicillin works: How bacteria become resistant to ampicillin:  Two Modes of Resistance First mode: Enzymatic hydrolysis of the beta-lactam ring. – beta-lactamase or penicillinase break open the beta-lactam ring of the antibiotic The genes encoding these enzymes present on the bacterial chromosome or acquired via plasmid transfer. – bla gene encodes beta-lactamase, which provides ampicillin resistance The bla gene is present in many cloning vectors and allows ampicillin resistance in E.coli, including pGLO (used in BIOL2281). Beta-lactamase enzyme activity not found in mammalian cells  Two Modes of Resistance Second mode: – possession of altered penicillin binding proteins. Beta-lactams cannot bind effectively to altered PBPs (transpeptidases that finish the formation of peptidoglycan layer, known as penicillin binding proteins) – This mode of resistance is found in Staphylococcus aureus (MRSA) and penicillin-resistant Staphylococcus pneumoniae.  Tetracyclines Discovered in 1940 A family of antibiotics that inhibit protein synthesis by binding of aminoacyl-tRNA to the ribosomal acceptor (A) site Tetracyclins are broad-spectrum agents, against Gram-positive and Gram-negative bacteria There is one high affinity binding site for tetracyclines in the ribosomal 30S subunit  Tetracyclines Mode of resistance: Two gene families, tet and otr, have 80% amino acid similarity – Total 29 tet genes and 3 oxytetracycline resistance (otr) genes have been characterized Some tet and otr genes code for efflux pumps – 18 tet genes and 1 otr gene Some tet and otr genes code for ribosomal protection proteins – one of the otr genes otr(A) All the tet efflux genes code for membrane-associated proteins which export tetracycline from the cell. Export of tetracycline reduces the intracellular drug concentration and thus protects the ribosomes within the cell. Determining LacZ phenotype  Beta-Galactosidase breaks down lactose Beta-galactosidase splits lactose to glucose + galactose 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal): splits into galactose and “X” group “X” group dimer has blue color  Beta-Galactosidase also breaks down X-gal substrate X-gal: 5-bromo-4-chloro-3- indolyl-beta-galactopyranoside  The Week In Review Experiment 1 – Sterile techniques: Inoculating liquid medium with bacteria Streaking for singles – Phenotyping: Patching bacterial strains on selective media plates  Be Able To’s Know the major factors that influence microbial growth Know the common laboratory sterilization techniques and under what conditions they are used Be able to identify bacterial strains based on their growth pattern in the presence of different antibiotics.  Patching LB Amp Amp + XGAL 1 1 1 2 2 2 6 6 6 3 3 3 5 5 4 5 4 4 Tet Kan Amp + Tet 1 1 1 2 2 2 6 6 6 3 3 3 5 4 5 4 5 4 What will be completed this week Lecture 1 on E1 Quiz 1 on E1 Demo videos: – Sterile technique – Culture inoculation technique – Streaking for singles Phenotype testing by patching Group Work on E1  BIOL4380 Gel electrophoresis and Plasmid mapping Lecture 2: E2  Gel electrophoresis (GE) Separation by charge: – Allows separation of charged molecules in an electric field Separation by size – Separates charged molecules by size using molecular sieve Separation in electric field – The strength of electric field Separation in buffer – Movement of charged particles to anode/cathode  Rate of migration Rate of migration is dependent upon: – Electrical field strength The stronger the current, the faster the movement – Net charge of particle – Viscosity of electrophoretic medium In a liquid medium In semisolid matrix – Conformation of molecule of interest Linear or circular Interaction with another molecules  Separation of DNA fragments by GE Electrical field strength Separation by size As the voltage applied to a Molecular sieve gel is increased, DNA – Is determined by the range fragments migrate faster. of sizes that need to be – The best resolution of separated fragments larger than ~2 kb Creates certain size pores is attained by applying no – Starch gels more than 5 volts per cm to – Acrylamide gels the gel – Agarose gels the cm value is the distance between the two electrodes, not the length of the gel  Gel electrophoresis equipment Power supply Gel box Gel tray Combs Staining – – EtBr, ethidium bromide – GelRed Gel electrophoresis equipment http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html  DNA Migration on Agarose Gel Because of an equal charge to mass ratio (one negative charge for each nucleotide), the DNA fragments are separated based upon their size and shape. – Linear DNA fragments migrate differently than circular DNA http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/supercoils.jpg "Mice" run faster through the forest than "elephants" "Mice" run faster through the forest than "elephants"  Structure of Agarose Gel http://openwetware.org/images/7/7b/Be109agarosegelEM.jpg  Gel electrophoresis – separation by size DNA is a large molecule In order to separate on the gel, needs to be digested to smaller fragments Enzymes that naturally occur in bacteria: restriction enzymes  Restriction Enzymes Proteins that recognize specific sites in DNA molecule Restriction enzymes recognize 4-8 basepair palindromes – Base sequence reads the same forwards & backwards. – EcoRI: G AATTC - SmaI: CCC GGG CTTAA G GGG CCC “sticky ends” “blunt ends”  Restriction Endonucleases Palindromic DNA sequences Blunt ends Sticky ends http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.gif Restriction enzyme sources, cleavage sites, and cleavage products Dubey (2014) A Textbook of Biotechnology  Buffers Tris-Borate-EDTA (TBE) TBE buffer has a superior Tris-Acetate-EDTA (TAE) buffering capacity – pKa of Tris (8.08) is closer to – 10X stock prepared pKa of boric acid (9.24) while – dilute to 1X for working acetic acid pKa is 4.76 solution – Is not the best choice for large _____ is commonly used, easy DNA fragments to prepare and store – Good for smaller sizes, interact with agarose more tightly, – TAE should be used when effectively forming smaller DNA is to be purified from pores the gel. – Current Protocols Essential Laboratory Techniques, 2nd TBE/agarose interaction will edition, Gallagher and Wiley reduce the amount of DNA purified.  Electrophoresis Buffer DNA fragments will migrate at different rates in TAE vs. TBE due to differences in ionic strength. Buffers not only establish a pH but provide ions to support conductivity. – If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! – If you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it. http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html  Buffers Loading Buffer: what is the function? Provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated Contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer. Running Buffer: What is the function? Contains ions that allow the conduction of the DNA molecules. Maintains the pH Prevents temperature change Prevents DNA degradation  A Dye is Used to Visualize Nucleic Acids in an Agarose Gel EtBr Intercalation We will use gel red, a non-carcinogenic fluorescent DNA dye. http://www.madsci.org/posts/archives/1999-02/919869466.Mb.r.html http://www.company7.com/uvp/graphics/ethidiumBromideGel_frmTrans182182.jpg  A Plasmid is an extrachromosomal Loop of DNA http://www.agen.ufl.edu/~chyn/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/14_1.jpg  Circular and Supercoiled DNA Migrate Differently From Linear DNA http://web.virginia.edu/Heidi/chapter12/chp12.htm  Circular and Supercoiled plasmid Migrate Differently From Linear DNA http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/images/m688d0036.jpg  The Percentage of the Agarose Gel Determines What Size Molecules Can Be Separated Which of these gels should be used for a 100bp fragment? http://openwetware.org/images/5/54/100_bp_DNA_ladder.jpg  Estimation of DNA Fragment Sizes Using Agarose Gel Electrophoresis - eyeballing http://www.nature.com/cmi/journal/v7/n4/images/cmi201016f2.jpg  Estimation of DNA Fragment Sizes Using Agarose Gel Electrophoresis Size (bp or kbp) Distance migrated (mm or cm) http://csm.jmu.edu/biology/courses/bio480_580/mblab/agarose2003.html  Visualization of DNA Ethidium bromide: intercalates with the DNA molecules, fluoresces under UV light, carcinogenic GelRed: non-carcinogenic alternative https://biotium.com/product/gelred-nucleic-acid-gel-stain/  Restriction Endonucleases Cut DNA at Highly Specific Sites http://www.allanwilsoncentre.ac.nz/massey/fms/AWC/recreate_the_resource/RFLP1.png Separation by size: How can we determine the DNA fragment size? Molecular ladders: Known size fragments used as a reference – Bacteriophage λ digest one of the first created Molecular Markers, BioRad  Molecular Markers – determining DNA size Run size standards in the gel along with the samples of interest A linear relationship is obtained if the logarithms of the sizes (in base-pair units) of the DNA fragments are plotted against the distance traveled through the gel http://www.bx.psu.edu/~ross/workmg/Struc_Nucleic_Acids_Chpt2.htm  Molecular Markers – determining DNA size logM d Construct a calibration curve for distance run by the fragment of certain size (d vs. logM, M = molecular weight, i.e., fragment length) The size of the samples of interest can be determined by measuring the distance run d and reading M from the calibration curve. http://www.bx.psu.edu/~ross/workmg/Struc_Nucleic_Acids_Chpt2.htm  DNA migration in a gel Calibration curve DNA conformation Electrophoretic mobility of DNA restriction fragments in an agarose gel. The DNA is from bacteriophage lambda digested with the restriction enzyme HindIII. The graph shows migration distance (cm) vs. size of the restriction fragment in bp.  You need to plot the known data from the MW ladder onto semi- logarithmic graph paper.  Don’t forget to include a title! You can interpolate from the standard curve to estimate the size of the band of interest. 40 30 20 10 MW ladder Distance (kb) (cm) 23.7 0.75 9.46 1.35 6.75 1.85 4.26 2.6 2.26 4.05 1.98 5.3  Gel picture and caption Agarose gel electrophoresis of uncut and digested DNA. The gel was run at 120 V in Tris/borate/EDTA Buffer for 1 hour, stained with ethidium bromide and viewed under UV light. Lanes 1 and 8 – molecular marker, lambda DNA digested with EcoRI. Lanes 2 – vector undigested, lane 3 – vector digested with EcoRI. Lanes 4 – 7: recombinant vectors A or B undigested, digested with EcoRI or HindIII/SalI http://homepages.strath.ac.uk/~dfs99109/BB211/ResMap.html Determine the size of the fragment Ladder MW ladder Distance (kb) (cm) 23.7 0.75 9.46 1.35 6.75 1.85 4.26 2.6 2.26 4.05 1.98 5.3 3.1 cm = ___ kb? http://www.mun.ca/biology/scarr/Gel_Electrophoresis.html  Practical use: restriction mapping One of the first applications: restriction maps of plasmids http://www.bio.miami.edu/~cmallery/150/gene/c7.20.8.electrophoresis.jpg  Practical use: restriction mapping One of the first applications: restriction maps of plasmids Lambda DNA http://www.apsnet.org/education/k-12plan  Restriction Mapping Why should you care? Can find RE sites which might be useful for cloning Verify your inserted DNA fragment Verify/check identity of a plasmid Human genome mapping – finding genes on chromosomes (historically) Awesome tool for genetic manipulation!  Restriction Mapping Where does it cut? What is the size of the fragment/s cut? How many fragment/s are made by the RE? Restriction Digest of pSR4380 https://www.youtube.com/watch?v=v2T8Y3-8674  Restriction Digest of pSR4380 E = EcoRI H = HindIII B = BamHI E H B E/H H/B E/B E/B/H Ladder (kb) 6 5 4 3 2 1.5 0.5 6 kb 6 kb 5 kb 4.5 kb 3.5 kb 3 kb 2 kb 1 kb 1.5 kb 1.5 kb 2 kb 1.5 kb, 1.5 kb 1 kb 1 kb 1 kb  Restriction Digest of pSR4380 EcoRI HindIII 0/6 kb 0/6 kb E H B E/H H/B E/B E/B/H Ladder (kb) 6 5 6 kb 4 pSR4380 6 kb pSR4380 3 2 1.5 BamHI 0/6 kb 1 kb 0.5 BamHI 4.5 kb 3.5 kb 3 kb 2 kb 1kb 6 kb 6 kb 5 kb 1.5 kb 1.5 kb 2 kb 1.5 kb, 1.5 kb 6 kb 1kb 1 kb 1 kb 1 kb pSR4380 5 kb  BamHI 0/6 kb Restriction Digest of pSR4380 1 kb BamHI 6 kb E H B E/H H/B E/B E/B/H Ladder (kb) 6 5 5 kb 4 3 BamHI 0/6 kb 2 1 kb 1.5 BamHI 3 kb 6 kb 0.5 6 kb 6 kb 5 kb 4.5 kb 3.5 kb 3 kb 2 kb 2 kb 1.5 kb 1.5 kb 2 kb 1.5 kb, 1.5 kb 5 kb 1kb 1 kb 1 kb 1 kb EcoRI  Restriction Digest of pSR4380 BamHI 0/6 kb 1 kb E H B E/H H/B E/B E/B/H Ladder (kb) BamHI 6 5 3 kb 6 kb 4 3 2 kb 2 5 kb EcoRI 1.5 BamHI 0/6 kb 1.5 kb 1 kb 0.5 BamHI 6 kb 6 kb 5 kb 4.5 kb 3.5 kb 3 kb 2 kb 1.5 kb 2 kb HindIII 6 kb 1.5 kb 1.5 kb, 1.5 kb 1kb 1 kb 1 kb 1 kb 1.5 kb 2 kb 5 kb EcoRI  Mapping Guidelines Label Lanes Determine how many times each enzyme cuts plasmid Add up fragment sizes of each lane – Are some bands “doublets”? Determine total size of a plasmid Start a map – pick 12 o’clock position – hold constant! Compare double digests that use the 12 o’clock reference Compare triple digest See https://www.youtube.com/watch?v=v2T8Y3-8674 for another example  The Week in Review Experiment 2: Gel Electrophoresis of Pre-digested DNA Watch video demonstrations of loading samples on a gel and running the gel. You will be loading pre-digested samples on a gel Based on the pattern of the bands on the gel, you will determine the map of the plasmid.  Be able to’s Describe the scientific basis behind the separation and visualization of DNA on agarose gel Create a calibration curve for DNA separation and determine a size of a fragment on a gel Draw a plasmid map based on the DNA pattern on agarose gel. Draw a DNA pattern on agarose gel given a plasmid map and various restriction digest conditions  What we are doing in lab this week Take quiz Gel electrophoresis using pre-digested samples Mapping  Cloning experiment Restriction digest and ligation Lecture 3  Molecular cloning Molecular cloning is a set of experimental methods in molecular biology that are used: – to assemble recombinant DNA molecules – to direct their replication within host organisms Cloning: involves the replication of one molecule to produce a population of cells with identical recombinant DNA molecules. Molecular cloning methods are central to many contemporary areas of modern biology and medicine – Patten CL, Glick BR, Pasternak J (2009). Molecular Biotechnology: Principles and Applications of Recombinant DNA. Washington, D.C: ASM Press. ISBN 978-1-55581-498-4.  Cloning of DNA fragments DNA manipulation – Identification of gene of interest (GOI) – Isolating a DNA fragment containing the GOI using restriction enzyme digestion – Using a vector to multiply DNA – Identifying a vector carrying _______ Sequencing Gene expression Gene analysis http://www.wix.com/samemali/bacterial-transformation  Cloning Purposes How to make many copies of one gene? – Often genes of interest are present in a genome in a single or low copy number – Hard to manipulate – Cloning in a vector To study a gene function To express a product of certain gene Important to consider promoters when designing a cloning experiment http://www.scfbio-iitd.res.in/tutorial/promoter.html  Cloning Purposes: Promoters Clone ______ gene to study structure or for expression (with the endogenous promoter) Clone _________ region for expression (under another promoter) Clone _________ regions (promoter) to study function or further use for expression http://www.scfbio-iitd.res.in/tutorial/promoter.html  Process of cloning Find a system that will allow gene to make many copies of itself – through replication Bacteria – convenient system – Contains bacterial chromosome with origin of replication – Contains plasmids with their own origins of replication – Plasmids carry genes that can be used as ___________markers (antibiotic resistance) Use plasmids as vectors Process of cloning: Think, pair, share How to introduce gene of interest into bacterial plasmid? How to introduce recombinant plasmid into bacterial cell? How to find bacterial cells with recombinant plasmids?  Functions of DNA cloning Creating DNA libraries Analyzing unknown DNA fragment Cloning for gene expression Identification and use of promoters – Cloning under a promoter  Plasmids Plasmids are circular extra-chromosomal DNA that can be replicated by the host replication machinery Plasmids are used in recombinant DNA technology to transfer pieces of DNA (genes) from one organism to another http://en.wikipedia.org/wiki/Plasmid  Cloning Guide: Creating a useful vector A useful vector will be able to: – Replicate ________________________ – Allow insertion of DNA fragment ________________________ – Allow identification of a recombinant vector http://www.neb.com/nebecomm/tech_refere nce/restriction_enzymes/cloning_guide.asp ________________________  https://www.neb.com https://youtu.be/KpTrEKfHhVo  Vectors Naturally occurring DNA fragments that are capable of replicating in certain live organisms – Bacterial plasmids – Yeast plasmids – Bacteriophages – Cosmids: hybrid plasmid that contains a Lambda phage cos sequence (cos site + plasmid = cosmid) – Bacterial artificial chromosomes (BACs): based on functional F plasmid – Yeast artificial chromosomes (YACs)  pBR322 pBR322 was the first popular modern cloning vector, constructed by Ray Rodriguez and Herbert Boyer from 3 basic components: 1) the tetracycline resistance gene segment was derived from the plasmid pSC101 (constructed by Stanley Cohen) which was, in turn, derived from the broad host range conjugative plasmid, R6. 2) an ampicillin resistance gene segment was obtained from the transposon, Tn3. 3) the origin of replication segment was obtained from pMB9 (constructed by Mary Betlach), which was, in turn, derived from the colicin plasmid, ColEI. http://www.ncbi.nlm.nih.gov/pubmed/3034735 http://www.mun.ca/biochem/courses/4103/topics/plasmids.html  Plasmid maps pUC The pUC series of plasmids were constructed by Jeffrey Vieira and Joachim Messing, and were derived from pBR322. The tetracycline resistance gene region of pBR322 has been replaced by a gene segment coding for part of the Beta- galactosidase enzyme into which a series of unique restriction sites have been designed (the multiple cloning site, MCS). pUC19 has high copy number inside cell.  Origins of replication http://mikeblaber.org/oldwine/BCH4053l/Lecture07/lecture07_files/image002.jpg  Origins of replication http://mikeblaber.org/oldwine/BCH4053l/Lecture07/lecture07_files/image002.jpg  Origins of replication http://mikeblaber.org/oldwine/BCH4053l/Lecture07/lecture07_files/image002.jpg  Origin of Replication Low copy ______ copy 1-10 copies per 50-150 copies per cell cell E. coli ColE1 pUC ori Yeast Cen/ars 2 micron (2µ) Centromere/ autonomously replicating sequence If DNA extraction is done on 2 cell cultures A containing plasmid with ColE1 ori and B containing plasmid with pUC ori, which will yield higher amount of plasmid DNA?  DNA manipulation Restriction enzymes – Non-directional cloning – Directional cloning Modifying enzymes – Exonuclease I E. coli – T7 exonuclease – DNA polymerase I, Large (Klenow) fragment – Alkaline phosphatase Ligation enzymes – T4 ligase  Cloning Tools: Restriction enzymes Restriction enzymes Enzymes that recognize and cut – To open the vector double-stranded DNA at specific – To digest DNA molecule into nucleotide sequences – smaller fragments palindromes – Example: EcoRI – Escherichia coli Need to have compatible RY 13 ends – Recognition sequence: – One enzyme: 5’ – gagctt G AATTCtgtact-3’ fragment can insert 3’ – cacgaaCTTAA Gacatga-5’ two ways ________________ Produces 5’ overhang, sticky ends 5’ – gagctt G AATTCtgtact- – Two different 3’ 3’ – cacgaaCTTAA Gacatga-5’ enzymes: only one position of the insert _________________  Restriction Enzymes Certain enzymes will generate: – Blunt or flush termini – 3’ extensions or overhangs – 5’ extensions or overhangs http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.gif  Working with Enzymes Optimal pH – buffers Optimal temperature Deactivation – if necessary Restriction digest Amount of DNA Total volume of the reaction Amount of restriction enzyme Buffer Optimal temperature Is it necessary to remove or stop the enzyme for the next steps? How?  Restriction digest table Stock Reaction 1 Negative Control DNA, 0.2 ug/ml (Need 1 ug total) 5 ul Enzyme 10U/ml (Need 10U/ug) 1 ul Buffer, 10X 2 ul (Need 1X in reaction tube) Water (ddH2O) to 20 ml 12 ul Total volume: 20 ul 20 ul DNA: 1 ug = 5 ml 0.2 ug/ml Enzyme needed 10 U * 1 ug = 10 U Vol. of enzyme = 10 U = 1ul ug 10 U/ul Buffer: 10X*V1 = 1X*20 ml = 2ul Common reasons that restriction enzyme digests fail: - Incorrect _______________composition (e.g., wrong pH) - Incorrect incubation ______________ - Incorrect incubation ______________ – For example, star activity may occur if the reaction conditions are suboptimal for that restriction enzyme. Star activity leads to the relaxation or alteration of the specificity of the restriction enzyme cleavage of DNA, – DNA methylation: The methylation (epigenetic) status of the DNA of interest may preempt restriction enzyme digestion. –  DNA Polymerase I Large (Klenow) Fragment Klenow fragment is a proteolytic product of E. coli DNA polymerase I which retains 1) ____________ polymerase activity 2) ____________ exonuclease activity Has lost 5’  3’ exonuclease activity Klenow retains the polymerization fidelity of the holoenzyme without degrading 5’ termini  Alkaline Phosphatase (Apase) Catalyzes the removal of 5’ __________from DNA, RNA and ribo-deoxyribonucleoside triphosphates. Brown. Gene Cloning and DNA Analysis, 2006.  Why do we use alkaline phosphatase? Since dephosphorylated fragments lack the 5’ phosphoryl termini required by ligases, they cannot ______________ – This property can be used to decrease the vector background in cloning strategies – Alkaline Phosphatase needs to be heat-inactivated before ligation! Brown. Gene Cloning and DNA Analysis, 2006.  T4 DNA Ligase Catalyzes the formation of a phosphodiester bond between juxtaposed 5’- phosphate and 3’ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt-end and cohesive end termini as well as repair stranded nicks in duplex DNA, RNA or DNA/RNA duplex https://di.uq.edu.au/community-and-alumni/sparq-ed/sparq-ed-services/dna-ligation  Ligation reaction In cloning, you need to ligate your vector with the desired DNA fragment After digestion, ligation reaction is set up. Important to consider: – The amount of vector DNA and insert DNA – Recommended molar ratio may be 1:1, 1:3, or 3:1 vector:insert DNA (see product guidelines) length of insert = amount of insert x molar ratio length of vector amount of vector  Ligation reaction length of insert (in kb) * ng of vector * molar ratio of insert to vector length of vector = ng of insert needed for a 1:1 ratio (in kb) Example: How much 500 bp insert DNA needs to be added to 100 ng of 3 kb vector in a ligation reaction for a desired vector:insert ratio of 1:1? (500 bp = 0.5 kb) ___kb insert X___ ng vector X molar ratio of 1 = ___ ng insert ___ kb vector  Ligation reaction length of insert * ng of vector * molar ratio of insert to vector length of vector = ng of insert Example: How much 500 bp insert DNA needs to be added to 100 ng of 3 kb vector in a ligation reaction for a desired vector:insert ratio of 1:3? (500 bp = 0.5 kb) ___kb insert X___ ng vector X molar ratio of 3 = ____ ng insert ___ kb vector  Identifying a vector carrying GOI Selectable markers Antibiotic resistance – Drug markers: require drug Blue-white selection sensitive strains Reporters Nutritional markers: require auxotrophic strains – LEU2, URA3, TRP1, HIS3 Blue-white selection: activity of beta-galactosidase – requires cloning into the coding sequence, interrupts gene, stops expression LacZ+  LacZ- Selectable Markers: blue-white selection Blue-white selection: activity of beta- galactosidase – requires cloning into the coding sequence, interrupts gene, stops expression – therefore stops blue color LacZ+  LacZ- Which of the plates have recombinant bacteria? http://oregonstate.edu/instruction/bb350/textmaterials/13/Slide19.jpg  Our cloning procedure General cloning Our cloning Isolate DNA fragment of interest DNA fragment is already cloned in a plasmid Insert in a vector Will re-insert in a different vector (subclone) Transform into a host Will transform into bacteria Identify cells containing recombinant Will use selectable markers plasmid Will isolate plasmid DNA – Use selectable markers Transfect mammalian cells – Use blue-white selection Use reporter gene to see gene – Use reporters activity Isolate plasmid DNA for further manipulations  Our cloning procedure We want to (sub)clone beta-galactosidase open reading frame from a donor plasmid pTETbeta, containing a bacterial promoter, to the receiver plasmid pTUD, which contains a mammalian promoter (CMV promoter). The main goal is to see if a bacterial protein can be expressed under a mammalian promoter in animal cells.  Our cloning procedure pTETβ – donor plasmid pTUD – vector (receiver) Standard Cloning Strategy pTETbeta pTUD SacII digest SacII digest Day 1 Cloning Lab 3 Gel check Gel check APase Ligation Transformation into bacteria Day 2 Cloning Lab 4 Day 3 Cloning Lab 5 DNA isolation, Analysis of clones Transfection into mammalian cells Day 4 Cloning Lab 7  Cloning Day I Restriction digest Restriction digest of a donor plasmid of a receiver plasmid Check digest on agarose gel Check digest on agarose gel Purify vector DNA Purify insert fragment Dephosphorylate Ligate vector and insert  Begin gene cloning Subclone beta-gal from pTETbeta to pTUD Tet SacII pTET beta SacII 8.9 kb bla pTUD Beta-galactosidase ORF 3.7 kb SacII  Step 1: SacII digestion Tube 1: Tube 2: Tube 3: Tube 4: pTETbeta SACII pTETbeta pTUD pTUD digest control digest control  Step 1: SacII digestion Tet SacII pTET beta SacII 8.9 kb bla pTUD Beta-galactosidase ORF 3.7 kb SacII pTET beta backbone (8.9 – 3.5) = 5.4 kb Linear pTUD 3.7 kb Beta-galactosidase ORF (3.5 kb)  Step 2: Dephosphorylation pTET beta backbone (8.9 – 3.5) = 5.4 kb Linear pTUD 3.7 kb P OH OH P Beta-galactosidase ORF (3.5 kb) P OH Linear pTUD 3.7 kb OH  Step 3: Ligation pTET beta backbone (8.9 – 3.5) = 5.4 kb P OH Beta-galactosidase ORF (3.5 kb) Linear pTUD 3.7 kb OH P OH Control Experimental  The week in review Cloning I: restriction digest and ligation – Prepare a restriction digest table showing how you would perform four restriction digests: two experimental digests of pTET beta and pTUD two controls – Check your digests on an agarose gel – Understand the rationale for Performing a dephosphorylation reaction on pTUD vector digest Performing a “dirty” ligation reaction of digested vector receiver plasmid and donor plasmid  Be able to’s Be able to design a basic cloning strategy to move one DNA fragment from one plasmid to another (subcloning) Know the minimum requirements for a useful plasmid Know and be able to use the various plasmid’s ori and selectable markers in reference to a cloning strategy Be able to explain for what purpose a plasmid can be used based on a plasmid map  Be able to’s Be able to set up a basic restriction digest table Know the common reasons why restriction digests fail Be able to correctly handle enzymes Be able to recognize and know the function of various restriction and modifying enzymes discussed  Transformation Lecture 4 Transferring Plasmid DNA into Bacterial Cells  Cloning process & purpose Cloning of an unknown DNA sequence Cloning for gene expression: – Cloning of a known sequence with a promoter Subcloning: transferring a cloned fragment into another plasmid – Cloning of an open reading frame under a promoter for future expression Cloning of a regulatory fragment (e.g., promoter region) to study its function Gene structure: Promoters are important elements for gene expression Promoter – Prokaryotic promoters – Eukaryotic promoters Coding sequence – Open reading frame http://www.scfbio-iitd.res.in/tutorial/promoter.html  Cloning process: vectors Vectors are specifically designed for a certain cloning purpose – Gene expression Expression under endogenous promoter Expression under an inducible promoter Expression in a bacterial cell Expression in a yeast cell (shuttle vectors) Expression in mammalian cells – Study of a regulatory sequence reporters http://carrot.mcb.uconn.edu/~olgazh/bioinf2010/images/09-04.jpg Gene ORF Promoter  Cloning of an unknown DNA fragment or cloning of a gene with an endogenous promoter Using blue-white selection Old vectors (Clontech) Important elements: Origin of replication Unique restriction sites Selectable markers http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture4/Lecture4.html Cloning for gene expression in bacteria Each is an important element in a plasmid Plus elements for gene expression: – Promoter – Ribosome binding site – Sequence for protein tagging http://wolfson.huji.ac.il/expression/vector/Doron.jpg  Expression Vectors: pRSET T7 (Invitrogen) Are pUC derived Designed for high-level protein expression and purification from cloned genes in E.coli Have T7 promoter to make high levels of expression possible www.invitrogen.com  Cloning for gene expression in yeast: shuttle vectors, pYES A shuttle vector is constructed such that it can propagate in 2 different host species The pYES2-DEST52 Gateway® destination vector is designed for rapid cloning – expression in S. cerevisiae. www.lifetechnologies.com  Carries the promoter and enhancer sequences for ed (galactose-inducible) protein expression in S. cerevisiae 2µ origin of replication for high-copy replication and maintenance in yeast URA3 gene for auxotrophic selection of yeast transformants on minimal medium pUC origin for high copy replication and maintenance in E. coli Bla gene for AmpR in E. coli www.lifetechnologies.com  Additional Reporter Vector Examples pCMV – luciferase pGFP http://www.addgene.org/17234/ www.neb.com Think/Pair/Share: Think about the following cloning experiment for a minute. Then talk to a neighbor about your answer for a minute. Then share it with the class You ligate a GOI into the PstI restriction enzyme cut site in the pBR322 ‘old’ vector. Then you transform bacteria with your recombinant plasmid. You know that transformation is inherently inefficient. How can you determine which bacterial cells (clones) contain your recombinant DNA? http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture4/Lecture4.html  Our cloning pTETβ – donor plasmid pTUD – vector (receiver) Tet SacII SacII pTET beta bla pTUD 8.9 kb 3.7 kb Beta-galactosidase ORF SacII  Dirty and Clean ligation “dirty” ligation: means that you performed your ligation with all of the pieces of digested donor plasmid. “clean” ligation: would mean that you purified and isolated the section of the digested plasmid that you want to ligate, prior to the ligation. This would be more efficient for the ligation experiment but would take longer overall and the reagents would cost more.  Our cloning We want to clone beta-galactosidase open reading frame under a mammalian promoter We want to see if a bacterial protein can be expressed under a mammalian promoter in animal cells We will do a subcloning of beta-galactosidase open reading from pTETbeta to pTUD under CMV promoter We are going to analyze a regulatory sequence  Standard Cloning Strategy pTETbeta pTUD SacII digest SacII digest Day 1 Cloning Gel check Gel check SAPase Ligation Transformation into bacteria Day 2 Cloning Lab 4 DNA isolation Day 3 Cloning Analysis of clones. Transfection into mammalian cells Day 4 Cloning Clockwise (CW) and Counterclockwise (CCW) Orientation SacI (0.535) Sac II, (7.9 kb) SacI (0.535) SacII (0.820) SacII (0.820) 1.4 kb 1.4 kb Sac I pTET beta pTUD beta 2.1 kb Sac I (6.5 kb) pTUD beta 8.9 kb CW CCW 2.1 kb Sac I 1.4 kb 2.1 kb SacII Sac II (4.4 kb) Sac II  Ligation Results Resulting Viable AmpR TetR Selective fragment media Amp pTUD + B-Gal CW pTUD + B-Gal CCW pTUD + pTET pTETbeta pTUD B-gal by itself pTET no beta  Methods of Transferring DNA between Organisms Viral transfer – lambda phage Bacterial conjugation – F+  F- Electroporation Chemically – PEG polyethylene glycol/LiAOc – heat shock – CaCl2 – heat shock, 42⁰C, container, time important  Methods of Transferring DNA between Organisms Chemical transformation with CaCl2: – Pre-incubation of cells with CaCl2 concentration of cells and growth stage important – Incubation of “competent” cells with plasmid DNA Concentration of DNA important – Heat shock 42⁰C Time of heat shock vs container important – Recovery Grow in LB at 37⁰C with shaking – Plating on selective media  Transformation Efficiency Calculations Transformation efficiency = Total number of cells growing on the agar plate/Amount of DNA spread on the agar plate (in μg) – Basically, number of colonies per 1 μg DNA transformed Transformation efficiency (transformants/µg) is calculated as follows: # colonies on plate/ug of DNA plated Unit of transformation efficiency is CFU/ug Transformation Efficiency Calculations 70ul of plasmid (concentration: 4x10-3ug/ul) 350ul total volume 50ul spread on plate 140 colonies observed What is the transformation efficiency? FORMULA: # colonies on plate/ug of DNA plated Unit of transformation efficiency is CFU/ug  Answer: 70ul of plasmid (concentration: 4x10-3ug/ul) 350ul total volume during recovery 50ul spread on plate 140 colonies observed Total amount DNA used: Fraction of DNA used: DNA plated: Transformation efficiency:  OR: 70ul of plasmid (concentration: 4x10-3ug/ul) 350ul total volume during recovery 50ul spread on plate 140 colonies observed Initial amount of DNA = DNA concentration before plating = Final amount DNA plated = Transformation efficiency =  Transformation efficiency calculations: Keep in mind the total volume and how much plasmid DNA you have added to the tube. You need to know how much you plated and how many colonies you obtained from that. Take into account any dilutions you made before plating You simply divide colonies obtained by the AMOUNT (ug) of DNA spread. Always report in CFU/ug (not ng)  Ligation reaction In cloning, you need to ligate your vector with the desired DNA fragment After digestion, ligation reaction is set Important: – The amount of vector DNA and insert DNA – Recommended molar ratio is 1:1 or 1:3 vector:insert length of insert (in kb) x ng of vector length of vector = ng of insert needed for a 1:1 ratio (in kb)  Ligation efficiency Optimization of amount of DNA for vector:insert ratio 1:1 or 3 insert:1 vector Calculation of ligation efficiency based on results of your transformation Compare to positive control (plasmid DNA control) Transformation Plating Plate Medium A 5 µl water – no DNA control 1 LB (plate 100 µl) 2 LB + Amp DH5alpha AmpS B 5 µl plasmid control 3 LB+Amp mixture of pTUD C Your ligation control: 4 LB+Amp (100 µl) 5 µl vector + water ligation D Your ligation: 5 LB+Amp+X-gal (100 µl) 5 µl vector + insert ligation  Plating mixture Plate Expected Results Plating Expected Results A-1 No DNA LB A-2 No DNA LB+Amp B-3 Plasmid control pTUD LB+Amp C-4 Ligation Control LB+Amp Vector+ water D-5 Experimental ligation: vector LB+Amp+ and insert X-gal  Week In Review Make bacterial cells “competent” by pre-incubating cells in calcium chloride. Perform transformation of competent cells Allow bacteria to recover by growing in LB Spread bacteria onto selective media  Week In Review View results: day before E5 lab – Growth/no growth – Number of colonies in control and experimental plates If growth is seen, then two 5 ml cultures should be started for next week’s plasmid isolation lab.  Be able to’s Know the basic steps in bacterial transformations Be able to predict the possible recombinants from a ligation and how to select for or against certain recombinants Be able to suggest a vector for a certain cloning procedure Know the gene structure and be able to explain the function: a promoter, a coding region, an open reading frame  ANOTHER Transformation Efficiency practice calculation Example: How much DNA is plated? 0.1 ng of control DNA is added to 100 µL of competent cells. 900 µL of SOC medium is added prior to expression. 10 µL is then diluted in 990 µL SOC and 100 µL is plated If 100 colonies are counted on the plate, the transformation efficiency (transformants/µg) is: # colonies on plate/ug of DNA plated DO THE CALCULATION YOURSELF BEFORE CHECKING THE ANSWER… Plasmid DNA Isolation and plasmid mapping Lecture E-5 Steps in traditional Mini-Prep Protocol 1. GTE solution 2. SDS/NaOH solution 3. KOAc 4. Isopropanol 5. Cold 70% EtOH 6. TE  Steps in alkaline lysis miniprep protocol 1. GTE solution – resuspends cell pellet – Glucose: maintains osmotic pressure – Tris: buffer – EDTA: 1) ___________, thus inhibiting nuclease activity 2) binds bivalent cations in the lipid bilayer, thus weakening cell membrane 2. SDS/NaOH solution – breaks up cells and denatures DNA – SDS: ionic detergent, _________________, denatures and solubilizes cellular proteins – NaOH: generates alkaline condition, ____________, degrades RNA  Alkaline Conditions + SDS Lyse Bacterial Cells Berg, J.M., J.L. Tymoczko, and L. Stryer (2002). Biochemistry (5th Ed.). New York, NY: W.H. Freeman and Company. http://learn.genetics.utah.edu/content/labs/extraction/howto/images/6.jpg  Steps in alkaline lysis miniprep protocol 3. KOAc solution, pH5.2 -- lowers pH to neutral, allowing plasmid DNA to renature K+: ________________ OAc-: ___________________ Plasmid DNA – circular, intertwined, smaller, _________________ Chromosomal DNA – larger, attached to cell wall, reanneals slower, _______________ with KDS Centrifugation after addition of potassium acetate solution will precipitate chromosomal DNA + KDS (potassium salt of dodecyl sulfate) Differences in Renaturation Allow Separation of Plasmid and Chromosomal DNA after adding KOAc solution. The chromosomal DNA caught within the SDS/lipid precipitate is pelleted by centrifuging. The plasmid DNA is found in the lysate. http://bioinfo2010.files.wordpress.com/2009/07/isolation.jpg?w=463&h=791 Steps in alkaline lysis miniprep protocol 4. 100% Isopropanol – precipitates DNA In the presence of alcohol, cations interact with negatively- charged DNA backbone, which causes DNA to become insoluble and ________________________ Isopopanol – add one volume 90% ethanol – add 2.5 volume Centrifugation after adding isopropanol will precipitate nucleic acids into a pellet. Steps in alkaline lysis miniprep Protocol 5. Cold 70% EtOH – washes nucleic acids pellet to remove remaining salts 6. TE – dissolves DNA – Tris: maintains pH – EDTA: binds Mg2+, thus inhibiting nucleases (DNA can also be dissolved in Tris or water– not very stable) Steps in Qiagen spin Mini- Prep Protocol  Steps in QiaPrep Spin miniprep protocol 1. Buffer P1: resuspends cells 50 mM Tris-HCl pH 8.0 10 mM EDTA 100 μg/ml __________ 2. Buffer P2: breaks up cells and denatures DNA 200 mM ________ 1% ______ 3. Buffer N3: neutralizes pH and reanneals plasmid DNA – 4.2 M Gu-HCl (guanidine hydrochloride) – 0.9 M potassium acetate, pH 4.8 Centrifugation after adding N3. ________________ is then passed through a _________ membrane which selectively bind to plasmid DNA under high salt condition. Steps in QiaPrep Spin miniprep protocol 4. Buffer PB: __________________ – 5 M Gu-HCl – 30% ethanol 5. Buffer PE: washes membrane and removes salt – 10 mM Tris-HCl pH 7.5 – 80% ethanol 6. Buffer EB: ____________ DNA from membrane – 10 mM Tris-HCl pH 8.5 http://openwetware.org/wiki/Miniprep/Qiagen_kit Standard Cloning Strategy pTETbeta pTUD SacII digest SacII digest Day 1 Cloning Lab 3 Gel check Gel check SAPase Ligation Transformation into bacteria Day 2 Cloning Lab 4 DNA isolation Day 3 Cloning Lab 5 Analysis of clones Day 4 Cloning Lab 6 Transfection into mammalian cells pTETβ: donor plasmid, pTUD: receiver plasmid  Cloning strategy Receiver plasmid Donor plasmid (Vector, receiver for insert) (Source of insert)  Ligation Results Resulting Viable AmpR TetR Selective fragment media Amp pTUD + B-Gal CW Yes it has promoter Yes No Yes pTUD + B-Gal Yes Yes No Yes CCW pTUD + pTET No because it will Yes Yes No have 2 origins of replication pTETbeta Yes No Yes No pTUD Yes Yes No Yes B-gal by itself No because no origin No No No of replication pTET no beta Yes No Yes No Calculate SacI fragment size for pTUD + β (CW) or (CCW) 1.4 kb 2.1 kb 1.4 kb 0.3kb 2.1 kb SacI 0.3kb SacI 2.1 kb 1.4 kb β (CW) β (CCW)  How can we determine the insert orientation? SacI (0.535) 0.3 kb SacII (0.820) 3.4 kb 2.1 kb SacII (4.4 kb) pTUD beta CW (3.7 + 3.5) kb 1.4 kb SacI SacI (6.5 kb) 1.4 kb pTET beta 8.9 kb SacII 2.1 kb Predicted DNA fragment sizes after SacI digest: SacII, (7.9 kb) 5.4 kb Predicted DNA fragment sizes after SacI digest: SacI (0.535) 0.3 kb SacII (0.820) 3.4 kb SacI (0.535) 1.4 kb pTUD beta SacII (0.820) CCW SacI (3.7 + 3.5) kb pTUD 2.1 kb (3.7 kb) SacII Predicted DNA fragment sizes after SacI digest: Predicted DNA fragment sizes after SacI digest:  Conclusions After using selective media, we will get rid of all ____variants There will be at least three possible ____ variants To distinguish between them, we will isolate DNA from bacterial cultures We will digest each DNA with one restriction enzyme to find the recombinant plasmid we need The enzyme chosen should cut once in a vector and once in an insert Should not cut an insert in two equal parts We will be able to distinguish three variants: _______ __________ _____________ Gel electrophoresis of Sac I digest MW pTUD pTUDβ pTUDβ Ladder /SacI CCW/SacI CW/SacI Transformation results Transformation Plates Expected results A Water control A1: LB A2: LB+Amp B Positive control B3: LB +Amp +X-gal Plasmid DNA C Water + insert ligation C4: LB+Amp Dephosphorylation control D Ligation vector + insert D5: LB + Amp + X- gal E Ligation vector + insert D5: LB + Amp + X- gal Transformation Transformation efficiency Number of colonies per microgram DNA Calculations: TE = # of colonies/mg transformed DNA Should be above 108 CFU/ug Formula : ANOTHER Transformation Efficiency practice calculation Example: How much DNA is plated? 5ul (0.1 ng) of control DNA is added to 100 µL of competent cells. 900 µL of SOC medium is added prior to expression. 10 µL is then diluted in 990 µL SOC and 100 µL is plated If 100 colonies are counted on the plate, the transformation efficiency (transformants/µg) is: # colonies on plate/ug of DNA plated  Transformation Efficiency practice 5ul (0.1 ng) of control DNA is added to 100 µL of competent cells. 900 µL of SOC medium is added prior to expression. 10 µL is then diluted in 990 µL SOC and 100 µL is Initial amt. of DNA? plated Total transformation vol. ? If 100 colonies are counted on the plate, what is TE? Initial amt. of DNA = Dilution? Vol. plated/Total transformation vol. = Dilution fold = Fraction of DNA = Amt. of DNA plated = How much TE =________CFU/ ________ug = ____CFU/ug plated?  Transformation Efficiency practice 5ul (0.1 ng) of control DNA is added to 100 µL of competent cells. 900 µL of SOC medium is added prior to expression. 10 µL is then diluted in 990 µL SOC and 100 µL is plated Initial amt. of DNA? If 100 colonies are counted on the plate, what is TE? Total transformation vol. ? _____ in ____ SOC Initial amt. of DNA = ____________ Dilution? Initial conc. Of DNA = _______ug/ul Dilution fold = Final conc. Of DNA = _____________ = ____ ug/ul Final amt. of DNA plated = ________________= _____________ How much TE =______CFU/ ___ug = _____CFU/ug plated? This Week In Review Check results of your transformation Prepare two 3 ml overnight (O/N) cultures Isolate DNA from the cultures using commercial kit Set up a digest to analyze the presence of insert and its orientation The Be Able To’s Be able to describe methods for miniprep DNA isolation Know the purpose of all steps and chemicals used in plasmid DNA isolation Be able to use restriction enzymes to determine the orientation of an insert in a plasmid Be able to predict the sizes of plasmid fragments after restriction digest using plasmid map