Activity 4 & 5 slides.pdf

pGLO Bacterial Transformation BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. All trademarks used herein are the property of their respective owner. © 2020 Bio-Rad Laboratories, Inc. explorer.bio-rad.com Genetic transformation / Recombinant DNA Technology / Genetic Engineering / Molecular or DN...

pGLO Bacterial Transformation BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. All trademarks used herein are the property of their respective owner. © 2020 Bio-Rad Laboratories, Inc. explorer.bio-rad.com Genetic transformation / Recombinant DNA Technology / Genetic Engineering / Molecular or DNA Cloning → The process of making cells/organisms acquire/take up and express a new piece of foreign genetic material/DNA explorer.bio-rad.com Transformation – One of three types of horizontal/lateral gene transfer that naturally occurs in bacteria Horizontal/Lateral Gene Transfer – Process in which an organism transfers genetic material to another organism that is not its offspring. explorer.bio-rad.com Transgene – Foreign DNA introduced into an organism Recombinant plasmid – Plasmid artificially inserted with foreign DNA segment Vector/Construct – DNA molecule used as a vehicle to carry a particular DNA segment (the foreign DNA) into a host cell (E.g., plasmid, virus DNA) Recombinant protein – Protein produced by the inserted foreign DNA Transgenic – Unicellular or multicellular organism whose genome has been artificially altered by the introduction of one or more foreign DNA sequences from another organism; “genetically modified organism” ❖ Transduction – Foreign DNA in viral vector is introduced to prokaryotic or eukaryotic cell ❖ Transfection – Various artificial means of introducing recombinant plasmid to animal eukaryotic cells; “Transformation” is the preferred term for the technique to artificially introduce recombinant plasmid to bacterial and plant cells Transformed cell – Cell that has taken up the recombinant plasmid and has acquired a new trait or phenotype after expressing the transgene Benefits from transgenic or genetically modified organisms Modified animal models for research Cancer, obesity, heart disease, etc. Disease/drought/pest resistance. Increased nutrition explorer.bio-rad.com Modified mosquitoes to fight disease Drug production like insulin, hormones, vaccines, and anti-cancer drugs. Overview: How can we produce the protein of interest? 1. Identify a gene for a protein. explorer.bio-rad.com Gene Overview: How can we produce the protein of interest? 1. Identify a gene for a protein. 2. Put the gene into bacteria. explorer.bio-rad.com E. coli Overview: How can we produce the protein of interest? 1. Identify a gene for a protein. 2. Put the gene into bacteria. 3. Grow lots of the bacteria. explorer.bio-rad.com Overview: How can we produce the protein of interest? 1. Identify a gene for a protein. 2. Put the gene into bacteria. DNA mRNA protein 3. Grow lots of the bacteria. 4. The bacteria transcribe and translate the gene — mini protein factories! explorer.bio-rad.com Overview: How can we produce the protein of interest? 1. Identify a gene for a protein. 2. Put the gene into bacteria. 3. Grow lots of the bacteria. 4. The bacteria transcribe and translate the gene — mini protein factories! explorer.bio-rad.com Overview: How can we produce the protein of interest? 1. Identify a gene for a protein. 2. Put the gene into bacteria. 3. Grow lots of the bacteria. 4. The bacteria transcribe and translate the gene — mini protein factories! 5. Purify the protein of interest. explorer.bio-rad.com Genetic engineering using plasmids 1. Make a plasmid with your gene. 2. Do bacterial transformation. This is what you’ll do in this activity. Plasmid explorer.bio-rad.com E. coli Genetic engineering using plasmids Bacteria often have plasmids — extrachromosomal circular loops of DNA Bacteria can also take in new plasmids Chromosome Bacteria Plasmids explorer.bio-rad.com Genetic engineering using plasmids Scientists can modify or engineer plasmids for specific purposes. Origin of replication Let’s the bacteria make copies of the plasmid Antibiotic resistance Allows transformed bacteria to survive on plates with antibiotic explorer.bio-rad.com Gene/s of interest Genes for producing the desired protein or trait/phenotype Green fluorescent protein (GFP) The jellyfish Aequorea victoria has a gene for green fluorescent protein which glows green under UV light. Under visible light explorer.bio-rad.com Under ultraviolet (UV) light Green fluorescent protein (GFP) explorer.bio-rad.com Discovery of GFP Originally Isolated from the jellyfish Aequorea victoria Naturally occurring in many bioluminescent jelly fish, reef corals and marine crustaceans Recombinant GFP has 239 amino acids Expressed as a 26,870 Dalton (Da) protein Barrel structure with the fluorescent chromophore at center of the protein “Nobel prize-winning” molecule! What is a chromophore? A group of atoms forming part of an organic molecule that causes it to appear colored The GFP chromophore is comprised of three adjacent amino acids. These amino acids are enzymatically converted to an active cyclic chromophore GFP Chromophore Absorbs at 395 nm Emits at 509 nm In vitro, UV light is used to excite the GFP chromophore, absorbing light at a wavelength of 395 nm, and emitting at a longer wavelength of 509 nm visible fluorescent green light Using GFP as a biological tracer or reporter protein http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html Real-world applications GFP is a visual marker – Study of biological processes (example: synthesis of proteins) – Localization and regulation of gene expression – Cell movement – Cell fate during development – Formation of different organs – Screenable marker to identify transgenic organisms www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm → Caption for previous slide o Examples only, no need to memorize Mouse blood vessels (green-GFP) in tumor (red-DsRed). Mouse with brain tumor expressing DsRed (another fluorescent protein). A transgenic Arabidopsis seedling expressing GFP (green fluorescence). Sentinel plants will turn green when they detect chemical or biological agents that can be used in biological warfare. A mouse with different neuron colors inherited from its two parents can reveal how neurons network in the brain. www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm Plasmid containing GFP gene fused to Nup98 gene was inserted into cultured mammalian cells Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152321#sec002 pGLO plasmid The pGLO plasmid is engineered to have the GFP gene from Aequorea victoria → 3 genes present in the pGLO plasmid: GFP, bla, and araC ori Beta-lactamase (bla) Allows transformed bacteria to survive on plates with ampicillin. explorer.bio-rad.com pGLO araC Gene for the protein AraC that controls the GFP gene like and ON/OFF switch. GFP Gene for green fluorescent protein. Selectable marker: ampicillin resistance pGLO plasmid explorer.bio-rad.com pGLO plasmid Multiple Cloning Site (MCS) bla gene explorer.bio-rad.com Components of the pGLO plasmid ❖ ori — Origin of replication; DNA sequence where replication of the plasmid begins (Without the ori, the cell cannot make copies of the plasmid) ❖ bla — The antibiotic resistance gene (Selectable marker); gene that encodes βlactamase, an enzyme that breaks down the antibiotic ampicillin; transformants expressing the bla gene can be selected by placing ampicillin in the growth medium ❖ Multiple cloning site (MCS) — A region containing restriction sites (e.g., NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion of foreign DNA in the plasmid ❖ GFP — Jellyfish gene that encodes green fluorescent protein (GFP); the foreign DNA (gene) inserted in the pGLO plasmid ❖ pBAD promoter — Promoter sequence for the inserted GFP gene o Without arabinose in the growth medium, araC blocks this sequence, and RNA polymerase cannot bind and initiate transcription of the GFP gene (No GFP protein produced) explorer.bio-rad.com Components of the pGLO plasmid ❖araC — Gene that encodes the regulatory protein that prevents RNA polymerase from binding to the pBAD promoter in the absence of arabinose o Only when arabinose is present and binds to the araC protein will the production of GFP protein be switched on NOTE: gfp, araC, and bla gene in the pGLO plasmid have different promoter sequences; only the promoter (pBAD) for gfp is usually shown in the diagrams of the pGLO plasmid explorer.bio-rad.com Plasmid: inserting the transgene Multiple Cloning Site (MCS) → The foreign DNA and the plasmid should be cut with the same restriction enzyme so that the foreign DNA can be inserted in the plasmid o Restriction enzymes differ in the DNA sequences that they can cut → DNA ligase connects the backbone of the foreign DNA to the backbone of the plasmid pGLO Plasmid: inserting the transgene (gfp) → araB, araA, & araD genes replaced with the gfp gene → Operon: set of related genes transcribed from the same promoter; common in bacteria Replaced with gfp gene pGLO Plasmid: inserting the transgene (gfp) → Structure of araC monomer ❖ In the absence of the sugar arabinose, araC protein causes DNA folding which prevents RNA polymerase from binding to the pBAD promoter → araBAD genes (replaced by gfp gene in the pGLO plasmid) cannot be transcribed pGLO Plasmid: inserting the transgene (gfp) ❖ When the sugar arabinose is present, it binds to araC protein, causing the protein to change conformation → DNA looping doesn’t occur, and RNA polymerase can bind to the pBAD promoter → araBAD genes (replaced by gfp gene in the pGLO plasmid) are transcribed Constitutive genes are always expressed (e.g., araC gene, bla gene) Inducible genes are normally off, but can be turned on (e.g., gfp gene in the pGLO plasmid) Repressible genes are normally on, but can be turned off pGLO Plasmid is an example of an expression vector Expression vector – Recombinant plasmid has origin of replication, multiple cloning site, and selectable marker (antibiotic resistance gene), AND promoter sequence for the foreign gene Inside the cell, plasmid is replicated and the foreign gene is expressed (Protein of the foreign gene is produced) Cloning vector – Recombinant plasmid has origin of replication, multiple cloning site, and selectable marker (antibiotic resistance gene), but no promoter sequence for the foreign gene Inside the cell, plasmid is replicated but the foreign gene is not expressed Making cells competent for transformation ❖ In nature, competent cells are those that can take up plasmid/DNA from the environment ❖ In the lab, we are artificially making cells competent so that they can take up the recombinant plasmid Bacterial membrane E. coli Plasma Membrane Non-polar explorer.bio-rad.com Polar Making cells competent for transformation Add plasmid Negative charges on DNA backbone E. coli Plasmid DNA Plasma Membrane explorer.bio-rad.com Making cells competent for transformation Add transformation solution (CaCl2) E. coli Ca2+ shields charges on DNA to make it less polar explorer.bio-rad.com Making cells competent for transformation Heat shock E. coli Add heat to create pores in the membrane explorer.bio-rad.com Making cells competent for transformation Heat shock E. coli Add heat to create pores in the membrane explorer.bio-rad.com Making cells competent for transformation Heat shock E. coli explorer.bio-rad.com Plasmid enters cell through pore Making cells competent for transformation Recovery on ice, 2 min Pores close E. coli explorer.bio-rad.com Add LB broth, to allow cell recovery and initiate gene expression E. coli explorer.bio-rad.com LB broth LB (Lysogeny broth or Luria Bertani) broth is like chicken noodle soup for bacteria. It has all the nutrients bacteria need to grow: o Carbohydrates o Amino acids o Nucleotides o Salts o Vitamins explorer.bio-rad.com Add LB broth, to allow cell recovery and initiate gene expression AraC, regulatory protein E. coli Beta-lactamase, ampicillin resistance explorer.bio-rad.com GFP, only if arabinose is in the media LB –pGLO explorer.bio-rad.com LB/amp –pGLO LB/amp +pGLO LB/amp/ara +pGLO Only cells that have taken up the plasmid can grow in culture media with ampicillin (Antibiotic) Beta-lactamase, ampicillin resistance explorer.bio-rad.com Selective media – Ampicillin Ampicillin on the plate Bacteria without the plasmid cannot grow in the presence of ampicillin explorer.bio-rad.com Selective media – Ampicillin Transformed cells (with the plasmid) will make betalactamase , which breaks down ampicillin. This enables them to grow on ampicillin plates Bacteria without the plasmid (NOT transformed) cannot grow on plates with ampicillin explorer.bio-rad.com Cells with the plasmid can survive AND produce GFP in culture media w/ ampicillin AND arabinose AraC, regulatory protein E. coli explorer.bio-rad.com Cells with the plasmid can survive AND produce GFP in culture media w/ ampicillin AND arabinose Arabinose (a sugar) works like a switch Without arabinose, the switch is OFF. AraC blocks RNA polymerase , and the GFP gene is not transcribed With arabinose , the switch is ON. AraC changes shape and RNA polymerase transcribes the GFP gene explorer.bio-rad.com AraC RNA polymerase No GFP produced Cells with the plasmid can survive AND produce GFP in culture media w/ ampicillin AND arabinose Arabinose (a sugar) works like a switch Without arabinose, the switch is OFF. AraC blocks RNA polymerase , and the GFP gene is not transcribed With arabinose , the switch is ON. AraC changes shape and RNA polymerase transcribes the GFP gene explorer.bio-rad.com AraC Arabinose RNA polymerase GFP produced Cells with the plasmid can survive AND produce GFP in culture media w/ ampicillin AND arabinose E. coli GFP, only if arabinose is in the media explorer.bio-rad.com Transformation summary 1. CaCl2 transformation solution Shields negative charge on DNA → Allows plasmid to more easily bind to and pass through the cell membrane 2. Pre-heat shock incubation on ice Slows fluid plasma membrane for greater shock 3. Heat shock Increases permeability of cell membranes 4. Post-heat shock incubation on ice Restores cell membrane 5. Incubation at room Allows cell recovery and beta-lactamase temperature with LB broth expression so bacteria can grow on plates with ampicillin 6. Spread on culture plates explorer.bio-rad.com Selects for transformed bacteria and allows formation of colonies Plates –pGLO LB Components Bacteria Plasmid Ampicillin Arabinose Grow? Glow? explorer.bio-rad.com –pGLO LB/amp +pGLO LB/amp +pGLO LB/amp/ara Large amounts of bacteria (Lawn) explorer.bio-rad.com No bacteria White-colored bacteria (Under UV light) Green-colored bacteria (Under UV light) Hydrophobic Interaction Chromatography (HIC) GFP Chromatography Kit What is Chromatography? A set of methods for separating molecules based on their physical or chemical properties Used to purify or isolate a single protein of interest (e.g., GFP) from over 4,000 naturally occurring E. coli gene products GFP Purification by HIC: Overview Nutrient broth (Supernatant) Pellet of intact bacterial cells GFP Purification by HIC: Overview Nutrient broth (To be discarded) (Intact bacterial cells) GFP Purification by HIC: Overview → Discard liquid broth (Supernatant) before resuspending pellet of intact bacterial cells with TE buffer Supernatant containing the bacterial and GFP proteins Supernatant containing the bacterial and GFP proteins Pellet of cellular debris Hydrophobic Interaction Chromatography (HIC): 1. Add bacterial lysate to column matrix in high salt buffer (i.e., Binding buffer) → Target protein (e.g., GFP) is a hydrophobic protein 2. Wash less hydrophobic proteins from column in medium salt buffer (i.e., Wash buffer) →HIC column matrix is hydrophobic 3. Elute GFP from column with Low or no salt buffer (i.e., Elution buffer) → Protein sample is added to the column in high salt buffer Equilibration Buffer = Binding Buffer (Same salt concentration as E.B. after mixing with equal →Proteins are eluted from the column in order of increasing hydrophobicity volume of sample) > Wash Buffer > Elution Buffer → HIC Column preparation: Equilibration buffer (2 M (NH4)2SO4) without proteins added to the column [Salt in the E/B/W buffers – Ammonium sulfate] GFP proteins Protein sample in binding buffer (4 M → 2 M (NH4)2SO4) added to the column Elution buffer (No salt or low salt concentration) added to the column Wash buffer (1.3 M (NH4)2SO4) added to the column Hydrophobic Interaction Chromatography Step 1: Hydrophobic bead Add bacterial lysate to column matrix in high salt buffer (Binding buffer) – Hydrophobic proteins (i.e., GFP) interact with the hydrophobic HIC column → Collection tube 1 Hydrophobic Interaction Chromatography Step 2: Hydrophobic bead Wash or elute the less hydrophobic proteins from column with medium salt buffer (Wash buffer) – Salt ions interact with the less hydrophobic proteins and H2O – Less hydrophobic proteins fall from column – GFP remains bound to the column O -O S OO → Collection tube 2 Hydrophobic Interaction Chromatography Step 3: Hydrophobic bead Elute GFP from column by adding a no salt or low salt buffer (Elution buffer) – GFP released from column matrix → Collection tube 3 High salt concentration strengthens the hydrophobic interaction between GFP and the HIC column → Polar water molecules bind to other polar molecules/atoms → (+) and (-) ions from a salt (e.g., NaCl) → Water forms solvation/hydration shell/sphere around polar molecules/atoms, such as the ions of NaCl High salt concentration strengthens the hydrophobic interaction between GFP and the HIC column o At moderate salt concentration, ions of salt bind to the polar amino acid residues of the protein, and this also enhances interaction of water molecules with the protein → protein solubility increases (Salting in) o At higher salt concentration, there are too many salt ions and most of the H2O molecules interact with the salt ions → protein solubility decreases (Salting out) o At moderate salt concentration, ions of salt bind to the polar amino acid residues of the protein, and this also enhances interaction of water molecules with the protein → protein solubility increases (Salting in) o At higher salt concentration, there are too many salt ions and most of the H2O molecules interact with the salt ions → protein solubility decreases (Salting out) → (+) and (-) ions of salt → Water molecule → Protein Salting In Salting Out → Buffers are added to the column in order of decreasing salt concentration o Equilibration buffer = Binding buffer > Wash buffer > Elution buffer → Proteins are eluted from the column in order of increasing hydrophobicity o Ex. Chymotrypsin > Lysozyme > RNAse A > Cytochrome c Orange color indicates higher hydrophobicity Orange line in the graph – salt concentration Orange line – salt concentration Orange line – salt concentration HIC Column Column Chromatography Types of chromatography used for protein purification – Size Exclusion Chromatography – Ion Exchange Chromatography – Affinity Chromatography – Hydrophobic Interaction Chromatography Size Exclusion Chromatography → Smaller molecules elute later than larger molecules → Column is made of a porous resin Size Exclusion Chromatography → Smaller molecules elute later than larger molecules → Column is made of a porous resin Size Exclusion Chromatography → Smaller molecules elute later than larger molecules → Column is made of a porous resin Ion Exchange Chromatography o Cation Exchange Chromatography Column – negatively charged Target protein – positively charged o Anion Exchange Chromatography Column – positively charged Target protein – negatively charged Ion Exchange Chromatography o Cation Exchange Chromatography Column – negatively charged Target protein – positively charged o Anion Exchange Chromatography Column – positively charged Target protein – negatively charged Affinity Chromatography Mobile Substance S (binding partner of target sample protein) in the buffer added to elute target protein Stationary Substance S (binding partner of target sample protein) bound to the column Protein-ligand binding: This technique relies on the specific and reversible binding of a protein to a matrixbound ligand or binding partner Lock and Key mechanism Affinity Chromatography Affinity Chromatography The bound protein is eluted with a buffer containing a competing molecule or that can disrupt protein-ligand interactions Affinity Chromatography In this affinity chromatography column, immobilized antibodies are used that bind only to the target protein in the sample Experiment Tips: Add a small piece of paper to collection tube where column seats to ensure column flow Rest pipette tip on side of column to avoid column bed disturbance when adding solutions