Lecture 4 - BIO 311 Ligation and Transformation I Summary...

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Unformatted text preview: BIO 311 Ligation and Transformation I Summary of the specific cloning steps 1. 2. 3. 4. 5. 6. V Use restriction enzymes (EcoRI and BamHI) to cut the EGFP DNA fragment (I) Cut a receiving vector (pET28, V) with the same restriction enzymes Join the insert to the vector using DNA ligase to make pET28bEGFP Transform bacteria with the recombinant plasmid Select the transformed bacteria on agar with appropriate antibiotics Analyze colonies for desired recombinant plasmid *The antibiotic resistance genes are always ON pET28b-EGFP Connecting two pieces of DNA: EGFP GGATCC CCTAGG GGATCC CCTAGG BamHI digest GATCC G G CCTAG pET28 Plasmid Add BamHI digested plasmid G CCTAG GATCC G pET28 Plasmid G*GATCC CCTAG*G G*GATCC CCTAG*G The nick in the backbone 5' P Phosphodieste r backbone 3' * Endonuclease Restriction Site * S 3' 5' Creation of pET28bEGFP Clone through directional cloning r fo NA P cD EGF R Bam H1 cut T4 DNA ligase Eco R1 joining Eco R1 cut AMP KANR T7 promoter Bam H1 joining pEGFP vector cut with Bam H1 and Eco R1 to excise cDNA for EGFP pET28b vector cut with Bam H1 and Eco R1 accepts cDNA for EFGP Step 3: DNA ligation http://content.answers.com/ 5' and 3' of DNA bases http://content.answers.com/ http://www.msu.edu/ Biochemistry of DNA ligation + ATP DNA ligase enzymes: Catalyze covalent bond formation in the DNA backbone DNA ligase activity is energy (ATP) dependent Works on doublestranded DNA molecules In nature involved in DNA repair and replication http://www.biochem.arizona.edu/ T4 DNA ligase The smallest ATP dep. DNA ligase is from T4 bacteriophage (41 kDa) One of the most popular ligases used in the laboratories The T4 phage http://www.biochem.ucl.ac.uk Alkaline phsphatase (AP) enzymes remove 5' phosphates Step 4: Transformation Genetic Transformation: alteration of a cell (bacterial, animal or plant) through uptake of foreign genetic material http://www.coolestgadgets.com/ Types of Gene Transfer Horizontal Gene Transfer Vertical Gene Transfer Example: retroviral infection Example: parents to progeny Types of Gene transfer Horizontal gene transfer Vertical gene transfer http://en.wikipedia.org Bacterial Horizontal Gene Transfer Conjugation (happens in nature) Chemical transformation (lab procedure) Electroporation (lab procedure) Bacterial conjugation http://io.uwinnipeg.ca http://en.wikipedia.org/ Chemical transformation http://www.dnai.org/b/index.html Cold competent cells have fragile cell membranes Calcium ions (+++) complex with negatively charged phosphates in the DNA Shielded DNA can move through adhesion zone upon thermal imbalance on either side of the bacterial membrane Limited by the size of DNA fragment to be transformed Electroporation 1. Suitable for variety of cells 2. Effective for transformation with large plasmids. 3. Done in a short time (few seconds) 4. Similarly to chemical transformation, the exact mechanism is not known. http://www.bridgebio.com How many bacteria, on average, are being transformed? Chemical transformation: 1 cell in 1,000 Electroporation: 1 cell in 100 Step 5: Selection How do we distinguish between cells that receive plasmid to those that did not? Answer: Antibiotic resistance The difference between antibiotic+ and antibiotic growth conditions While all bacteria will grow on media lacking antibiotics, only bacteria with antibiotic resistance will grow on growth media containing an appropriate antibiotic http://sciencemole.files.wordpress.com/ Antibiotics Are Essential For Selection of transformed Bacteria Bacteriostatic antibiotics: Temporarily inhibit bacterial growth. Example: ampicillin, inhibits cell wall synthesis Bacteriocidal antibiotics: Kill bacteria. Example: metronidazole, inhibits DNA metabolism Antibiotic modes of action Penicillin Family: inhibits cell wall synthesis (eg. Ampicillin*) Tetracycline Family: inhibits 30S ribosomal subunit (eg. Doxycyline) Aminoglycoside Family: inhibits 30S ribosomal subunit (eg. Kanamycin*) Macrolide Family: inhibits 50S ribosomal subunit (eg. Erythromycin) Fluoroquinolone Family: Inhibits bacterial DNA gyrase (eg. Ciprofloxacin) (* We will use these in lab) Ampicillin mode of action Ampicillin inhibits Interbridge formation -lactamase, the product of the AMP resistance gene, cleaves ampicillin rendering it inactive Peptidoglycan Bacterial Cell Wall Brock Microbiology 1998 Ampicillin resistance conferred by lactamase Active ampicillin Amp resistance gene http://www.bio.davidson.edu/ Betalacamase Amp resistance gene product lactamase Inactive ampicillin http://perso.orange.fr Kanamycin mode of action Inhibition of Protein Synthesis Kanamycin resistance The kanamycin resistance (encoded by NPTII enzyme) inactivates a wide range of aminoglycoside antibiotics, including neomycin and kanamycin by phosphorylation. The product of the KAN resistance gene is a phosphotransferase that adds phosphate groups to Kanamycin and prevents its binding to the 30S subunit of the bacterial ribosome http://www.bioscience.org Step 6: Analyzing results Lab C http://www1.istockphoto.com Methods OF purifying and Analyzing DNA constructs DNA constructs introduced into bacteria can be easily purified and analyzed by a variety of methods Colony Polymerase Chain Reaction Restriction digest Sequencing Protein expression and characterization Any other DNA and protein manipulation... I Summary of the specific cloning steps 1. 2. 3. 4. 5. 6. V Use restriction enzymes (EcoRI and BamHI) to cut the EGFP DNA fragment (I) Cut a receiving vector (pET28, V) with the same restriction enzymes Join the insert to the vector using DNA ligase to make pET28bEGFP Transform bacteria with the recombinant plasmid Select the transformed bacteria on agar with appropriate antibiotics Analyze colonies for desired recombinant plasmid pET28b-EGFP ...
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This note was uploaded on 03/28/2011 for the course BIO 311 taught by Professor Staff during the Fall '08 term at SUNY Stony Brook.

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