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Cloning Using Plasmid Vectors Cloning – when one gene isn't enough... Cloning = transfer of a DNA fragment of interest to a selfreplicating genetic element such as a plasmid (genomics.energy.gov) Vector = a molecule used as a vehicle to carry foreign DNA into a host cell Simplest vector = plasmid Features of Plasmids Size Functions encoded Structure Nomenclature Micrograph taken by Dr. Jack Griffith from: http://www.accessexcell ence.org/RC/AB/WYW/ cohen/cohen_3.php. Why are plasmids suitable cloning vectors? Generally do not kill host cell Relatively easy to purify Can be made small Why not use naturally occurring plasmids? Replication Plasmid must contain a replicon Replicon - an origin of replication (oriV) plus associated cis-acting elements Functions of the ori region Host range Narrow vs Broad Copy number – eg - pUC vs pET Incompatibility Conjugation/mobilisation Desireable Features of Plasmid Cloning Vectors High copy number Small Selectable Genes Unique restriction sites May have additional features such as mob sites, RNA polymerase promoters, etc. pBR322 1973-1978 Bolivar and Rodriguez derivative 322 4.36 Kb; ~16 copies per cell Narrow host range Encodes resistance to ampicillin and to tetracycline How can we tell if plasmid contains DNA of interest? Insertional inactivation Use BamHI site in Tetr gene for cloning Transform Plate cells on? Confirm by replica-plating on? Early 1980s – pUC series Features of pUC Plasmids Small Very high copy number (temperature dependent) No insertional inactivation Blue-White Selection Vector contains first 146 aa of the β−galactosidase gene (lacZ) (α−peptide) MCS embedded within this region Blue-White Selection Continued Host cell encodes carboxy terminal portion of lacZ Neither host nor plasmid encodes for entire protein Together produce enzyme that can cleave Xgal to produce blue precipitate http://www.tciamerica.com/ product/bio-chem/B030.shtml What if foreign DNA inserted into MCS? Foreign DNA will contain a termination codon in the same reading frame as the α−peptide No α−peptide therefore no β−galactosidase and no blue coloured colonies www.sigmaaldrich.com Additional Features Origin of DNA replication from ss filamentous phage such as F1 or M13 Phagemid vector Not commonly used anymore T7 and SP6 promoters RNA production Sequencing primers www.promega.com Cloning in Plasmid Vectors Plasmid and DNA of interest cleaved with restriction enzyme(s) forming compatible ends Ligation Cloning – Easiest method 5' 3' GAATTC CTTAAG 3' 5' EcoRI cleavage creates 5' Sticky ends 5'P 3'OH 3'OH G CTTAA 5'P 5'P AATTC G 3'OH 5'P Inter- and Intramolecular Reactions vector insert Intermolecular reaction vector insert Intramolecular reaction Formation of a noncovalently bonded circular molecule Enzyme Choice Determined by Vector MCS from pUC19 from Fermentas.com Enzyme Choice Also determined by: Insert sequence NEB Cutter: http://tools.neb.com/NEBcutter2/index. php Cloning PCR Products Mix PCR product and linearised vector and carry out blunt end ligation Will this work? Sometimes, sort of Problems? Strategies Incorporate restriction enzyme recognition sequences into PCR product OR Create specialised cloning vectors Introduction of Restriction Sites via PCR Restriction site sequence Primer 3' 5' Template DNA (http:www.fermentas.com/techinfo/re /restrdigpcrii.htm) Steps Involved Choose restriction enzyme sequences and build in to primers Carry out PCR Digest product and vector with complementary enzymes Ligate Digestion of PCR Products May be necessary to add additional bases in order to digest linear DNA (ie, restriction enzymes may not digest at ends of DNA molecules) NEB, Stratagene, Fermentas all have online resources to consult pGEM-T (Promega) Why Use? Allows H-bonds to form to stabilise hybrid molecule Advantages Disadvantages Other Options? www.fermentas.com Cloning 16srDNA Through the Fermentas web site, download instructions for CloneJET system Wed – plan on gel extraction, quantify DNA via spec and gel (if time permits – if not, finish Friday) Determine amount of PCR product to add for a 3:1 insert to vector molar ratio