Molecular Cloning Biology 20L Spring 2003. Overview of Molecular Cloning Restriction digest of plasmid pUC19 and phage –GOAL: Linear pUC19 DNA and several.
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Overview of Molecular Cloning Restriction digest of plasmid pUC19 and phage –GOAL: Linear pUC19 DNA and several fragments of phage DNA Ligation reaction –GOAL: pUC19 recombined with one or more fragments Transformation reaction –GOAL: Use a bacterial host create multiple copies of our new DNA construct (cloning)
Overview of Molecular Cloning Identification of candidates –GOAL: Determine which bacteria have the desired product (plasmid pUC19 with fragment insert) Re-isolation of plasmid DNA –GOAL: Obtain multiple copies of the desired DNA construct Restriction map of plasmid DNA –GOAL: Determine which fragment was inserted in pUC19
Restriction Digest of plasmid pUC19 and phage Use the restriction enzyme Hind III to cut both plasmid pUC19 and phage. The recognition sequence for Hind III is: AAGCTT. This specific sequence occurs once in pUC19, and occurs seven times in phage. One site in pUC19 creates one linear piece of DNA when cut. Seven sites in create 8 fragments when cut. The DNA fragments will be separated and analyzed with gel electrophoresis.
pUC19 a genetically engineered plasmid 2.7 Kb (small size allows lots of room for inserting DNA) Circular non-genomic DNA. –Phage DNA is linear and much larger (48.5 Kb). Has an origin of replication, and a high copy number. (200+/cell) Ampicillin resistance gene – Codes for an enzyme that binds and degrades ampicillin. Lac Z gene (part of the Lac operon) –Codes for ß-galactosidase –An enzyme that breaks down lactose into glucose and galactose. –Polylinker cloning site within LacZ Contains recognition sequences for several restriction enzymes. A disruption at this site prevents the production of ß-galactosidase.
Ligation Reaction Hind III breaks covalent bonds at the recognition sequence. –AAGCTT AAGCTT –T TCGAATTCGA A The complimentary or “sticky” ends can readily form H -bonds. During the ligation reaction, the linearized plasmid and fragments are combined. –Most of these DNA fragments have “sticky” ends. However, because is originally linear, the fragments cut from each end do not have overhangs complementary to the HindIII cuts. When combined, the pieces form H-bonds in various configurations. The enzyme DNA ligase is used to form new covalent bonds. –ATP drives this reaction.
Some possible ligation reaction products: Recombinant No insert Fragments No ligation
Transformation Using bacterial cells to amplify the DNA of interest. Competent cells are able to take up foreign DNA and acquire genetic information. Ordinary Escherichia coli cells can be made competent through a treatment with Ca 2+. –Competent cells have very fragile cell walls, and must be handled gently. During the transformation reaction: –Competent cells are combined with the ligation products. –Incubated on ice (DNA sticks to the outer cell walls.) –Heat Shocked (Membranes become more porous and allow DNA to enter.) Not all the competent cells will take up DNA. We will determine the frequency. –Incubated in LB broth at 37ºC for ≈ 45 min. Long enough to allow transcription and translation of ampicillin resistance gene.
Some possible products of the transformation reaction: Plasmid w/ insert Ampicillin resistant Nonfunctional LacZ Plasmid w/o insert Ampicillin resistant Functional LacZ No plasmid No ampicillin resistance No LacZ gene Non circular DNA gets degraded within the cells. Bacterial cellGenomic DNA
Candidate Identification The transformation culture is plated on special media to help identify which cells have received the recombinant plasmid. Two types of media: LB + X-gal, & LB+ X-gal + amp Selection: –Cells with the plasmid can grow on ampicillin media. –Cells without the plasmid cannot grow on ampicillin media. Screening: –Cells with a functional LacZ gene can convert X-gal to X + gal. X-gal -------------------------------> X + galactose –Colorless ß-galactosidase Blue BLUE coloniesCells which produce ß-galactosidase form BLUE colonies. WHITE coloniesCells which are able to grow on ampicillin without ß-galactosidase production form WHITE colonies. (Suspect fragment insert)
Some possible products of the transformation reaction: Plasmid w/ insert Ampicillin resistant Nonfunctional LacZ White colony on LB+X-gal+amp. Plasmid w/o insert Ampicillin resistant Functional LacZ Blue colony on LB+X-gal+amp. No plasmid No ampicillin resistance No LacZ gene No growth on Ampicillin Bacterial cellGenomic DNA
Isolating plasmid DNA Transformed cells are grown in LB + ampicillin to amplify the target DNA Selective pressure is important. –E. coli has no instructions for passing the plasmid to the next generation during cell division. –The high number of plasmids within each cell slows growth and lowers the ability to compete. Plasmid DNA is amplified in two ways: –Cell division (Cells multiply in culture) –High copy replication (multiple copies per cell) A plasmid miniprep will be performed on cell cultures to extract and purify plasmid DNA. –Cells are disrupted chemically, and the plasmid DNA is separated from genomic DNA and cellular debris.
Final restriction digest Plasmid DNA isolated from transformed cells will be digested with Hind III, and compared to a known Hind III marker. Digests will be analyzed with gel electrophoresis for identification of cloned inserts.
UncutBlue CutBlue UncutWhite CutWhite /Hind III / BstE II
Transformation success Frequency of transformation = # Transformed cells Total # of cells in the culture Transformation efficiency = # Transformed cells Amount of DNA in g