1 / 13

Transforming E. coli with a Recombinant Plasmid

Transforming E. coli with a Recombinant Plasmid. What is biotechnology?. Employs use of living organisms in technology and medicine Modifying living organisms for our use Mass production Recombinant DNA Recombine DNA fragments from various sources. Introduction. Restriction enzymes

braima
Download Presentation

Transforming E. coli with a Recombinant Plasmid

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Transforming E. coli with a Recombinant Plasmid

  2. What is biotechnology? • Employs use of living organisms in technology and medicine • Modifying living organisms for our use • Mass production • Recombinant DNA • Recombine DNA fragments from various sources

  3. Introduction • Restriction enzymes • Cut DNA molecules from various organisms and recombine pieces • Recombinant DNA • Restrict the growth of viruses in bacteria • Digest the DNA molecule at specific nucleotide sequences • Restriction fragments • DNA fragments • Sticky ends • Allow annealing and recombination of DNA fragments from different sources

  4. BamH I sticky end Hind III sticky end Hind III sticky end BamH I sticky end Engineering the Plasmid: ligation of rfp gene into p-ARA Bruce Wallace

  5. Introduction • Bacterial plasmids • Circular pieces nonessential DNA found in bacteria • Can be engineered to carry genes • Express proteins encoded by these genes

  6. pGLO Plasmid • Contains: • Green Fluorescent Protein gene • Codes for green fluorescent protein • Ampicillin resistance gene • Great example of : DNA RNA PROTEIN TRAIT

  7. Introduction • Purpose: • Get the pGLO plasmid containing the gfp gene into bacterial cells • Get those cells to express the gfp gene and make the mutant fluorescent protein • Transformation • Process of taking up foreign pieces of DNA • In this case, a plasmid • Not very common in mature! • Can occur under experimental conditions (1 cell in a thousand!)

  8. Plasmid DNA Insertion

  9. Transgenic Colony Allowed to Grow

  10. Introduction • What are competent cells?!?! • Cells ready to receive plasmids through lab procedures • Soaked in calcium chloride • PM and DNA negatively charged • Calcium ions (Ca++) neutralize charges to allow for plasmid to pass through PM • Need to “heat shock” cells • Creates pressure differences to allow cell membrane to be permeable to DNA • Cold then hot • Then feed and recover!!

  11. Media • Spread cells on various plates (3) • LB plate • Only bacterial food • “control” • LB/amp plate • Contains ampicillin • Antibiotic prevents bacteria from forming CW • Cells that contain ampr produces protein that destroys ampicillin • “-” and “+” • LB/amp/ara plate • Contains arabinose • Needed to express the gfp gene • If take up plasmid, this helps with transcription of gene

  12. Predictions…… -pGLO +pGLO -pGLO +pGLO LB LB/amp LB/amp/ara LB/amp

  13. Predictions…… -pGLO +pGLO -pGLO +pGLO + + + - LB LB/amp LB/amp/ara LB/amp

More Related