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What are Plasmids?

What are Plasmids?. Plasmids are circular pieces of bacterial DNA that often contain genes not related to basic life functions Often contain antibiotic resistances Humans often cut open plasmids…attach a desired gene…reinsert the plasmid to the bacteria. What are restriction enzymes?.

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What are Plasmids?

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  1. What are Plasmids? • Plasmids are circular pieces of bacterial DNA that often contain genes not related to basic life functions • Often contain antibiotic resistances • Humans often cut open plasmids…attach a desired gene…reinsert the plasmid to the bacteria

  2. What are restriction enzymes? • Enzymes that bacteria use to fight off viruses • Restriction enzymes are like an immune system for bacteria • They cut DNA at very precise locations Virus Virus DNA bacteria v i r u D N A s

  3. Genetic Engineering • Humans are learning to manipulate DNA • We use restriction enzymes to cut open a bacterial plasmid… • We use the same restriction enzymes to cut out a human gene… • Once removed, we can insert the human gene into a bacterial plasmid Insulin gene Insulin gene

  4. Stage 1: Prepare the plasmids to be cut by restriction enzymes • Obtain the plasmids (pKAN and pAMP) P stands for plasmid pKAN = plasmid with resistance to kanamycin pAMP = plasmid with resistance to ampicillin pKAN pKAN pAMP pAMP

  5. Stage 1: Prepare the plasmids to be cut by restriction enzymes • Mix plasmids with… • restriction enzymes BamH1 and Hind III… • or water K- A- K+ A+ Bam HinD Bam HinD H2O H2O pKAN pKAN pAMP pAMP

  6. Plasmid w/ kanamycin resistance (pKAN) Restriction enzyme: Hind III (cuts @ bp 234) pKAN = 4194 bp Restriction enzyme: Bam HI (cuts @ bp 2095) 1861 bp restriction fragment 2333 bp restriction fragment

  7. There are thousands of plasmids in our microdrop sample

  8. K–(uncut plasmid)- means the restriction enzymes not added K+(digested plasmid)+ means the restriction enzymes were added Small (1861bp) Big (2333bp) How many plasmid fragments? How many plasmid fragments?

  9. Stage 2: Check to see if the restriction enzymes worked • DNA electrophoresis • Plasmid fragments are loaded into a gel • Connected to a power supply • Separates fragments based on their sizes • Smaller fragments travel further through the gel

  10. We will then micropipette the plasmids

  11. Load the plasmids into an electrophoresis chamber

  12. Connect the electrophoresis to a power supply…DNA has a negative electric charge.

  13. Electrophoresis: sizes DNA fragments K+ K- A+ A- Known DNA markers How many marks will appear in the K- and A- lanes? 10,000bp 8,000bp 6,000bp What just happened? Why not just 1 band? 5,000bp 4,000bp 3,000bp 2,000bp Look at the lab handout and let’s predict the A+ fragments 1,500bp 1,000bp positive 500bp

  14. K+ K+ K+ K+ A+ A+ A+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

  15. 2011 Class Data K- M A+ K+ A+ K+ A+ K+ A+ K+ A- M 10,000 10,000 8000 8000 6000 6000 5000 5000 4000 4000 3000 3000 2000 2000 1500 1500 1000 1000 500 500

  16. So now what??? • Plasmids have been engineered for human uses. The human gene for insulin (red) can now be added to the plasmid. The bacteria will produce insulin for diabetics!

  17. So now what??? The bacteria with the recombinant DNA replicates, thus passing the insulin gene onto its offspring. Each cell now will produce insulin for humans to harvest and use.

  18. K+ K+ K+ K+ A+ A+ A+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

  19. K+ K+ K+ A+ A+ A+ K+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

  20. K+ K+ K+ K+ A+ A+ A+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

  21. K+ K+ K+ A+ A+ A+ K+ A+ K- A- Marker DNA Marker DNA 10,000bp 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

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