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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 resistance genes Humans often cut open plasmids…attach a desired gene…reinsert the plasmid to the bacteria

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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 resistance genes • Humans often cut open plasmids…attach a desired gene…reinsert the plasmid to the bacteria • When complete, the bacteria will contain a new gene (instructions) to create a desired protein such as insulin.

  2. What are restriction enzymes? • Enzymes that bacteria use to fight off viruses • Restriction enzymes are like an immune system for bacteria • They cut virus 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 can use a restriction enzyme to cut open a bacterial plasmid… exposing sticky ends. • We would use the same restriction enzyme to cut out a human gene…exposing matching sticky ends • We can then insert the human gene into the bacterial plasmid. The sticky ends of each will match and bond.

  4. The bacteria will hopefully absorb the recombinant DNA and replicate. Each time passing the insulin gene onto its offspring. But how do we know if the bacteria absorbs the plasmid? Plasmids often contain antibiotic resistance genes Ex: pKAN resists kanamycin If the plasmid is known to resist kanamycin, the scientist will grow the bacteria in kanamycin If the bacteria lives… it must have absorbed the plasmid. If the bacteria dies… it did not absorb the plasmid. Each bacteria cell now will produce insulin that can eventually be harvested for diabetics. So now what???

  5. 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

  6. 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

  7. Plasmid w/ kanamycin resistance (pKAN) Restriction enzyme: Hind III pKAN = 5512 bp Restriction enzyme: Bam HI 807bp restriction fragment 4705bp restriction fragment

  8. There are thousands of plasmids in our microdrop sample

  9. K–(uncut plasmid)- means the restriction enzymes not added K+(digested plasmid)+ means the restriction enzymes were added Small (807bp) Big (4705bp) How many plasmid fragments? How many plasmid fragments?

  10. 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

  11. We will then micropipette the plasmids

  12. Load the plasmids into an electrophoresis chamber

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

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