Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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. What are restriction enzymes? • Enzymes that bacteria use to fight off viruses Virus Virus DNA vi u r s DNA bacteria • Restriction enzymes are like an immune system for bacteria • They cut virus DNA at very precise locations 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. So now what??? • 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. 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 Stage 1: Prepare the plasmids to be cut by restriction enzymes • Mix plasmids with… – restriction enzymes BamH1 and Hind III… – or water K+ K- A+ A- Bam HinD H2O Bam HinD H2O pKAN pKAN pAMP pAMP Plasmid w/ kanamycin resistance (pKAN) Restriction enzyme: Hind III pKAN = 5512 bp Restriction enzyme: Bam HI 4705bp restriction fragment 807bp restriction fragment There are thousands of plasmids in our microdrop sample K+ (digested plasmid) K– (uncut plasmid) + means the restriction enzymes were added - means the restriction enzymes not added Big (4705bp) Small (807bp) How many plasmid fragments? How many plasmid fragments? 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 We will then micropipette the plasmids Load the plasmids into an electrophoresis chamber Connect the electrophoresis to a power supply…DNA has a negative electric charge.