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? Enzymes that bacteria use to fight off viruses Restriction enzymes are like an immune system for bacteria They cut DNA at very precise locations bacteria Virus Virus DNA viru s DNA

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

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 pAMP pKAN pAMP

Stage 1: Prepare the plasmids to be cut by restriction enzymes Mix plasmids with… –r–restriction enzymes BamH1 and Hind III… –o–or water pKAN pAMP Bam HinD Bam HinD pKAN pAMP H2OH2O H2OH2O K+ K- A+ A-

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

There are thousands of plasmids in our microdrop sample

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

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.

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

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

2011 Class Data MA+K+A+K+A+K+A+K+ K- A-M , ,

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!

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.

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

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