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 Restriction enzymes are like an immune system for bacteria They cut DNA at very precise locations Virus Virus DNA bacteria v i u r D N A s

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.

How do we know if the process worked? Insulin gene Penicillin resistance gene How do we know if the process worked? Scientists use plasmids that possess antibiotic resistance genes Sometimes the bacteria absorbs the engineered plasmid Sometimes the bacteria does NOT absorb the engineered plasmid So how do we know if the bacteria absorbed the plasmid? Penicillin environment Insulin gene Penicillin resistance gene Insulin gene Penicillin resistance gene If the plasmid was NOT absorbed, the bacteria will… If the plasmid was absorbed, the bacteria will…

How do we know if the process worked? Scientists use plasmids that possess antibiotic resistance genes Sometimes the bacteria absorbs the engineered plasmid Sometimes the bacteria does NOT absorb the engineered plasmid So how do we know if the bacteria absorbed the plasmid? Penicillin environment

Stage 1: Prepare the plasmids to be cut by restriction enzymes Obtain the pARA plasmid pARA pARA

Stage 1: Prepare the plasmids to be cut by restriction enzymes Mix plasmids with… restriction enzymes BamH1 and Hind III… or water A+ A- Bam HinD H2O pARA pARA

A+ (digested plasmid) + means the restriction enzymes were added A– (uncut plasmid) - means the restriction enzymes not added BamHI 4495bp 4872bp 4872bp 377bp HindIII How many plasmid fragments should we be able to find? 2

There are thousands of plasmids in our microdrop sample

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 micropipette the A+ and A- samples.

Load the samples into an electrophoresis chamber

Connect the electrophoresis chamber to a power supply.

Electrophoresis: separates the plasmid fragments by their size Marker A+ A-

+ ― Electrophoresis: separates the plasmid fragments by their size Marker A+ A- DNA has a negative charge and will be pulled through the gel towards the positive electrode. +

+ ― Marker A+ A- What is the Marker? 10 precut segments of DNA. A+ fragments 4495 bp 377 bp + Marker A+ A- 4872 Multimer A- plasmid 4872 bp 10,000bp 4872 Nicked-circle 8,000bp 4872 Supercoiled 6,000bp 5,000bp What is the Marker? 4,000bp 3,000bp 10 precut segments of DNA. They have been cut to a specific size. 2,000bp Without the Marker, it would be impossible to determine the size of the fragments. We wouldn’t know if the enzymes properly cut the plasmids. 1,500bp 1,000bp ― 500bp

Common error #1

Common error #2

Common error #3

Perfect! 4495 bp 377 bp

#1) What likely went wrong? Marker A+ A- 10,000bp 8,000bp 6,000bp Restriction enzymes were accidentally added to the A- tube. 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

#2) What likely went wrong? Marker A+ A- 10,000bp 8,000bp 6,000bp Restriction enzymes were never added to the A+ tube. 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

#3) What likely went wrong? Marker A+ A- 10,000bp 8,000bp 6,000bp A+ and A- were accidentally placed in the wrong lanes. 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp 500bp

#4) What likely went wrong? Electricity was left on for too long. Marker A+ A- 10,000bp Electricity was left on for too long. 8,000bp 6,000bp 5,000bp 4,000bp 3,000bp 2,000bp 1,500bp 1,000bp