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

v i ru s D N A

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

Insulingene

Insulingene

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 pAMPpKAN pAMP

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

• Mix plasmids with…– restriction enzymes BamH1 and Hind III…– or water

pKAN pAMP

BamHinD

BamHinD

pKAN pAMP

H2O H2O

K+ K- A+ A-

Restriction enzyme: Hind III

(cuts @ bp 234)

Restriction enzyme: Bam HI

(cuts @ bp 2095)

Plasmid w/ kanamycin resistance (pKAN)

pKAN = 4194 bp

1861 bp restriction fragment2333 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? 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

Kno

wn

DN

A

mar

kers

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

Mar

ker

DN

A

K+

K-

A+

A-

2,000bp

500bp

Mar

ker

DN

A

K+

K+

K+

A+

A+

A+

2011 Class Data

M A+ K+ A+ K+ A+ K+ A+ K+ K-A- M

500

1000

1500

2000

300040005000

10,00080006000

500

1000

1500

2000

300040005000

10,00080006000

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

Mar

ker

DN

A

K+

K-

A+

A-

2,000bp

500bp

Mar

ker

DN

A

K+

K+

K+

A+

A+

A+

10,000bp

8,000bp

6,000bp

5,000bp

4,000bp

3,000bp

1,500bp

1,000bp

Mar

ker

DN

A

K+

K-

A+

A-

2,000bp

500bp

Mar

ker

DN

A

K+

K+

K+

A+

A+

A+