Ch. 13.1: BIOTECHNOLOGY
Objectives:
1. Explain how the
use of bacteria has
contributed to the
development of
DNA technology.
2. List some recent
research trends in
recombinant DNA
technology.
Vocab:
 Biotechnology
 Recombinant DNA
technology
Biotechnology
= Use of organisms
to perform
practical tasks.
Examples:
 Use bacteria to make proteins/drug therapies
 Engineer plants with natural resistance to
insects, drought, fungi, etc.
 Develop vaccines.
Recombinant DNA
= DNA from 2
different
sources
combined.
13.2: Bioengineering
Objectives:
1. Explain the role of plasmids in
2.
3.
4.
engineering bacteria.
Explain how biologist “cut and
paste” DNA.
Describe the procedure used in
cloning a specific gene.
Identify the usefulness of
recombinant microorganisms.
Vocab:
Plasmid
Genomic library
Restriction enzyme
Nucleic acid probe
Bacteria: Work horses of Biotech.


Used to mass produce
useful genes +
proteins.
Simple organisms




1 chromosome.
Plasmids
Reproduce rapidly
Easy to manage in a
lab.
Bacteria Plasmids






Small, circular DNA
Separate from
chromosome.
Contains a few genes.
Make copies of itself
Can be shared/ transferred
b/w bacteria.
Exchange of plasmids is how
bacteria build genetic variation.
Genes for antibiotic resistance
are shared this way :(
Making Recombinant DNA
1. Remove desired gene
from donor cell.
2. Remove bacteria
plasmid.
3. Insert gene into
plasmid.
4. Return plasmid to
bacteria.
5. Gene is transcribed
and translated into
protein product.
Recombinant DNA
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio38.swf::Early
Restriction Enzymes
= Enzymes that CUT foreign DNA
sequences.
Each enzyme is cuts a specific
sequence (CCCGGG or GAGCT)
Cuts sugar-phosphate backbone of
DNA
Make staggered cuts.
Leave “sticky ends” on cuts.
Evolved in bacteria to protect
bacteria fr. invading viruses.
Restriction Enzymes: Cut DNA
into fragments
BLUNT ends =no staggered cuts;
not as useful
Sticky ends = made by staggered
cuts; unpaired bases; useful b/c
they h-bond w/ complimentary
bases in other fragments.
Helps to “sew” fragments
together & make recomb. DNA.
Ligase: Glues DNA fragments together
Genetic Cloning = Copies of recomb. DNA (and resulting
proteins) are made by reproducing organisms
Genomic Library
= Complete collection of cloned DNA
fragments from an organisms.
When you use restriction enzymes they cut
up the donor DNA into MANY fragments.
Each fragment is incorporataed into a
plasmid.
You need to figure out which bacteria has
desired recomb. Plasmid!
Identifying Desired Recomb. DNA
1. Use nucleic acid probe


Radioactively labeled
complimentary sequence
(TAGGCT will find and bind to
ATCCGA when strands are
separated).
2. Insert desired DNA into plasmid sequence for
antibiotic resistance.
Recomb. plasmids will lose resistance to antibiotics
and will NOT survive when exposed to antibiotic.
Task: Diagram Steps in Creating
recombinant DNA clones
Use and illustrate the following key terms…
 Host DNA
 Bacteria Plasmid
 Vector
 Restriction Enzyme
 Sticky Ends
 Ligase
 Bacteria reproduces/plasmid replication
 Recombinant DNA
 Clones
Lab: Recombinant Paper Plasmids
Goal: Insert human gene for insulin production into
bacteria plasmid.
1. Construct bacteria plasmid. Color code sequences for
antibiotic resistance.
2. Label 3’ and 5’ ends of restriction enzymes and
plasmid DNA.
3. Identify restriction enzymes that will cut plasmid in
sequence for antibiotic resistance. Mark these
locations and label the enzyme used.
Lab: Recombinant Paper Plasmids
Goal: Insert human gene for insulin production into
bacteria plasmid.
•
Label 3’ and 5’ of human DNA
•
Determine the enzyme that will allow for removal of
human insulin gene AND match up with the sticky
ends on the plasmid.
•
Create the recombinant plasmid.