Professional Documents
Culture Documents
THE TEST?
39%
A. Too hard
33%
24%
C. Good
D. A little too easy
3%
E. Too easy
0%
A.
B.
C.
D.
E.
Great work!
Bacterial wheels
Chloroplasts
Holes
Plasmids (DNA)
Plasmids (RNA)
4%
A.
4%
B.
2%
0%
C.
D.
E.
BIO215, Spring
2015; Marco
Gallio
A.
B.
C.
D.
E.
90%
Genetic
engineering
USES
Isolate genes to better study the proteins they
encode, understand their function in the cell
Isolate and study genes that are causing human
diseases - screen drugs, design rational
therapies
Manipulate genes to improve crops, produce
useful proteins (insulin), create useful bacteria
for bioremediation
DEFINITIONS:
Recombinant DNA
Recombinant DNA molecules are formed by
laboratory methods of genetic recombination to
bring together genetic material from multiple
sources, creating sequences that would not
otherwise be found in biological organisms.
Molecular Cloning
is a set of experimental methods used to
direct the replication of recombinant DNA
molecules within host organisms [bacteria, yeast].
cloning refers to the fact that the method
involves the replication of a single DNA molecule
starting from a single living cell to generate a large
population of cells containing identical DNA
molecules
GOAL
Make fluorescent fish!
STEP1:
Isolate the green fluorescence gene =
encodes the Green Fluorescent Protein
GFP
STEP1:
Replicate the GFP gene in bacteria
to study it
= cloning
EcoRI
EcoRI dimer
Structure of the homodimeric
restriction enzyme EcoRI (cyan
and green cartoon diagram)
bound to double stranded DNA
(brown tubes). Two catalytic
magnesium ions (one from each
monomer) are shown as
magenta spheres and are
adjacent to the cleaved sites in
the DNA made by the enzyme
(depicted as gaps in the DNA
backbone)
DNA
RESTRICTION ENZYMES CUT DNA AT SPECIFIC SEQUENCES
BIO215, Spring 2015; Marco Gallio
RESTRICTION
ENDONUCLEASES
NotI site
NotI is also a common
RESTRICTION ENZYME
CLONING SCHEMATIC
insert
vector
CLONING=
Each colony contains
Identical bacterial
descending
From1 cell = clones.
If transformed each will
have the same plasmid
Genomic library
(many plates like this)
38%
B. NO
C. Dunno
5%
A.
BIO215, Spring 2015; Marco Gallio
B.
C.
WE NEED A VECTOR
With a bacterial promoter for expression
Which
components
do we need
in a
plasmid?
98%
B. No
C. GFP must have
no introns
0%
GF
P
m
us
t
ha
v
en
in
tro
ns
No
Ye
s
2%
Pre-mRNA
STAGE 1
Reverse
transcriptase
Complementary
DNA is an
artificial DNA copy
of each RNA
STAGE 2
DNA
Polymerase
cDNA Library
STEP1:
Discover the green fluorescence gene =
encodes the Green Fluorescent Protein
GFP
Here we will
assume we know
the genome
sequence of the
jellyfish
MICROARRAY
tentacles
hood
BIO215, Spring
2014; Marco Gallio
GFP SEQUENCE
cycle number
BIO215,
Spring 2015;
Marco Gallio
GFP coding
CLONING SCHEMATIC
RT-PCR
insert
vector
GFP STRUCTURE
SEQUENCING
QUESTIONS?