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HOW WAS

THE TEST?
39%

A. Too hard

33%

B. A little too hard

24%

C. Good
D. A little too easy
3%

E. Too easy

0%
A.

B.

C.

D.

E.

BIO215, Spring 2015; Marco Gallio

BIO215, Spring 2015; Marco Gallio

Great work!

More Lac Operon

what is this recombinant dna you speak of


BIO215, Spring 2015; Marco Gallio

WHAT ARE THESE THINGS?


these

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

Isolate, study, manipulate genes


from any organism
Key to modern molecular
genetics, medicine, biotechnology

BIO215, Spring 2015; Marco Gallio

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

BIO215, Spring 2015; Marco Gallio

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

BIO215, Spring 2015; Marco Gallio

NOT THE SAME!

OUR GENETIC ENGINEERING


PROJECT

BIO215, Spring 2015; Marco Gallio

BIO215, Spring 2015; Marco Gallio

GOAL
Make fluorescent fish!

BIO215, Spring 2015; Marco Gallio

BIO215, Spring 2015; Marco Gallio

STEP1:
Isolate the green fluorescence gene =
encodes the Green Fluorescent Protein
GFP

BIO215, Spring 2015; Marco Gallio

STEP1:
Replicate the GFP gene in bacteria
to study it

= cloning

How do we get the GFP gene?


BIO215, Spring 2015; Marco Gallio

BIO215, Spring 2014; Marco Gallio

1.1 SYSTEMATICALLY CUT THE DNA

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

1.1 SYSTEMATICALLY CUT THE DNA

One of the more common


RESTRICTION
ENZYMES

BIO215, Spring 2015; Marco Gallio

RESTRICTION
ENDONUCLEASES
NotI site
NotI is also a common
RESTRICTION ENZYME

A MULTIPLE CLONING CASSETTE (MCS) CONTAINS MULTIPLE RE SITES

BIO215, Spring 2015; Marco Gallio

1.2 INSERT THE DNA INTO A VECTOR


With a bacterial promoter for expression
Which
components
do we need
in a plasmid
vector?

BIO215, Spring 2015; Marco Gallio

Clone EcoRI fragments Here

CLONING SCHEMATIC

insert

vector

BIO215, Spring 2015; Marco Gallio

1.3 TRANSFORM BACTERIA

BIO215, Spring 2015; Marco Gallio

1.3 TRANSFORM BACTERIA

BIO215, Spring 2015; Marco Gallio

CLONING=
Each colony contains
Identical bacterial
descending
From1 cell = clones.
If transformed each will
have the same plasmid

Genomic library
(many plates like this)

BIO215, Spring 2015; Marco Gallio

WOULD THE JELLYFISH


PROMOTER WORK IN
BACTERIA?
57%
A. Yes

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?

BIO215, Spring 2015; Marco Gallio

Clone cDNAs Here

WOULD BACTERIA KNOW


HOW TO SPLICE GFP?
A. Yes

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%

BIO215, Spring 2015; Marco Gallio

Pre-mRNA

BIO215, Spring 2015; Marco Gallio

REVERSE TRANSCRIPTION POLYMERASE CHAIN


REACTION
(RT-PCR)

STAGE 1

Reverse
transcriptase

Complementary
DNA is an
artificial DNA copy
of each RNA

STAGE 2

DNA
Polymerase

BIO215, Spring 2015; Marco Gallio

cDNA Library

BIO215, Spring 2014; Marco Gallio

BIO215, Spring 2015; Marco Gallio

WHAT IF WE KNEW THE


JELLYFISH GENOME
SEQUENCE?

BIO215, Spring 2015; Marco Gallio

STEP1:
Discover the green fluorescence gene =
encodes the Green Fluorescent Protein
GFP

BIO215, Spring 2014; Marco Gallio

Here we will
assume we know
the genome
sequence of the
jellyfish

MICROARRAY

A DNA microarray (also commonly


known as DNA chip) is a collection of
microscopic DNA spots attached to a
solid surface
Each spot (25bp) is
seeded with a known
sequence that
corresponds to a gene

tentacles

hood

BIO215, Spring
2014; Marco Gallio

Gene prediction in the genome

BIO215, Spring 2015; Marco Gallio

GFP SEQUENCE

BIO215, Spring 2015; Marco Gallio

REAL TIME or quantitative PCR


We could use this to
confirm expression

cycle number

You can follow the


amplification curve in real
time: quantification of
initial amount of mRNA is
much more accurate
BIO215, Spring 2015; Marco Gallio

BIO215,
Spring 2015;
Marco Gallio

(WHAT IF WE DO NOT FIND THE RIGHT SITES


FLANKING THE GFP GENE?)

GFP coding

We can add them to the


primers when we amplify the
GFP from jellyfish by RTPCR: theyll get incorporated
into the PCR product

BIO215, Spring 2015; Marco Gallio

Electrophoresis in an agarose gel

BIO215, Spring 2015; Marco Gallio

CLONING SCHEMATIC

RT-PCR

insert

vector

BIO215, Spring 2015; Marco Gallio

BIO215, Spring 2015; Marco Gallio

GFP STRUCTURE

BIO215, Spring 2015; Marco Gallio

SEQUENCING

BIO215, Spring 2015; Marco Gallio

QUESTIONS?

BIO215, Spring 2015; Marco Gallio