Transcript Document

BIOLOGY 3020 Fall 2008
Recombinant DNA Technology
(DNA Cloning)
DNA and p53 Transcription Factor
How many transcription
factors (TFs) in Corn?
Lecture Outline
1. Traditional Cloning Method
2. New Gateway Cloning Method
3. Class Project
- The Keys of Corn
4. Class Project Part 1 - Cloning a
TF ORF into a Gateway entry
vector
5. Transformation of DNA into E.
coli
6. Sterile Technique (working with
E. coli)
1: Traditional Cloning
DNA Cloning
(many identical copies
of specific DNA
molecules)
is NOT the
same as
Organismal
Cloning
(identical genetic
copies of specific
individuals)
General Cloning Strategy for DNA
With Restriction enzymes
With Restriction
enzymes
with DNA ligase enzyme
(Genetic transformation)
See Chapter 17
of Lecture Text
(Klug and Cummings
Essential s of Genetics)
Traditional cloning of DNA using enzymatic
restriction and ligation
Genetic Map of Lambda Phage
Head
proteins
Tail
proteins
Intregration
Excision
Control
0
Lysis
48kb
EcoRI Restriction Sites
Cut with EcoRI
(‘sticky ends’)
pUC18
Mix and ligate together with DNA Ligase
Typical Cloning Strategy to make Library
Cut with restriction enzymes, mix and ligate
EcoR1
Transformation of E. coli
EcoR1
Nonrecombinant
EcoR1
Recombinant
Characterize
insert (“clone”)
Blue Colonies (Discard)
White Colonies (Keep)
pUC18 - a common
cloning vector
Essential features
Polylinker
Selectable marker (Ampr)
Screenable Marker (LacZ)
Bacterial Origin of replication (oriR)
LacZ- a screenable marker
EcoR1
EcoR1
EcoR1
Lac Z gene
Interrupted
Lac gene
pUC18
pUC18
“Recombinant
Molecules”
Beta-galactosidase
NO Beta-galactosidase
Gal + X(Blue dye)
X-gal
White colonies
blue colonies
(colorless)
Allows for easy visual “screening” of bacterial
colonies that contain recombinant DNA molecules
Bacterial colonies transformed with pUC18
White colonies
(contain recombinant
DNA molecules)
blue colonies
(contain non-recombinant
DNA molecules)
Advantages of Traditional Cloning
1: Recombine DNA molecules from any source
“The Awesome Skill”
2: 100’s of different restriction enzymes available
Disadvantages of Traditional Cloning
1: Some restriction sites not present or present
where not desired
2: Careful planning of cloning strategy required and
many steps involved (including gel purification)
3: Transfer from one vector to another not
straightforward (e.g. to maintain reading frame)
2: A NEW way of cloning
- “Gateway Cloning”
1. Maximize compatibility and flexibility
2. Minimize planning
3. Maintain reading frame
4.Eliminate the need for restriction enzyme
digestion, gel purification and ligation.
5. Provide high-throughput compatibility–
reactions are simple and robust
www.invitrogen.com/Content/Online
%20Seminars/gateway/1.htm
How to avoid restriction enzymes - take
advantage of Site specific recombination
Phage l recombination in E. coli
Phage l
E. coli
attP 243bp
attB 25bp
1.
Site-specific recombination
mediated by phage lambda
recombination proteins.
2. The reaction is specific and
directional: attB x attP
⇔attL x attR.
3. Each reaction is mediated by
a different combination of
enzymes.
attL 100bp
Integrated prophage
attR 168bp
Summary of Site Specific Recombination in E. coli
In lambda, the integration site is known as attP, in E. coli the site is
attB. The attB site is short, only 25 bp, keep this in mind as it will be
important later. The att sites contain the binding sites for the proteins
that mediate l recombination. The integration reaction (attB x attP)
is mediated by the proteins integrase (Int) and host integration factor
(IHF). When integration occurs, two new sites are created, attL and
attR, flanking the integrated prophage, with no loss of DNA
sequence.
All the att sites are alike in that they contain a 15-bp recognition
sequence for the recombinase (integrase). The reaction can also go in
the opposite direction (excision). When attL x attR recombine
(mediated by the proteins integrase, host integration factor and
excisionase [Xis]), the lambda -DNA is excised from the E. coli
genome, recreating the attB site in E. coli and the attP site in lambda
The GATEWAY™ Cloning System
The entry vector is recombined with a destination vector
using lambda recombination enzymes
Gene
attL1
attL2
Entry
Clone
attB1 x attP1
Km ccdB
attR1
attR2
Destination
Vector
Transform
E. coli
Amp
attR2
BP
Clonase
ccdB
Co-integrate
LR
Clonase
attP1
attB1
Gene
attL1 x attR1
attL2
Amp
Km
> 99% correct entry clones in Km r colonies next day
The intermediate cointegrate is resolved (2nd
recombination event) to leave an expression clone and
a by product. Select for the former on Ampicillin plates.
attB1
Amp
attR2
Gene
attB2
attB2 x attP2
BP
Clonase
Expression
Clone
LR
Clonase
Amp
ccdB
ccdB
Co-integrate
attB1
Gene
attP1
attL2 x attR2
attL2
Km
Transform
E. coli
>99% correct expression clones
in Ap r colonies next day
attP1
ByProduct
Km
attP2
3: Class Research Project Overview
Transcription Factors - The Keys of Corn
Corn which was
domesticated by Native
Americans has become the
most important cereal crop
worldwide
In 2005 nearly 700 million
metric tons of corn were
harvested worldwide
Plant scientists aim to
understand corn as much as
doctors understand humans
Corn genome is underway
Avg Annual US Usage
0.2% Hybrid Seed
1.4% Food
1.8% Starch
3.7% Alcohol
5.8% Sweeteners
44.7% Animal
Feed/Residual
16.8% Exports
25.6% Ending Stocks
(Buffer against a bad
crop)
Oil is extracted from the
germ (embryo) for cooking,
Starch in building materials
or intravenous solutions, the
shell (hull) is used in animal
feed
Source: National Geographic
June 1993 p91-117
All of the major crops worldwide are
cereals (grasses)
Maize
Wheat
Rice
Barley
Sorghums
Millets
Oats
Rye
Grain 2005 (Mt)
694,575,552
628,101,035
618,534,989
137,302,263
58,620,842
27,388,444
23,972,508
15,605,370
1961 (Mt)
205,004,683
222,357,231
215,654,697
72,411,104
40,931,625
25,703,968
49,588,769
35,109,990
Knowledge of one grass species helps immensely in
the breeding of other grass species
Known for some time that
Transcription Factor (TF)
proteins are molecular
machines that turn on and of
genes - like the keys of a car
Estimate that about 10% of
all genes encode TFs - about
3000 in humans and maybe
6000 in corn
Scientific American (February 1995, pp. 54-61)
Class Project is to begin
cloning all the TFs in maize as
a basis for further study of
global gene regulation - (Field
of Regulomics)
A set of Entry clones will be
made that can be used to
make many diffenret
constructs for molecualr
biology investigation.
With the Keys in hand, the
pace of discovery will quicken
Overview of Keys of Corn Project Strategy
Identify full length
TF clones in Genbank
Design PCR primers
to amplify ORF from
flcDNA clones
Transfer clone into a
variety of Gateway
Destination vectors
Sequence and verify
Entry clones
Produce blunt-end
PCR products of TF
ORFs
Select colonies and
isolate plasmid DNA
Mix PCR product with
pENTR TOPO Vector
Transform into
competent E. coli cells
4: Cloning full length Corn TF ORFs
1: Start with a partial sequence of an isolated corn
TF cDNA (see list at end of lecture) - (cDNA should
show some homology to known TFs)
2: Perform BLAST search with sequence to identify
closely related overlapping sequences in Genbank
database (>97% identity)
3: Organize different sequences into a contig using
ContigExpress program in Vector NTI
4: Translate long contig to identify if start and stop
codons are present - compare to known TFs
5: Choose the most 5’ clone and order from clone
repository (e.g. Arizona Genomics Institute)
6: Design PCR primers suitable to clone into
pENTR/D vector
7: Amplify the Open Reading Frame (ORF)
for each gene (Lab 10 Oct 30th/31st)
1 2
3
4
5
6
Lane 1 1kb DNA Ladder
Lane 2 DV535460 687 bp
Lane 3 DR972034 1302 bp
Lane 4 EE024212 831 bp
Lane 5 EE173988 1941 bp
Lane 6 DV532714 1434 bp
PCR products like these will be cloned into pENTR/D
(Lab 10 Nov 4th/5th) PCR of Corn TF
You will be provided with
1: a plasmid containing a TF flcDNA (this is the
template)
2: PCR primers to amplify the ORF of the cDNA
(designed by the course instructor and your TA)
3: Taq Polymerase, buffer, Mg solution and an
optional “PCR enhancer” solution
You will set up a few PCR reactions to find the
optimal Mg concentrations needed to amplify your
TF gene of interest.
Next week you will clone the PCR product (if
successful into a cloning vector (PENTR/D)
(Lab 10 Nov 4th/5th) PCR of Corn TF
PCR Optimization
Each PCR reaction must be optimized. Factors
such as annealing temperature, Mg ion
concentration, and Polymerase stabilizing agents
all affect PCR.
Each PCR reaction is different because of the
different primers that are used.
You will set up 4 reactions.
1:
2mM MgCl2,
2:
3mM MgCl2,
3:
2mM MgCl2 + Enhancer,
4:
3mM MgCl2 + Enhancer,
9: Clone PCR product into Cloning Vector
(Lab 11) Nov 18th/19th)
Topoisomerase speeds up Cloning
Topoisomerase has ligase activity. Kit provides linear
pENTR vector with Topo covalently bound near end ready to ligate in insert ($20 per ligation)
Mix PCR product
with pENTR/TOPO
Incubate 5
minutes at room
temperature
Place on ice
Ready for
transformation
into E. coli
PCR product ligated into pENTR/D
The GTGG overhang is displaced and the insert
is directionally cloned into the entry vector
(i.e. start codon is near attL1 region)
5: Genetic transformation of E. coli
E. Coli is naturally unable to take up DNA efficiently
By treating rapidly growing E. coli cells with ionic
solutions (CaCl2 and MgCl2) the cells are made
“competent” to take up DNA.
The competent cells can be frozen at -70°C for
later use (but they are very fragile and must be
pipetted very slowly).We will use One Shot Competent
cells.
Incubate thawed cells with DNA, then “heat-shock”
at 42°C for 30 seconds (DNA is taken up by cells).
Transfer to nutrient broth (S.O.C) and allow cells to
recover for 1hr.
Spread plate out on appropriate selection media
Heat shock
transformation of E. coli
1: Transfer 2ul of
TOPO cloning rxn
to One Shot cells
2: Keep on
ice for 30
min
3: Heat
shock at
42°C 30 sec
4: Back
on ice
Heat shock
transformation of E. coli
During 1hr incubation, the
kanamycin resistance gene is
expressed
5: Add 250 ul of
SOC nutrient
medium
6: Shake
transformed
cells at
37°C for 1
7: Plate out cells on
Kanamycin selective
medium
Performing the
Spread Plate
method I
1: Choose appropriate
nutrient agar plate with
the correct antibiotic
(and X-gal) if visual
screening
2: Using sterile technique
transfer a loopful of bacteria
from a culture tube onto
plate (or 100l of bacterial
culture using a pipette)
Performing the Spread
Plate method II
“Spreader” or
“Hockey Stick”
70% EtOH
Keep flame
away from
alcohol !!
3: Dip glass “hockey stick” in
70% ethanol. Holding it
DOWNWARDS flame until
alcohol is burned off. DO NOT
put back into alcohol
4: Remove lid of petri dish. With one
hand rotate dish. With other hand
move hockey stick lightly over surface
to spread the inoculum evenly
After Incubating the
plate overnight at
37°C- individual
colonies of
transformed bacteria
should be seen
Each team will pick
two individual colonies
(clones) and streak on
a new plate (single
colony purification)
for next week
Sterile technique
When handling E. coli and other bacteria it is essential
that the live cultures do not become contaminated with
other bacteria or fungi. The set of procedures used to
accomplish this are known as “sterile technique”
General Points
1: Keep vials or plates containing bacteria open for a
minimum amount of time.
2: Use sterilized instruments when handling the bacteria
3: Discard all bacteria in “biohazardous” waste - this
will be destroyed later
4: When using an open flame never leave it unattended
Streak Plate method to
Purify Single bacteria
Principle This is essentially a method to dilute the number of
organisms, decreasing the density - individual colonies to be
isolated from other colonies. Each colony is "pure," since
theoretically, the colony began with an individual cell
1. Begin with inoculating the first, or primary, quadrant of the
agar plate. Use a light touch. Don't penetrate or scrape the
agar surface. Cover plate with lid.
2. Flame the loop, cool by touching an uninoculated portion of
the surface.
3. Now rotate the plate. Open lid and streak again, remember:
you are picking up growth from quadrant one, and using this
as your inoculum for quadrant two.
4. Flame loop; rotate plate, and repeat procedure for
quadrants three and four.
Performing a Plate Streak I
1: Flame metal
inoculating loop,
let cool
momentarily.
2-3: Using sterile
technique transfer a
loop of bacterial
culture or single
colony onto loop
4: With one hand remove
lid of dish. With other
hand lightly brush the
loop back and forth on one
quadrant of the dish
Performing a Plate Streak II
8 :Incubate o/n at 37°C
4: Reflame metal
inoculating loop,
let cool
momentarily.
5,6,7: Rotate petri dish 90° Use 1st streak
as inoculum for 2nd streak (only pass the loop
through the 1st streak once). Repeat once
more rotating dish 90° and sterilizing loop
again
Plate Streak Method
This is an example of a good streak for isolation
using the "four corners" method.
This is not a great streak
plate but it is serviceable,
as there are a few
isolated colonies. - would
have been better if the
loop had been flamed
between each sector.
This is an example of how
NOT to streak for isolation.
Scribbling is not streaking,
and most likely will not result
in isolated colonies.
Final Caution!
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Cloning corn genes
may be hazardous
to your health don’t let this
happen to you!
