BIOLOGY 3020 Fall 2008Recombinant DNA

Technology(DNA Cloning)

How many transcription factors (TFs) in Corn?DNA and p53 Transcription Factor

Lecture Outline1. Traditional Cloning Method2. New Gateway Cloning

Method3. Class Project

- The Keys of Corn4. 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)

DNA Cloning (many identical

copies of specific

DNA molecules) is NOT the same as

Organismal Cloning

(identical genetic copies of specific

individuals)

1: Traditional Cloning

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

EcoRI Restriction Sites

0 48kb

Genetic Map of Lambda PhageHead proteins

Tailproteins

Intregration Excision Control Lysis

Cut with EcoRI(‘sticky ends’)

Mix and ligate together with DNA Ligase

pUC18

Typical Cloning Strategy to make Library

Non-recombinant

Blue Colonies (Discard)

Cut with restriction enzymes, mix and ligate

Characterize insert (“clone”)

EcoR1EcoR1 EcoR1

Recombinant

White Colonies (Keep)

Transformation of E. coli

Essential features

Polylinker

Selectable marker (Ampr)

Screenable Marker (LacZ)

Bacterial Origin of replication (oriR)

pUC18 - a common cloning vector

Lac Z gene

Beta-galactosidaseX-gal(colorless)

Gal + X(Blue dye)blue colonies

White colonies

NO Beta-galactosidase

EcoR1

InterruptedLac gene

EcoR1 EcoR1

pUC18 pUC18

“Recombinant Molecules”

LacZ- a screenable marker

Allows for easy visual “screening” of bacterial colonies that contain recombinant DNA molecules

Bacterial colonies transformed with pUC18

blue colonies(contain non-recombinant DNA molecules)

White colonies(contain recombinant DNA molecules)

Advantages of Traditional Cloning1: Recombine DNA molecules from any source

“The Awesome Skill”

2: 100’s of different restriction enzymes available

Disadvantages of Traditional Cloning1: 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

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.

Phage

E. coli

attP 243bp

Integrated prophage

Phage recombination in E. coli

attB 25bp

attL 100bp

attR 168bp

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 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

Summary of Site Specific Recombination in E. coli

attL1 x attR1

attB1 x attP1

> 99% correct entry clones in Km r colonies next day

TransformE. coli

attL1

Gene

attL2

Km

EntryClone

ccdB

attR1 attR2

Amp

DestinationVector

LR Clonase

BPClonase

Co-integrate

Gene

ccdB

Amp

Km

attB1

attP1

attL2

attR2

The GATEWAY™ Cloning SystemThe entry vector is recombined with a destination vector

using lambda recombination enzymes

Co-integrate

Gene

ccdB

Amp

Km

attB1

attP1

attL2

attR2

Gene

Amp

ExpressionClone

attB1 attB2

ccdB

Km

By-Product

attP1 attP2

LR Clonase

BPClonase

>99% correct expression clones in Ap r colonies next day

Transform E. coli

attL2 x attR2

attB2 x attP2

The intermediate cointegrate is resolved (2nd recombination event) to leave an expression clone and a

by product. Select for the former on Ampicillin plates.

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

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)

Avg Annual US Usage

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

Grain 2005 (Mt) 1961 (Mt)Maize 694,575,552 205,004,683 Wheat 628,101,035 222,357,231 Rice 618,534,989 215,654,697 Barley 137,302,263 72,411,104Sorghums 58,620,842 40,931,625Millets 27,388,444 25,703,968Oats 23,972,508 49,588,769Rye 15,605,370 35,109,990

All of the major crops worldwide are cereals (grasses)

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

Produce blunt-end PCR products of

TF ORFs

Mix PCR product with pENTR TOPO

Vector

Transform into competent E. coli

cells

Select colonies and isolate plasmid

DNA

Sequence and verify Entry clones

Transfer clone into a variety of Gateway Destination vectors

4: Cloning full length Corn TF ORFs1: 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)

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

1 2 3 4 5 6

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,

Topoisomerase speeds up CloningTopoisomerase has ligase activity. Kit provides linear pENTR vector with Topo covalently bound near end - ready to ligate in insert ($20 per ligation)

9: Clone PCR product into Cloning Vector

(Lab 11) Nov 18th/19th)

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. coliE. 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

100

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

100

5: Add 250 ul of SOC nutrient medium

6: Shake transformed cells at 37°C for 1 hr

7: Plate out cells on Kanamycin selective medium

Heat shock transformation of E.

coli

During 1hr incubation, the kanamycin resistance gene

is expressed

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 100l of bacterial culture using a pipette)

100

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

Performing the Spread Plate method II

70% EtOH

“Spreader” or “Hockey Stick”

Keep flame away from alcohol !!

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

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

Sterile technique

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.

Streak Plate method to Purify Single bacteria

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

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

8 :Incubate o/n at 37°C

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.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Final Caution!

Cloning corn genes may be hazardous to your health -

don’t let this happen to you!