1

Plant Biotechnology PLNT 2530

Lab Manual

Table of Contents

Page

Laboratory Schedule and Write-Up Due Dates 2

Overview 3

Format of Lab Reports 3

Useful Websites 4

Sample Dilution Calculation 5

Lab 1: Sterilization and Aseptic Technique 6

Lab 2: Agrobacteria Mediated Plant Transformation (Part I) 11

Lab 3: Plant Regeneration 15

Lab 4: Plasmid Construction 18

Lab 5: Bacterial Transformation 23

Lab 6: Isolation and Characterization of Plasmids 26

Lab 7: Gene Expression in E. coli and Bioassay 32

Lab 8: Isolation of Genomic DNA and PCR 36

Lab 9: Agrobacteria Mediated Plant Transformation (Part II) 43

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LABORATORY SCHEDULE

(Monday, Tuesday Rm 342)

Date /2019 Lab Title Write-up Due Date

Week of

Jan 14th

Sterilization and Aseptic Techniques/Computer Orientation none

Jan 21st Agrobacteria Mediated Plant Transformation (Part I) Apr 8th

Jan 28th Plant Regeneration Mar. 4th

Feb. 4th Plasmid Construction (Feb. 25th)

Feb. 11th Bacterial Transformation Feb. 25th

Feb. 25th Isolation and Characterization of Plasmids Mar 11th

& Mar. 4th

Mar 11th Gene Expression and Bioassay Mar 18th

Mar 18th Isolation of Genomic DNA and PCR Apr. 1st

& March25th

April 1st Agrobacteria Mediated Plant Transformation (Part II) [attached to Part I]

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NOTE: Last lab report must be handed in on or before April 8, 2019

Late lab reports will be docked 20% per day

NOTE: Most labs will require 2½ - 3 hrs to complete

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PLANT BIOTECHNOLOGY PLNT 2530

The purpose of a laboratory is to expedite the understanding of course material through

demonstration and hands-on experience. While it is not possible to cover all aspects of a course

in a lab, aspects such as developmental changes, principles and techniques can be demonstrated.

A demonstration is only as effective as its observers are observant. One of the most

IMPORTANT aspects of any laboratory experience is disciplining oneself to OBSERVE. To be

observant take time, concentration, knowing what to look for and how to record it for later

reference and comparison if an extended time frame is involved. Knowing what to look for

means being prepared by careful reading of the lab handouts and related readings before coming

to lab and taking the time to think about the lab.

Your lab report, above and beyond any definitive results of a given experiment, should

demonstrate that you have been observant. When experiments go wrong "for no apparent

reason" (a euphemism for "no one observed what happened") nothing is gained. The preparation

of, and participation in the labs takes time. To gain from the experience you must put an honest

effort into the labs!

Format of Lab Reports

Title:

Objectives: Outline both the general and specific objectives of the experiment.

Procedure: This should be essentially as described in the handout and need not be repeated.

However, if modification are incorporated (intentionally or accidentally, BE

OBSERVANT!) or some aspect was advanced beyond the lab outline, a complete

description of all steps and changes should be included.

Observations & Results: Describe concisely what you observe. For prolonged

experiments a log of the changes observed on a week to week basis should be

maintained. When a factor which will likely affect the experiment is changed,

very careful notes should be taken before and after the change to be able to report

the effect of the change. If things go wrong be observant of factors which may

have contributed to the problem. Tabulate in proper units and significant figures.

Discussion: Analyse your results. Compare what you get with what you might expected and

discuss reasons for differences if they exist. Cite reference material you have

used to establish your expectations. Answer the questions.

Reports should be carefully prepared, clearly written or typed and not exceed 5 pages of

text per lab. (Tables and Figures don’t count)

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STERILIZATION AND ASEPTIC TECHNIQUES

Sterilization Techniques

AUTOCLAVING: This is a very reliable method for sterilizing most materials. However, it is

not suitable for materials that are damaged by high temperatures. Some autoclavable substances

begin to breakdown with sustained sterilizing temperatures. Extended sterilizing of an agar

medium can result in pH changes of up to 0.5 unit, carbohydrates can be partially hydrolysed,

proteins can be denatured and inhibitory compounds formed by the combination of amino acid

and glucose units. Therefore the duration and temperature should be suited for the application.

The autoclave sterilizes the contents by raising the temperature to a point where contaminating

microbes and spores are killed. Increasing the atmospheric pressure inside the autoclave allows

the temperature to be raised above the normal boiling point of water without boiling occurring.

Steam can be used to quickly raise the internal temperature of the autoclave, heat conduction is

rapid and has great penetrating power. The temperature and duration required to sterilize a flask

of media with steam is shorter than that required by a 'dry' heat sterilizing treatment.

The duration of the heat treatment is important because it is essential that ALL contaminants are

killed; partial sterilization may leave viable microorganisms on lab material or in media.

REMEMBER: Check to see that the temperature and duration are set correctly. Frequently a

common setting is used if the volumes being autoclaved don’t vary greatly. However, larger

volumes require longer autoclaving times to allow heat to penetrate to the core of the liquid

(Table 1).

TABLE 1. Minimum exposure time at 121oC for a full load of the following volumes in

appropriate sized flasks.

Volume (ml) Time (min)

75 25

250 30

500 40

1000 45

1500 50

2000 55

Less than full loads will requires slightly less time. Longer times are required for heat to

penetrate to the core of larger volumes.

Bottles should be LOOSELY capped to allow for the equalization of air pressure during

sterilization, otherwise internal pressure may cause bottles to break. Media flasks should be

plugged with a foam stopper to allow equalization of air pressure and the foam plug and flask

neck wrapped with tinfoil.

Wrap lab utensils and equipment in tinfoil so that when they are removed from the autoclave

they will remain sterile.

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IMPORTANT: When opening the door of the autoclave after a sterilization run, allow the

temperatures between outside and inside the chamber to equalize for a short period of time by

unsealing the door slightly. DO NOT SWING THE DOOR WIDE OPEN, the sudden escape of

hot air from the chamber will drop the internal pressure and the hot liquids will boil over in the

reduced atmospheric pressure (you can break glassware, lose media and the cleanup is difficult).

MILLIPORE FILTRATION: Heat labile substances such as acids, vitamins, hormones and

antibiotics will be destroyed in a normal autoclaving cycle. These products can be sterilized at

room temperature by using a membrane filter.

The surface of the filter has very fine pores (see figure) that can prevent bacteria from passing

through. A 0.22-0.25 um pore

size will exclude all bacteria,

yeasts and fungal spores from

the filtrate. (see relative sizes

of common contaminants on

next page)

The filtrate can be added to

autoclaved material if desired

once the autoclaved material

has cooled.

The Millipore filters can be

bought in different pore sizes

and filter diameters according

to need and can be bought

pre-sterilized or unsterilized,

in which case they must be

sterilized by autoclaving

prior to use.

There are re-usable filter

chambers available that can

have the filters replaced and there are disposable filter chambers that can be used for small

volumes such as syringes.

REMEMBER: The filtrate must be collected in a container that is sterile.

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ULTRAVIOLET STERILIZATION: UV sterilization is

used for materials that otherwise cannot be treated (light

plastics, paper products etc). Ultraviolet sterilization is a

surface effect that requires direct illumination. Thus an object

to be sterilized should not be in the shadow of another object.

The materials should also be as clean as possible and dust free

before treatment because some bacteria can survive in the

'shadow' of dust particles.

The high energy radiation from the UV lamp (from 220-300

nm) is absorbed by many biomolecules including DNA, RNA

and proteins causing damage to these molecules which in turn

result in death to the micro-organisms. Exposure times vary

for different micro-organisms ranging from 9 seconds to 4

hours (Table 2).

CAUTION: High intensity UV radiation can generate ozone

gas which can be uncomfortable in a poorly ventilated room.

Long term exposure to ozone gas may be harmful to your

health. Damage to exposed skin can occur in as little as 90

seconds in the presence of a UV light. NEVER look at a

ultraviolet light source with the naked eye. Always wear

protective glasses or a face shield when working with UV

light.

DISINFECTANTS: Non-porous work surfaces can be

disinfected with a number of products. Of the more popular

ones, 70% ethyl alcohol is effective for both table tops and on

hands/forearms. Wiping non-porous surfaces down with

alcohol is effective and very common when you want to work

in a contaminant free space such as a flow hood.

CAUTION: Do not spray alcohol onto work benches in the

presence of open flames. Avoid inhaling the vapours. Ethanol

fumes may cause headaches.

Other products such as SAVLON, which is sold as a

germicidal soap for skin care, is also effective. The work

surfaces, hands, and forearms can be washed with savlon. Use

distilled water and paper towels to clean the area you wish to

work on. Savlon does not dry out the skin as much as ethanol.

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Table 2. ULTRAVIOLET RADIATION LETHAL DOSES

MICRO-ORGANISM

Lethal Dose For 180μW/cm

2

Radiation Intensity at Work Surface

90% Kill

99% Kill

99.99% Kill

Clostridium tetani

27.4 sec

54.8 sec

1.82 min

Bacillus anthracis (Spores)

25.1 sec

50.2 sec

1.67 min

Corynebacterium diphteriae

18.7 sec

37.4 sec

1.2 min

Staphylococcus aureus (Haemolytic)

14.4 sec

28.8 sec

57.7 sec

Escherichia coli

13.6 sec

27.2 sec

54.4 sec

Serratia marcescens

12.2 sec

24.4 sec.

48.9 sec

Streptococcus pyogenes

12.0 sec

24.0 sec

48.0 sec

Eberthella typhosa

11.9 sec

23.8 sec

47.7 sec

Streptococcus salivarius

11.1 sec

22.2 sec

44.4 sec

Streptococcus albus

10.2 sec

20.4 sec

40.9 sec

Spigellla paradysenteriae

9.3 sec

18.6 sec

37.3 sec

Yeast (Average)

22.2 sec

44.4 sec

1.48 min

Brewer’s Yeast

55.6 sec

1.6 min

3.7 min

Fungi (Moulds)

2.8-28 min

6-56 min

12-114 min

Protozoa

5.6-9.3 min

11.2-18.6 min

22.4-37.2 min

Algae, Blue-Green

28-56 min

0.9-1.9 hrs

1.9-3.7 hrs

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Aseptic Techniques

Laminar flow hoods are designed so that a positive flow of filtered (sterile) air passes over the

material in the hood. The filters (pre- and HEPA filter) are designed to eliminate particles of 0.3

microns or smaller (which eliminates most bacteria and particulate matter).

REMEMBER: Hands and forearms should be disinfected with 70% ethanol or Savlon before

working in the flow hood.

The working areas should be kept clean and free of particles.

The flow hood should be allowed to run for a few minutes prior to working with open sterile

media etc. so that the filters are passing sterile air across the bench.

The working area should be kept clear of unused items, do not place unsterilized objects 'upwind'

of open sterile media or critical items because contaminants may be blown onto the media.

Be aware that exposed skin is a source of contamination, skin cells are constantly being shed.

Try to avoid reaching over critical areas or exposed media to reach for objects. Do not allow lab

coat sleeves to drag across the bench.

Always place work away from you when talking (speak softly so that you do not spread bacteria

into the flow hood area). When sneezing or coughing turn away or remove yourself from the

flow hood area.

Remember to work as far INTO the flow hood as possible where the air stream is the strongest.

The positive pressure at the outside edge of the hood may not be enough to prevent

contamination from air borne particles.

Utensils such as scalpels, forceps etc, should be stored in 90% ethanol and flamed prior to use.

Be careful not to burn yourself - alcohol flames are invisible and very hot!

Do not flame a utensil and then place it back into the alcohol beaker if it is still flaming! This can

happen if you are not paying attention. If glassware containing alcohol should ignite, don't try to

pick up the beaker or the utensils, smother the flame with a large beaker placed over top of the

fire.

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AGROBACTERIA TUMEFECIENS MEDIATED PLANT

CELL TRANSFORMATION

(Part I)

One of several ways of introducing foreign DNA into plant systems (i.e. generation of transgenic

plants) is using the agrobacteria infections system. Agrobacteria strains such as A. tumefaciens

and A. rhizogenes are natural soil bacteria which parasitize susceptible plants by transferring a

specific segment of DNA, the tDNA region, of a bacterial plasmid (Ti plasmid) into plant cells.

This DNA is inserted into the nuclear DNA of the plant. Expression of the wild type genes

encoded on this tDNA segment can alter the normal development of the transgenic plant cells in

several ways including uncontrolled cell division (tumor production) and synthesis of unique

metabolites, opines, which the plant cannot use but which the invading agrobacteria can.

Agrobacteria do not efficiently infect and transform all plant species. In fact different strains of

agrobacteria exhibit different host ranges. The specific requirements for successful infection are

currently being elucidated. Agrobacteria invades a plant only at a site of injury. In this lab you

will create the injury by producing a cut surface. This wounding causes the release of specific

phenolic compounds at the wound sites. These compounds activate virulence genes in the

bacteria which in turn catalyse the binding of the bacterium to a plant cell wall and subsequently

the replication of the tDNA region and its transfer of the tDNA into the plant cell.

In this experiment the tDNA region of the Ti plasmid of an A. tumefaciens strain which you will

be using, has been engineered to remove most of the wild type genes (including those coding for

enzymes synthesizing an auxin and a cytokinin) and two new genes incorporated into this region.

The single wild type gene retained encodes octapine synthase, an enzyme responsible for the

synthesis of octapine, one of the unique metabolites mentioned above. The new genes encode

the enzymes ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII).

While the antibiotic kanamycin is toxic to most plant cells, those cells having and expressing the

NPTII gene are tolerant to this antibiotic by virtue of gene product's ability to phosphorylate

kanamycin and thereby detoxify it. Thus the presence of this gene allows one to differentiate

between transformed cells and non-transformed cells on the basis of their ability to grow on a

kanamycin-containing media. The ß-glucuronidase is utilized as a readily detectable marker as it

can catalyse the release of a fluorescent product from a non-fluorescent substrate. Because of the

linearity of the enzyme reaction and the low level of endogenous ß-glucuronidase activity in non-

transformed plant cells, the level of expression of the tDNA encoded genes can also be examined

using this enzyme.

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Procedure (Bring permanent ink marking pen to labs for labelling plates.)

Sterilization of Tissue and Preparation of Leaf Disks:

Tissue obtained from fully expanded green leaves of 1-2 month old N. tabaccum will be used as

explants with this transformation system. Leaves which have been removed from healthy plants

should be briefly washed with distilled water, followed by immersion in 70% ethanol (2 min).

Rinse the material with sterile distilled water and immerse in a 1% NaOCl solution (1/5 dilution

of commercial chlorox) for 10-20 min. If younger leaves are used the time of exposure to

hypochlorite may need to be reduced to avoid excessive bleaching of the tissue. In a laminar

flow hood rinse leave pieces several times with sterile distilled water and transfer to a sterile

petri plate.

Using a sterile hole punch cut approximately 25 discs into a sterile petri plate containing a small

volume of sterile water to prevent desiccation (avoid the midrib).

Infection with Agrobacteria:

A. tumefaciens strain MP90 which carries the engineered tDNA will be grown overnight at 28C

in LB media containing 50 μg kanamycin/ml. The cells will be measured at an optical density of

620 nm to determine the concentration (using 5 x108 cells/ml for 1 OD620) and then gently

centrifuged (5500xg, 10 min) to pellet the bacteria. Agrobacteria cells will be resuspended in

MS (Murashige & Skoog) complete media without hormones at a cell density of 109-10

10

cells/ml. This exchange of media removes the kanamycin containing bacterial growth media

which would inhibit callus development.

Transfer your leaf disks to a new petri plate containing the bacterial-MS media suspension. Float

the disks to incubate for approx. 2 min. Disks should then be individually removed, blotted

carefully on a sterile paper towel to remove excess liquid, and placed adaxial side (upper leaf

surface) up in petri plates (approx. 12 disks/plate; 2 plates/student) containing the following

medium:

Co-cultivation media

MS mineral salts

0.6g/l MES

B5 vitamins [Plates of all required media will be

3% sucrose prepared in advance by the demonstrator.]

0.8% agar

1.0 mg/l BAP (6-benzylaminopurine) (cytokinin)

0.1 mg/l NAA (α-naphthaleneacetic acid) (auxin)

100 μM acetosyringone

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The first students completed should cut additional disks for 3 control plates. These disks should

be immersed in MS media (NO agrobacteria) and blotted and plated as was done for the other

plates. These will be used for the regeneration of non-transformed tissue and plants. These

plates should be clearly marked as Controls.

Label all plates lids with initials, seal with a double layer of parafilm and incubate in the dark (a

drawer).

After 48 h. The explants treated with agrobacteria will show bacterial growth around the

edges by this stage. Both control and treated explants must be transferred to plates containing

the same medium supplemented with carbenicillin (500 mg/l). This antibiotic kills A.

tumefaciens as well as other frequent contaminants. (If the initial bacterial population is very

high, several subculturings may be required; check the cultures every 24 h and at the first signs

of bacterial growth, subculture onto fresh carbenicillin-containing media.) Explants will be

maintained on this media to induce the development of callus along the cut surfaces.

Selection and Shoot Development Media

MS mineral salts

MES

B5 vitamins

3% sucrose

0.8% agar

1.0 mg/l BAP

0.1 mg/l NAA

500 mg/l carbenicillin

50mg/ml kanamycin sulfate

The plate conditions are designed to i) select for transformed callus and ii) induce this callus to

undergo differentiation and shoot regeneration.

Note: Control disks should be transferred fresh plates of the same media except these should

contain no kanamycin

N. tabaccum cells are sensitive to kanamycin levels above 20-30 mg/l. (This can be

demonstrated by plating a small number of control explants on a kanamycin containing

plate.) Thus, this culture step allows the detection of non-transformed explants, which

are visible by the lack of growth or cell proliferation

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.

The calli that show shoot formation on their surface (these are easily recognized by the

development of green spots on their surfaces) should be excised and transfered onto fresh media.

(Ensure treated and control calli are place on the correct media! ie. +kan/-kan) Under

continuous light conditions, the development of green leaflets should be evident after one week.

Once shoot formation is well developed in transformed tissues carefully remove each developing

shoots from treated explant derived material and place them onto root induction medium. Note

that this media contains no exogenous hormones. If shoot development has occurred auxin will

be produced that should provide the necessary hormone for root formation.

Root Induction Media

M.S. salts

MES

B5 vitamins

0.8% agar

3.0% sucrose

50 mg/ml kanamycin

This medium supports the development of roots and plantlet growth, which is evident after 1-2

weeks. Plantlets showing root development are likely to be transformed and will be used for the

screening of the ß-glucuronidase activity. A small number of control plants might be placed on

this media to see the effect of kanamycin on normal plants. Other control plants should be

placed on this same media without kanamycin to allow normal rooting of the control plants.

Note: You are required to make weekly observations and comparisons of tissue development as

outlined under Lab Report at the end of Part II

Note: Students should be familiar with the types of changes to be anticipated at the various

stages of plant regeneration. A weekly log summarizing the OBSERVED changes should be

maintained, recording development as well as specific changes which occur following any

change in media. These changes will need to be summarized and discussed on a scale of days

post-infection. You will also be asked to discuss why the hormone composition of the media was

modified.

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PLANT REGENERATION

An important aspect of plant genetic engineering is the introduction of genes into plant cells and

recovery of transgenic plants. Such modified cells must be competent to undergo

dedifferentiation, cell division and the organization of organ structures. Although adventitious

organs often arise on intact plants, the most common technique in plant genetic manipulation is

the introduction of genes into explants, followed by in vitro culture on a medium for organ

regeneration. Explants from leaf, stem, root, petiole, immature embryos, hypocotyls,

cotyledons, microspore or thin cell layers are all utilized in this technique. Any tissue with living

cells capable of dedifferentiation can serve as an explant.

Under appropriate culture conditions such explants, or cultured cells and tissues, will organize

embryonic structures or primordia which can develop into shoots, roots, flowers or embryos. In

many cases these structures have a single cell origin. The term of totipotency is used to describe

cells which are capable of sustained cell division and organization of an intact plant or plant

organ. The earliest demonstration of cell totipotency was due to the work of Reinert 1958a and

Steward et al. 1958. They demonstrated the development of carrot plants from cultured cells by

the process of embryogenesis. The developmental pattern recapitulated all the stages

characteristic of zygotic embryogenesis.

The regeneration of plants through organogenesis, (organization of monopolar structures) was

demonstrated by White in (1939) and conditions for such regeneration from tobacco callus

Nicotiana tabacum L., were defined by Skoog and Miller in (1957). At least in this species

auxins and cytokinins regulate organ differentiation. High levels of auxin relative to cytokinins

promoted root differentiation, while the reverse favored shoot development. In addition, direct

differentiation of flower buds from epidermal cell layers of N. tabacum and other species was

demonstrated, Tran Thanh van K. 1973.

These examples clearly demonstrate the developmental plasticity of cells in tobacco explants or

callus. The discovery of the relationship between in vitro plant morphogenesis and plant

hormones and techniques for the in vitro culture of plant cells and explants are important factors

in the advances made in plant genetic engineering.

Objective

The objective of this exercise is to examine the relationship between auxins and cytokinins and

organ regeneration in leaf explants of Nicotiana tabacum L. cv Wisconsin #38.

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Procedure

Culture medium: You will be provided with culture medium containing the following

hormone combinations.

A 0.5 mg/l 2,4-D

B 2.0 mg/l BAP

C 2.0 mg/l BAP + 0.5 mg/l 2,4-D

D 2.0 mg/l 2,4-D + 0.5 mg/l BAP

E Control (no hormones)

Plant Material:

Sterilize the leaf material provided by first washing in distilled water for 1-2 minutes; then

immersing in 70% ethanol for 1 minute, with occasional agitation. Decant the ethanol and rinse

the tissue x 2 with distilled water. Immerse the tissue in 20% Javex containing 0.02% Tween 20

and place the container in the flow chamber. Agitate occasionally. After 20 minutes decant the

liquid under the flow chamber and rinse x 3 with sterile distilled water. These operations must

be performed carefully to avoid contamination. Once the material is placed in chlorox all

operations must be in the flow chamber.

NOTE: No antibiotics will be included in this medium; cf Exercise #1. With a sterile forceps

transfer the tissue to sterile petri plates and with a sharp scalpel remove all surfaces that are

damaged during sterilization. Cut the leaf into strips and discard the midrib. Cut remainder of

the lamina into small segments. With a sterile forceps transfer 4-5 segments to each type of agar

plate. Label as leaf explants and seal the dishes with parafilm. Place the cultures in a cupboard

at room temperature.

Observations

You will be required to observe the cultures weekly. Determine if there is bacterial or fungal

contamination. What is the source of contamination?

15

Lab Report

Determine the pattern of growth for each treatment. Record the time of organ initiation and the

number of organs per explant. Examine the pattern of callus development in each treatment and

compare the callus type (friable or compact).

Record the effect of auxin and cytokinins on organ initiation. Would you expect the hormone

requirement for organ induction to vary with species and genotype? Explain.

What are some of the cellular changes which are associated with callus formation from

specialized parenchyma cell?

From your observations, is callus development necessary for organ initiation? In terms of

genetic manipulation what do you think would be the advantage of enhanced callusing prior to

organ regeneration?

References

Thorpe T.A. (1980) Organogenesis in vitro. Structural, physiological and biochemical aspects.

Publ. Rev. Cytol. Suppl. 11A 71-112.

Schweiger H.-G. et al. (1987) Individual selection, culture and manipulation of higher plant cells.

Theor. Appl. Genet. 73:769-783.

Tran Thanh van K. (1973) Direct flower neo formation from superficial tissues of small explants

of Nicotiana tobacum L. Planta 115:87-92.

Steward, F.C., Mapes, M.O. and Mears, K. (1958) Growth and organized development of

cultured cells, II. Organization in cultures grown from freely suspended cells. Am. J. Bot.

45:705-708.

White, P.R. (1939) Controlled differentiation in a plant tissue culture. Bull. Torrey Bot. Club.

66:507-513.

Skoog F. and Miller, C.O. (1957) Chemical regulation of growth and organ formation in plant

tissues cultured in vitro. Symp. Soc. Exp. Biol. 11:118-131.

16

PLASMID CONSTRUCTION

Plasmids are small circular pieces of double stranded DNA which occur naturally as extra-

chromosomal elements in prokaryotic organism. They vary in size from 2.5 kbp up to more than

200 kbp. Encoded on a plasmid are normally one or more genes as well as a sequence that

serves as an origin of replication allowing the plasmid to replicate. Plasmid replication may be

either tightly controlled, occurring in synchrony with chromosomal replication, or under relaxed

control whereby plasmid replication occurs independent of chromosomal replication and

normally results in many copies of the plasmid being present in the cell.

Genetic engineering has taken great advantage of plasmids as a means of amplification of

selected DNA. Plasmids, which are used in genetic engineering or recombinant DNA, have been

extensively reconstructed using wild-type plasmids as well as sequences from other sources. The

resultant commercial plasmids combine a series of characteristics which allow them to serve as

useful DNA vectors - carriers of additional DNA.

In this lab you will isolate two pieces of DNA: i) a linearized plasmid vector, pSK and ii) ToxA,

a gene encoding a fungal toxin from Pyrenophora tritici-repentis. The toxin, Ptr necrosis toxin,

is a protein which causes necrotic lesions on sensitive wheat cultivars, and is a pathogenicity

factor in the disease known as tan spot. Your mission will be to isolate these two pieces of DNA

and join them together to create a recombinant plasmid. The linearized plasmid as well as the

ToxA DNA have been prepared from other recombinant plasmids by digestion with two

restriction endonucleases and the resultant digests separated on agarose gels. You will start by

isolating the DNA from the gel, purify the pieces away from other DNA and the agarose.

Note: The next four labs are sequential and depend on the material you have prepared in the

previous lab.

Procedure

You will be working in pairs with one person in each group being responsible for isolation of

one of the two pieces of DNA, ie. one of you will isolate the ToxA gene (900 bp), the other

person will isolate the linearized pSK vector (2964 bp).

Ten micrograms of DNA of starting plasmid has been used to generate each fragment. Each has

been digested with restriction enzymes EcoRI and XhoI. The products from this double digest

have been separated by electrophoresis in a 1% agarose gel containing Red Safe (final

concentration of 0.5 ug/ml). Red Safe is used because it binds to double stranded DNA/RNA

and will fluoresce under ultra-violet (UV) light thereby allowing you to see the separated DNA

fragments. It is safer to use than Ethidium Bromide which is a carcinogen. To assist in

identifying the piece of DNA you wish to isolate, a series of DNA fragments of known size (1 kb

ladder) has been run on the same gel for comparison.

Fragment Separation and Recovery

17

The demonstrator will remove your fragment from the electrophoretic gel.

i) Turn the power off, lift out the gel on its tray and examine the gel by UV light in

darkness. CAUTION: You must use protective eyewear when looking at UV light.

Because UV light damages DNA it is important that you minimize the time of exposure

of the fluorescent band containing your DNA. (less than 30 sec)

ii) Using a scalpel excise the appropriate band and place it in the labelled tube. Remember to

use the DNA markers as a guide to ensure that you are taking the right sample. Be

precise, do not take more agarose than is needed as this will decrease the concentration of

your recovered DNA.

A labelled microcentrifuge tube (pSK or ToxA) with a recovered gel slice will be

provided to you.

The DNA will be recovered and separated from the agarose gel by the Spin Column protocol.

Protocol

Overview: This protocol involves separating the DNA from the agarose gel and ethidium

bromide. The spin column is a plastic tube with a series of membranes and filters at its base.

These membranes and filters will retain the agarose and ethidium bromide while allowing the

DNA to pass through into a collection tube when centrifuged. The expected yield of recovered

DNA is 30 to 70%.

1) Add 450 μl of buffer QG to your tube containing your gel slice. Ensure that your

collection tube is appropriately labelled either pSK or ToxA.

2) Incubate at 50oc for 10 minutes (until gel dissolves) Check that your mix is yellow, if not

add 10 μl of 3M Sodium Acetate and mix.

3) Add 450 μl of Isopropanol to your tube and mix.

4) Apply your sample to the Quickspin column and centrifuge for 1 minute at 12,000 rpm.

Discard your flow thru and place Quickspin column back in the same collection tube.

5) Add 750 μl of Buffer PE to your Quickspin column and centrifuge for 1 minute at

12,000rpm. Remove flow thru and repeat spin.

6) Place your Quickspin column in a new 1.5 ml tube. Add 50 μl of Buffer EB to the center

of the column membrane and centrifuge for 1 minute at 12,000 rpm.

18

Quantification of Recovered DNA

You will determine the amount of DNA that you have recovered using a spectrophotometer

measuring absorbance at 260 nm. To measure your sample you will add 7 μl of your eluted DNA

to a new microcentrifuge tube containing 700 μl of water. The spectrophotometer will be blanked

using water first then you will measure your sample. Because glass absorbs UV light you will

need to use a quartz cuvette. Please be careful with these as they are expensive to replace!

Record your result.

Ligation of ToxA and pSK to yield pNEC

At this point you and your partner should have a tube with a ToxA and a tube with pSK and

know the concentration of each. What you are going to do is join the DNA in these two samples

together to construct the recombinant plasmid. To do this you will use two different molar ratios

of the vector:insert (ie. pSK:ToxA = 1:1 and 1:3).

To determine the molar ratios you don't need to actually calculate the molar amounts but simply

use the size ratio for the two molecules.

A 1:1 ratio pSK:ToxA = ____kb: ____kb

For your experiment you will be using a fixed amount of vector (pSK) 100 ng and varying the

amount of the insert put in the reaction.

100 ng pSK :____ng ToxA for a 1:1 ratio

100 ng pSK :____ng ToxA for a 1:3 ratio

Calculate the volumes of the pSK and ToxA solutions which you have prepared to yield the

required amounts for each reaction. Show solution concentrations and calculations to

demonstrator before proceeding to the next step.

19

Preparation of Ligation Reactions

Assemble the following in a sterile microcentrifuge tube on ice:

Vector/Insert ratio 1:1 1:3

pSK (vector) DNA (100 ng) X μl X μl

ToxA (insert) DNA Y μl 3Y μl

Ligase 5x buffer 2 μl 2 μl

T4 DNA Ligase 1 μl (1U) 1 μl

Sterile H2O to a final volume of 10 μl

Add the T4 DNA ligase last and stir the reaction with your pipet tip, mix well.

Transfer your ligation reactions to a styrofoam bath with water at 16oC. This bath is then moved

to a 4oC cooler where the reaction will continue overnight. Your reactions will be stored at -20

oC

until next week.

Lab Report -The lab report for this lab should be prepared together with that for Bacterial

Transformation.

20

21

BACTERIAL TRANSFORMATION

Bacteria provide researchers with a means of rapidly amplifying specific pieces of DNA. This is

possible because of the short regeneration time of bacteria and the fact that bacteria can be

induced to carry foreign DNA and replicate it as if it were native to the bacterium. One of the

most common ways of doing this is to insert the piece of DNA that you wish to amplify into a

vector (a carrier molecule) which will be retained and replicated by the bacteria. The vector

system you will be using in this lab is the plasmid you constructed in the previous lab. This is a

recombinant plasmid of pSK carrying the PtrNEC gene. An illustration of the pSK vector is

attached.

Transformation of bacteria is a process by which foreign plasmid DNA is taken up by bacteria.

For this to occur the bacterial cell membrane must be made leaky and this is done by treating the

cells with a mixture of divalent cations rendering their membranes temporarily permeable.

Bacteria cells which are treated this way and are then competent to take up DNA are called

competent cells. When competent cell are placed in solution containing plasmid, plasmid

molecules can pass through the cell membrane into the cells. The process is called

transformation due to the change (transformation) of the genetic makeup.

Even under ideal conditions only a fraction of the competent bacteria are successfully

transformed. Thus some means of selecting for the transformed cells (transformants) must be

used. A common mechanism which is widely used is based on antibiotic resistance. This is

possible by virtue of the original plasmid vector having a bacteria expressed gene which provides

resistance to any bacterium that carries the plasmid.

A second level of selection is sometimes also necessary. Non-recombinant plasmids can occur

when in the process of creating a recombinant plasmid some of the original plasmid is carried

over or reformed and thereby becomes part of the plasmid mixture. Upon transformation, the

bacterial population which carry plasmid (all of which should be antibiotic resistant) will consist

of two sub-populations - those carrying recombinant plasmids and those carrying non-

recombinant plasmids. To distinguish between these two groups a second selection mechanism

has been incorporated onto the basic plasmid vector. This mechanism is called insertional

inactivation and is based on the presence of an easily detectable expressed gene in the basic

vector being inactivated on insertion of foreign DNA. Inactivation occurs because the insertion

site for the foreign DNA is within this marker gene thereby resulting in an inactive gene product.

The gene in pSK which can be inactivated in this manner encodes the enzyme β-galactosidase.

The presence of this enzyme in bacterial colonies is determined by inclusion of a colourless

substrate, 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), on agar plates when the

transformation mixture is plated. If the enzyme is present (the gene was expressed and bacteria

must carry a non-recombinant plasmid) the substrate is cleaved to produce an insoluble blue

product resulting in a blue colony. If the enzyme is not present (gene was inactivated therefore

bacteria carry recombinant plasmid) the colony will be white.

22

Procedure

1) Your ligations from the previous week will be supplied on ice. Each group will do two

transformations using 10 μl and 1 μl of their ligated DNA which will have been diluted

10 fold (13 ng and 1.3 ng of DNA). Label two 10 ml sterile polypropylene tubes (on ice)

accordingly (name and amount of DNA).

2) A tube of commercially prepared competent E. coli cells will be supplied on ice. As soon

as this tube of cells thaws add 50 μl of cells to your chilled tubes. Add DNA to cells and

stir with pipette tip, gently shake for 5 seconds.

3) Incubate cells on ice for 30 minutes.

4) Heat shock cells 45 seconds in a 37oC water bath, do not shake.

5) Place on ice for 4 minutes.

6) Add 450 μl of room temperature YT media, incubate at 37o C for 40 minutes with

periodic gentle mixing.

During the 1 hour you will add the X-gal to your YT ampicillin (50 μg/ml) selection plates. You

will need 2 plates per transformation reaction. One group will make 2 extra plates for the

negative controls.

7) In the flow hood place the plate on a turn table. Flame the glass "hockey stick" and then

rest across second sterile beaker to cool. Pipet 20 μl of a 50 mg/ml solution of X-gal onto

the middle of the plate. Use the sterile "hockey stick" to spread the X-gal evenly across

the surface of the plate. Allow the plates to dry and the dimethylformamide to dissipate.

Label plates with name and DNA level (10 μl or 1 μl).

8) After the 1 hour incubation of bacteria dilute your reaction 1/10 in 1000 μl of YT media.

Plate out 20 μl of each diluted transformation reaction onto your YT, ampicillin, X-gal

plates. Be sure plates are labelled. When done incubate plates upside down at 37oC

overnight.

9) Your plates will be removed from the incubator for you the following day and stored at

4oC.

23

Results

You will need to count or estimate the number of blue and white colonies on each plate and

record relative to the level of DNA dilution and volumes plated.

Discussion

Using 130 ng as the starting weight of recombinant plasmid, and the dilutions which were

incorporated in this experiment, calculate the potential number of transformant you would expect

on your plates if you assume that every plasmid molecule finds a bacterial cell to transform.

How does this compare to the advertised transformation rate of the cells provided with pUC19 of

>107 transformants/ug of plasmid DNA?

Comment on possible reasons for the difference between what you calculated and what you

observed.

Comment on the frequency of blue/white colonies. Under what circumstances would you expect

to find a) higher and b) lower levels of blue colonies?

24

ISOLATION AND CHARACTERIZATION OF PLASMIDS

Plasmids are small, extra-chromosomal, circular pieces of double-stranded DNA which contain

an origin of replication and one or more genes. The origin of replication allows the plasmid to

be replicated independently from the chromosomal DNA and thus be maintained as an

independent entity. Plasmids are found within many prokaryotic hosts but they are found

infrequently in eukaryotic species. Plasmids frequently contain genes for resistance to one or

more antibiotics. These genes allow a host bacteria that is normally sensitive to these antibiotics

to grow in the presence of the drug(s). Any gene on the plasmid carrying an appropriate

bacterial promoter can be expressed in the bacteria.

Some plasmids exist at a level of a single copy per cell because their replication is tightly

coupled to replication of the host's own chromosome. Others are said to be under "relaxed"

control because plasmid replication, although it employs the host's DNA synthesizing machinery,

is not coupled to chromosome replication. Such plasmids frequently exist at a level of 20 to 50

copies per cell. This type of plasmid is commonly used in recombinant DNA work because of

the higher yield of plasmid obtained during isolation from a given amount of bacterial culture.

Plasmids are useful as cloning vehicles because they allow a cloned gene sequence to be

amplified by millions of times in bacteria. Purification of plasmid from a bacterial source

requires separating plasmid DNA from chromosomal DNA, as well as from RNA, protein and

other cellular components. Because of their small size and circular nature, plasmid molecules

can be extracted from cells under conditions in which they remain in a supercoiled form (more

resistant to denaturation) while the large chromosomal DNA molecules are denatured. In the

plasmid isolation procedure, you will use an alkaline solution of sodium dodecyl sulfate (SDS) to

disrupt cell membranes and produce conditions which result in the denaturation of chromosomal

DNA but not plasmid DNA. When the pH of this extract is rapidly lowered to near neutrality,

the chromosomal DNA precipitates, since it is largely in single-stranded tangles, while the

plasmid molecules remain in solution. Subsequent steps, including ammonium acetate

precipitation (to selectively remove protein) and alcohol precipitations (to precipitate the DNA,

allowing it to be concentrated), and treatment with the enzyme ribonuclease (to remove RNA),

allow a rather pure preparation of plasmid DNA to be obtained.

Three overnight liquid cultures of E. coli will be provided. Each culture will be of bacteria

carrying a different plasmid: pUC19, a standard plasmid cloning vector of 2686 bp; pNEC which

is a pSK vector (2958 bp) with a cloned necrosis toxin gene from Pyrenophora tritici-repentis;

total plasmid 3875 bp; and pBI121 a low copy plasmid carrying the GUS gene and kanamycin

resistance gene, 13 kb. Each student will extract and purify plasmid from two of these cultures.

In subsequent work you will evaluate the purity of the plasmid preparations and estimate

recovery. A demonstration of restriction enzyme digestion and agarose gel electrophoresis will

be carried out to examine the plasmids in more detail.

25

Hind III

26

Procedure

WEEK 1: Plasmid Isolation

Harvesting Cells

1) Five milliliter aliquots of each culture will have been centrifuged at 4000 x g for 5

min. to pellet the bacterial cells and the media decanted. These pelleted cells will be

provided on ice. Add 1 ml of TE buffer (10 mM Tris, 1 mM EDTA) pH 8.0, resuspend

the pellet, transfer to a microcentrifuge tube, recentrifuge and remove liquid by

aspiration.

2) Resuspend the bacterial pellet in 200 ul of ice-cold Solution I by vigorous vortexing.

Solution I 50 mM glucose

25 mM Tris-HCl, pH 8.0

10 mM EDTA, pH 8.0

3) Add 400 ul of freshly prepared Solution II. Close the tube tightly and mix the contents

thoroughly by rapidly inverting the tube 5-10 times (Do not shake). Make certain the

solution is completely mixed then place in ice for 2-5 min. Note how viscous the

solution is.

Solution II 0.2 M NaOH

1% SDS (sodium dodecylsulfate)

4) Add 300 μl of 3 M NaAc pH 4.8 to neutralize the alkaline solution. Close the tube and

again thoroughly mix by inversion and tapping the tube. The alkali-lysed cells will be

very viscous and it is important that the two solutions be uniformly mixed for effective

neutralization which leads to precipitation of the chromosomal DNA. However, overly

vigorous mixing such as vortexing can lead to shearing of the high molecular weight

DNA and subsequent contamination of the plasmid preparation. Store on ice for 5 min.

with 1-2 inversions at the mid point.

5) Centrifuge at 12,000g for 5 min in a microcentrifuge. Carefully transfer

approximately 700 ul of the supernatant to a clean microcentrifuge tube. Avoid

transferring any of the precipitated clot material.

6) Add 0.6 volume of isopropanol to precipitate the plasmid DNA, vortex and let stand at

room temperature for 10 min.

7) Centrifuge at 12,000g for 15 min and carefully remove the supernatant by aspiration.

8) Rinse the pellet by addition of 0.5 ml of 70% ethanol, mix and centrifuge at 12,000g

for 2 min. Remove the supernatant by aspiration without disturbing the pellet. Allow to

dry in air for 10 min.

27

9) Redissolve the pellet completely in 200 ul of TE buffer pH 7.4 containing 20 ug/ml

ribonuclease and incubate at 37oC for 1 h. Add 0.5 volume (100 ul) of 5.0 M ammonium

acetate and store at 4C overnight. This treatment should precipitate residual protein

without precipitating the plasmid DNA.

For time reasons the completion of step 9, and step 10 will be done for you.

10) Centrifuge (15 min, 12,000g) to pellet the precipitated material and carefully transfer

the supernatant to a clean microcentrifuge tube. Add 2 volumes of ethanol (95%) to

precipitate the plasmid and store at -20C until next lab.

WEEK 2: Agarose Gel Electrophoresis

DNA Quantification and Restriction Enzyme Digestion

Agarose Gel Electrophoresis

Agarose (a purified form of the same gelling agent that is used in culture plates) forms

gels with pores of a size which slightly impedes the migration of small DNA molecules like

plasmids. The pore size can be controlled by controlling the weight concentration of agarose

used to make the gel. Useful concentrations of agarose are in the range 0.6% to 3% -- the higher

concentrations would be used to separate DNA fragments of a few hundred base pairs (bp). For

plasmids and linear DNA's of 1 to 15 thousand bp (kb), 0.8% agarose is suitable. Typical

procedures involve making the gel in a moderately concentrated buffer (necessary to get

conductivity); the dye ethidium bromide is often included in the gel, since it forms a highly

fluorescent complex with DNA which allows the DNA to be visualized in the gel. It is easy to

detect less than 100 ng of DNA by examining a Red Safe-stained gel by UV light in the dark. If

Red Safe is not included in the gel when it is made, the gel is soaked in a solution of the dye

after electrophoresis is terminated. The purpose of this agarose gel is to look at the recovery and

purity of the uncut plasmid DNA. A subsequent gel will be run as a demonstration to show the

results of digestion of each plasmids with several different restriction enzymes.

Method

1) Agarose gel electrophoresis buffer consists of 0.04 M Tris, 0.001 M EDTA and glacial

acetic acid to a pH of 7.5-7.8.

2) Add 100 ml of diluted gel buffer from step 1 to 0.8 g of agarose in a 250 ml

Erlenmeyer (conical) flask. Melt the agarose completely on a stirring hot plate, taking

28

care not to splash yourself or others with the hot liquid.

3) When the agarose is completely melted (no specks of unmelted agarose still floating in

the melt), remove from heat, let the melt cool to about 60C, seal the edges of the

electophoresis apparatus and then pour the gel. Remember to install the plastic comb

which casts the sample wells. The gel will take about 30 min to solidify.

4) Centrifuge plasmid prep from first week (5 min, 12,000 g) to pellet DNA, rinse pellet

carefully with 200 ul of 70% ethanol, centrifuge for 3 min and remove supernatant.

Allow pellet to air dry for 10 min before re-dissolving the DNA in 150 ul of TE pH 8.0

buffer.

5) When the plasmid solution is ready quantify your plasmid DNA (below). Transfer 1ug

of plasmid DNA into a new microcentrifuge tube, add 2 ul of DNA sample buffer, and

mix thoroughly by vortexing and centrifuging the droplets down into the bottom of the

tube. Sample buffer is a mixture of bromophenol blue ("tracking dye", which moves

toward the anode at this pH but is unimpeded by the gel), Tris buffer, and sucrose to

make the sample dense.

6) When the gel is solid, remove the comb end plates and cover the gel surface to a depth

of a few millimetres with the same buffer used to cast the gel.

7) Load the samples into the wells, using a 20 ul automatic pipette. Be careful not to

puncture the gel at the bottom of the well. Connect the electrodes so that the DNA is

running the right way (towards the anode or positive pole). Run the electrophoresis for 1

hr at 100 volts, until the tracking dye has moved about 8 cm.

8) Turn off the power, lift out the gel on its glass plate, and examine the gel by UV light

in darkness. CAUTION: USE PROTECTIVE EYE-WEAR WHEN LOOKING AT

UV LIGHT.

Quantification of Plasmid DNA Recovered:

You will examine the plasmid DNA you have prepare by spectrometry, measuring the

absorbance at 230, 260 and 280 nm. To do this you will need to accurately dilute your sample.

Add 20 ul to 700 ul of TE buffer. Blank readings should be run with TE buffer as well. Because

glass absorbs UV light you will need to use a quartz cuvette. Please be careful with these as they

are expensive to replace!

29

Restriction Enzyme Digestion:

Once the amount and relative purity of the plasmid DNA preparations have been determined it

will be possible to do a restriction enzyme digest of the plasmids. This will be done as a

demonstration using your prepared plasmid DNA. Following digestion samples the DNA

fragments produced will be separated on an agarose gel. This will be done so that the gel can be

conveniently examined by the class. Markers of known size will be run for comparison to allow

size measurements to be made. Students should measure the migration distances (from the well)

of all visible bands.

REPORT

Using the photocopy of the electrophoretric gel results of both gels, explain what you have

observed and what you can interpret from the results.

Calculate the amount of plasmid DNA you recovered based on the absorbance results. Determine

the 260/280 and 260/230 ratios. Outline how the yield and purity of plasmid DNA might be

improved. What factors will influence the 260/280 ratio?

Plot the fragment length (in bases) against 1/mobility (cm) [band migration distance from the

well] of the molecular size markers (1 kb ladder) to create a standard curve. Using this curve

estimate the size of the inserts in the pNEC and pBI121 plasmids and compare the value you

obtain to the actual insert length outlined in introduction and figure, respectively.

Problem: Assume you have 8.4 ug of pBI121 plasmid DNA. The total amount is digested with

EcoRI to release the GUS gene with the CaMV 35 S promoter and the total digest separated by

electrophoresis into two components (GUS gene and residual plasmid). You purified the GUS

fragment from the agarose gel and recovered only 30% of potential DNA and this was recovered

in 20 ul. What would be the concentration of the DNA in solution?

30

GENE EXPRESSION IN E. COLI AND BIOASSAY

Many vector systems, be they plasmid or bacteriophage are constructed so that insertion of

foreign cloned DNA occurs into a region of an active gene in the vector. This insertion normally

results in inactivation of the resident gene (insertional inactivation) by disruption of the gene

product. However if the insertion site is near the 5' end of the vector resident gene and the

inserted DNA encodes an active gene without introns then it is possible that the inserted gene

will be expressed in the bacteria. This can occur because the resident gene promoter will still

promote transcription of the DNA downstream of it to produce a chimeric transcript (the product

of a chimeric gene which has been formed by joined two or more unrelated pieces of DNA).

Provided the reading frame of the inserted gene can be correctly read from an ATG start codon

(either that in the vector gene or the one in the inserted gene) then the product of the cloned gene

will be produced in the bacteria.

Expression of the foreign (inserted) gene can be useful from several perspectives. I) When you

have many different pieces of DNA cloned into identical vector molecules and carried by a

population of transformed bacteria (eg cDNA library), you can search for a vector carrying a

particular gene if you have a means of detecting the gene product specifically in a bacterial

lysate. Such is the case if you have an antiserum which will recognize and bind to a specific

protein gene product. This is a very valuable mechanism in screening cDNA libraries and is

referred to as immunoscreening. II) A second use is in the verification that a particular gene has

been cloned. One of the most convincing ways to prove you have a particular gene is to

demonstrate the particular biological activity associated with that gene. For example if you

wanted to convince someone that a gene you had cloned was in fact an alpha amylase gene, you

would want to show that the gene product had the ability to degrade starch. Even if you can

verify that the cloned DNA sequence is very similar to the sequence of a known amylase it does

not prove that the gene you have isolated encodes an active amylase - it could encode a defective

(inactive) gene product. III) A third use is the production of a gene product in large quantities.

Frequently this is the case when a particular gene product has a valuable function but it occurs

only at very low levels in its native organism. Human growth hormone and insulin which are

now produced in yeast are good examples of this application.

In this lab you will continue working with the transformants which you generated earlier. You

constructed a recombinant plasmid containing what should have been the ToxA gene. Bacterial

transformants were generated and screened using plate selection techniques to show that you had

bacteria with recombinant vectors. In this lab you will attempt to verify that the bacteria you

have isolated, carry an active ToxA gene by demonstrating the specific toxin activity is present

in lysates of bacteria which carry the ToxA gene (pNEC plasmid) but not lysates that carry an

identical plasmid except that it lacks this gene. The assumption is that the gene product has been

expressed in the cell and will be present in the lysate of appropriate cells. To demonstrate this

you will need to isolate bacterial lysate from bacteria carrying i) pNEC (pSK vector plus ToxA

gene) and ii) pSK. These lysates must be tested on toxin sensitive and insensitive wheat plants to

demonstrate not only necrosis inducing activity but also host specificity.

31

Procedure

1) Single bacterial colonies carrying either the pSK vector or pNEC will be used to

inoculate 10 ml LB cultures (50 μg/ml ampicillin). These cultures are grown overnight at

370C with shaking. The cells will be pelleted (1000 g, 10 min), then resuspended in 10

ml of fresh LB before 2 ml are used to inoculate 20 ml LB cultures (50 μg/ml ampicillin)

which will be shaken at 370C until cells reach mid log phase. At that point isopropyl β-

D-thiogalactopyranoside (IPTG) is added to a final concentration of 1 mM to induce

expression. Growth is allowed to continue for 4 hours at which point the cultures are

placed on ice for your use.

2) Each person will use their own culture of bacteria pNEC carrying bacteria. Control

cultures of pSK carrying bacteria will be prepared by the demonstrator. Transfer your

cultures to labelled 30 ml corex centrifuge tubes, balance your tubes in pairs and

centrifuge the cells at 2000 g for 5 min.

3) Decant the supernatant carefully into the flask provided, add 1 ml of lysis buffer (50 mM

Tris-HCl pH 8.0, 2 mM EDTA) and gently resuspend the cells. Centrifuge the cells again

at 2000 g for 10 min.

4) Again decant the supernatant, add 700 μl of lysis buffer and resuspend the cells. Transfer

your samples to labelled microcentrifuge tubes.

5) To each sample add 40 μl of a 100 mg/ml solution of lysozyme prepared in lysis buffer.

Mix and incubate cells at 37oC for 10 min. (Note the change.)

6) Chill your solution on ice then treat using a sonicator (this will be demonstrated prior to

use). Sonication will not only break the cells very effectively, but it will also shear the

high molecular weight DNA to reduce the viscosity of the solution. Sonicate using 3 x 5

sec pulses with 5-10 sec on ice in between. Continuous sonication can generate heat

which will denature (inactivate) proteins including the toxin protein if it is present.

7) Add 700 μl of water to your sample then centrifuge for 5 min at maximum speed to pellet

any insoluble material.

8) Label tags with your name and the culture lysate that you will be infiltrating.

9) You will now infiltrate your sample into the leaves of sensitive (Glenlea) and insensitive

(Erik) wheat cultivars using a Hagborg device. Your sample is drawn into a 1 ml syringe

and attached to the needle. Place leaf between rubber stoppers of the Hagborg device.

Clamp down on the leaf firmly, taking care not to damage the leaf tissue, and pressure

infiltrate the cell lysate sample into the leaf. Use of this apparatus will be demonstrated

for you. Practice with water until you are familiar with the technique before

attempting to infiltrate with your sample.

32

Purified Ptr necrosis toxin will also be infiltrated into leaves of both plants to provide a positive

comparison reaction. Note the stages of symptom development since different amounts of toxin

will cause the symptoms to develop more rapidly/slowly.

Observations and Results

Complete a table as shown below to record your results. You will have to return to check the

development of symptoms for the next three days and record the results.

Note if any change has occur and describe the nature of the change (greying, yellowing leaf

necrosis, shrivelling, etc).

Cultivar Sample Necrosis

24h 48h 72 h

Glenlea pSK

Glenlea pNEC

Glenlea Ptr toxin

Erik PSK

Erik pNEC

Erik Ptr toxin

Answer the following questions as part of your write up.

1. What reaction does the enzyme lysozyme catalyse and what is the purpose of incubating

the bacterial cells with lysozyme?

2. Sometimes when genes are inserted into a vector by the process you have used and

transformed into bacteria, no detectable expression product is observed based on

bioactivity. Explain some of the possible reasons why this may occur?

3. Discuss the function of the control reactions used in this infiltration experiment. The

goal of the experiment was to show that the plasmid you constructed, pNEC, contains a

gene which encodes an active form of the host specific toxin, Ptr necrosis toxin.

4. Why is IPTG added to the cell culture? Would your results have been different if IPTG

were not added? Explain.

33

5. Plasmids which have a cloning site in a marker gene sequence (to allow detection of

insertional inactivation) will have an ATG translational start codon upstream of the

insertion site (see diagram of pBluescript SK- the MET codon within the sequence

corresponding to the reverse primer). This allows for the expression of an inserted

foreign gene even if it is missing the 5' end of the gene including its ATG translational

start codon. If the foreign gene which is inserted into this site has a complete coding

sequence with its own ATG start codon, it is possible to get translation occurring (in a

transformed cell) from both of these start codons. Explain what is required of the

inserted sequence to obtain expression of the foreign gene from the endogenous plasmid

ATG codon. Illustrate you answer by assuming that you are inserting each of the two

genes below into the EcoRI site of the pSK (pBluescript SK) plasmid. Explain if and

why expression will or will not occur. The bolded ATG in the figures below represent

the translational start codon of each gene.

[EcoRI site]

Gene A 5' AATTCCATACATCCTCCGGAGGATGACC- - - - - - 3’ GGTATGTAGGAGGCCTCCTACTGG- - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - r est of gene A- - - - - - G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CTTAA

[EcoRI site]

[EcoRI site]

Gene B 5' AATTCTGCATAGGCCAAGGGAATGACG- - - - - - - 3’ GACGTATCCGGTTCCCTTACTGC- - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - r est of gene B- - - - - G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CTTAA

[EcoRI site]

34

ISOLATION OF GENOMIC DNA

Isolation of genomic DNA from plants is more difficult than from animal cells. Plant

tissues, with thick cell walls and high levels of phenolic compounds, have proven more

challenging to recover high molecular weight DNA from than either bacteria or mammalian

tissues. The thick cell walls must be broken effectively to release the DNA. However, the

mechanical breakage method cannot be so vigorous that it leads to shearing of the released DNA.

Similarly, once the cells are broken, partitioning of extracted phenolic materials and chlorophyll

away from the DNA must occur without damage to the high molecular weight DNA. The method

described below, which you will follow, is one of several (1,2) that have proven effective. One

way to minimize both of these problems is to use young etiolated (grown in the dark) seedlings.

The walls are less developed, and the cells have lower phenolics and chlorophyll.

Genomic DNA is isolated for various purposes including construction of genomic

libraries, PCR amplification of specific sequences and to search for the presence of specific

genes or sequences in the genome by Southern blot analysis. Your objective will be to isolate

clean, high molecular weight DNA from tobacco leaf tissue. Once the genomic DNA has been

isolated you will quantify the amount of DNA recovered. Small samples of the DNA will be

digested and the fragments generated will be examined by agarose gel electrophoresis. You will

also use your prepared DNA to PCR amplify a selected gene sequence during the second week.

Extraction of Genomic DNA from Plant Tissues

WEEK #1

1) Take 1cm of your tobacco leaf tissue. Place into a microcentrifuge tube and grind the

tissue to a fine paste using a Teflon pestle - the finer the better!

2) Immediately add 1.0 ml of (65C) CTAB buffer and grind the tissue again.

3) Transfer the homogenate to two microcentrifuge tubes and incubate the tubes at 65C for

20 minutes. At 5 min intervals gently mix the extraction mixture by several inversions.

(Vigorous mixing will shear extracted DNA.)

CTAB buffer

2 % CTAB (cetyltrimethyl ammonium bromide)

50 mM Tris-HCl, pH 8.0

20 mM EDTA (ethylenediamine tetraacetic acid)

1.4 M NaCl

35

4) Add an equal volume of chloroform:isoamyl alcohol (24:1). Mix for about 5 min by

repeated inversion. This step will result in the denaturation of protein and extraction of

any chlorophyll into the organic phase.

5) Centrifuge at 10,000g for 5 min at RT. Transfer the upper aqueous phase to a clean

microcentrifuge tube using a pipette (A wide mouthed pipette combined with slow uptake

and expulsion will minimize shearing of the high molecular weight DNA).

6) Repeat the chloroform extraction once. Transfer the aqueous phase to a clean

microcentrifuge tube.

7) To the aqueous phase add 0.4 volume of 5 M ammonium acetate, (mix gently) and 2

volumes of isopropanol (again mix gently). This procedure should cause the

precipitation of the DNA without precipitating protein. After 15 min on ice the DNA is

recovered by centrifugation at 12000g for 10 min. Wash the resulting pellet in 70%

ethanol (500 μl) to remove the isopropanol. Remove ethanol and allow tube to drain and

air dry.

8) Redissolve pellet in 100 μl of TE buffer.

36

Genomic DNA Part II

Quantitative Analysis, Digestion and Selective Amplification

Before proceeding to enzyme digestion and PCR amplification you will need to have a

good estimate of the amount of DNA in your sample. The quality of DNA can be measured by a

variety of techniques but will ultimately depend on what the intended use of the DNA will be.

The purity of the DNA preparation, un-complexed with polyphenolic material and free of

carbohydrates, is critical to enzymic manipulation or PCR amplification of the DNA.

WEEK #2

Quantitative Analysis of DNA

Spectrophotometric method The amount of DNA will be estimated by absorbance at

260 nm. Past experience has shown that you should recover approximately 100 ug of genomic

DNA from the 2.5 g of starting tissue, however an accurate value is required. Assume your

DNA has been dissolved in 100 ul of water. Calculate in advance of coming to lab what dilution

you will need to make to obtain an accurate estimate of the DNA concentration of this DNA

solution ie. calculate the dilution needed to get an absorbance (260 nm) of approximately 0.1.

Verify with the demonstrator how you will prepare 700 ul of the appropriate dilution before

actually making it. You will be shown how to run a scan of your sample with a

spectrophotometer to measure the absorbance at 230, 260 and 280 nm. This measurement may

be made later in the lab period.

EcoRI Digestion of Genomic DNA

1) From the concentration you determine in step 3), calculate the volume that will contain 5

ug of genomic DNA and carefully transfer this volume into a clean and labelled

microcentrifuge tube. This DNA will be digested with the restriction enzyme EcoRI.

Label (name, tube1/tube2) and prepare digests as follows in the order given:

Sterile water 35-N ul

DNA (your sample) N ul

10x EcoRI Digestion buffer 4 ul

EcoRI enzyme (10 units/ul) 1 ul

-----

40 ul

2) Mix the components carefully and incubate the digests at 37C for 1 hour.

37

Prepare a full sized agarose (1.6 g) gel in TAE (0.04 M Tris-acetate, 0.001 M EDTA)

electrophoresis buffer (200 ml) containing 0.5 ug/ml ethidium bromide. Follow the same

procedure you used previously to prepare the mini-gel for the analysis of your plasmid

preparation and allow it to gel while you wait for the digests.

3) When the digestions are completed add 5 ul of agarose gel loading buffer ( contains

0.25% bromophenol blue and 0.25% xylene cyanol as tracking dyes and 40% (w/v)

sucrose to make the solution dense to facilitate loading). Stir briefly to ensure complete

mixing and centrifuge for 10 seconds to drive all the liquid to the bottom of the tube.

Carefully load the complete digest into the wells in a recorded manner.

4) Connect the electrophoresis apparatus to the power supply in the correct orientation and

adjust the voltage to a constant output of 30 volts. This will be allowed to run overnight

to separate the DNA fragments in terms of size.

5) The following morning the power will be turned off and the gel examined on a UV light

box. A photograph may be taken to record the results but you are encouraged to examine

the gel yourself sometime the next day.

Polymerase Chain Reaction

The PCR technique allows a unique piece of DNA to be amplified through a cyclic

reaction involving a thermostable DNA polymerase, two primer molecules and the four

deoxynucleotides. If template DNA (dsDNA sequence containing the regions complimentary to

the two primers) is present in the reaction mixture then a product of defined size should be

produced. If the DNA which is present does not contain the appropriate sites for primer

annealing within ~3000bp of each other no products should be produced. The presence of an

appropriate sized product can be demonstrated by separating the reaction products on an agarose

gel (along with standard DNA sample markers of a known size) and visualizing the products

with ethidium bromide. (For small products, <300bp, a polyacrylamide gel is normally used)

In this test you are attempting to verify the presence of the neomycin phosphotransferase

II (NPT II) gene and of the β-glucuronidase (GUS) gene in your tobacco genomic DNA

preparation. The NPT II gene produces a product which is able to phosphorylate kanamycin and

thereby detoxify it. The GUS gene is a representative target gene in pBI121 that should be

present in successfully transformed tobacco plants. The sequence of the gene and the location of

the two primers you will be using are shown in Figure 1. Primers are typically 10-25 nt long with

longer primers providing a high degree of selectivity.

Procedure

1) From the concentration you determine, calculate the volume you will need to contain 100

ng of DNA. You may have to dilute your DNA in order to have a workable volume (a

38

volume less than 1 μl cannot be measured accurately). A good target concentration would

be 20 ng/μl allowing a 5 ul volume to be used.

2) Label the top of two 0.2 ml microcentrifuge tube with your name and add your genomic

DNA sample (100 ng in 5 ul) to each. Label one tube N (for NPT II) and label the

second tube G (for GUS). Place on ice.

3) To minimize pipetting error and simplify the addition process a master mix containing all

necessary buffers and reagents has been prepared and its relative composition is shown

below. GoTaq is a commercially available PCR mix containing Taq, dNTP’s and MgCl2.

Add 15 μl of the appropriate master mix to your tube, (always use a new pipet tip to

sample from the master mix to avoid contaminating the master mix). Important: Keep

your reaction tubes on ice!

Master mix Composition per 20 ul Reaction

GoTaq PCR 2xMix 10.0 μl

primer 1 (20 pmoles/μl) 0.4 μl

primer 2 (20 pmoles/μl) 0.4 μl

sterile H2O 4.2 μl

4) Master Mix N contains primers for the NPT II gene while Master Mix G contains primers

specific for the GUS gene.

5) Place tubes in thermocycler. The cycle you will run is:

95oC 3.0 minutes

95oC 0.5 minutes |

58oC 0.5 minutes | 40 cycles

72oC 1.0 minutes |

Hold at 4oC

This reaction will take about approximately 3 hours to run to completion. Your samples

will be analysed on a 1% agarose minigel with ethidium bromide. A copy of the photo of

this gel will be supplied for you to determine the result of your PCR reaction.

39

References

DNA extraction

1. S.O. Rogers and A.J. Bendich, 1988. Extraction of DNA from plant tissues. Plant

Molecular Biology Manual A6: 1-10.

2. M.G. Murray and W.F. Thompson, 1980. Rapid isolation of high molecular weight plant

DNA. Nucleic Acid Research 8: 4321-4325.

PCR

The Polymerase Chain Reaction. Ed K.B. Mullis, F. Ferre, R.A. Gibbs. 1994 Birkhauser

Boston

Discussion

Consider in looking at the electrophoresis results that the original chromosomes were greater

than 100,000,000 bp long. The more streaking of the DNA which occurs on the gel the greater is

the degree of degradation.

Calculate the recovery (ug/leaf tissue) and relative purity (260/280 ratio). Discuss these values

and the purity criteria you use in making your judgement. Discuss the isolation procedure and

your results in terms of the goal of obtaining clean, high molecular weight DNA and whether or

not you feel the DNA sample you have prepared would be suitable for creating a genomic

library.

Also answer the following questions as part of your discussion of the experiment.

Do you PCR results confirm that your tissue is transformed?

What would be the effect of having the annealing temperature (the low temperature in the cycle)

lower than the optimum? Discuss.

What are the appropriate PCR control reactions that should be run to ensure that the presence of

a band on a gel is indicative of the expected product?

40

Using the attached sequence information (Figure 1b) for NPT II provide the sequences for primer

#1 and primer #2 assuming perfect complementation.

Primer 1 is complementary to the lower strand of the NPT II gene from bp#11 to bp#31.

Primer 2 is complementary to the upper (coding) strand of the NPT II gene from bp#766 to

bp#786.

Sequence of Neomycin Phosphotransferase II

1 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg

51 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc

101 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca

151 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc

201 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc

251 gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc

301 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc

351 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca

401 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg

451 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc

501 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag

551 cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg

601 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa

651 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca

701 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag

751 cggccggaga acctgcgtgc aatccatctt gttcaatcat

41

Agrobacteria tumefaciens Mediated Plant

Cell Transformation (Part II, Confirmation of Transformation)

Part I of this lab began with the transformation of tobacco explants with two genes (see P 11 of

lab manual). Over the course of the term the transformed cells have been regenerated first into

callus then through changes in media induced to undergo organogenesis to regenerate whole

plants. The first gene (NPTII) provided the means to select for transformed cells by making them

immune to the effects of kanamycin, however survival and regeneration of non-transformed

“escapes” can happen. Today’s lab will provide a more direct method of testing if the recovered

plants are transformed or not. The confirmation of successful transformation is based on the

demonstration of the presence of the gene product from the second gene. The GUS gene product

is an enzyme (ß-glucuronidase), which is absent from untransformed tobacco. You will assess

the presence of the enzyme by demonstrating if a specific reaction is catalysed or not. In the case

of ß-glucuronidase the reaction being assessed is the cleavage of methylumbelliferyl ß-

glucuronide to methyl umbelliferone.

GUS Enzyme Reaction:

ß-glucuronidase (GUS)

methylumbelliferyl β-glucuronide (MUG)------------------------------->methyl umbelliferone (MU)

(non-fluorescent substrate) (fluorescent product)

Extraction:

(NOTE at least one control, non-transformed plant, should be assayed as well as putative

transformants)

1. Cut leaf samples (0.5 g) into small pieces and placed into a prechilled mortar (on ice).

2. In a fume hood add 2.0 ml of chilled GUS extraction buffer (50 mM NaPO4, pH 7.0, 10

mM ß-mercaptoethanol, 10 mM EDTA, 0.1% Triton X-100) and homogenize with a

pestle. It is very important to homogenize the leaf tissue well, as this results in

maximium cell breakage and release of the cytoplasmic contents (including the GUS

enzyme). If a sufficient fraction of the leaf cells are not broken the activity, which may be

present, will not be detected.

3. Centrifuge (10,000g, 5 min) the homogenate to remove cell debris. Recover the

42

supernatant and store on ice.

GUS Assay:

4. Assay the supernatant for GUS activity by preparing the following digest and blank

reactions. The methylumbelliferyl β-glucuronide (MUG) 0.5 mM 4-methylumbelliferyl

ß-D-glucuronide (MUG) is prepared in GUS extraction buffer. Prepare digest reactions

in duplicate with extracts from both non-transformed and transformed plants. Also

prepare blanks in duplicate.

MUG substrate extract supernatant GUS extraction buffer

Digest 1.0 ml 0.1 ml 0 ml

Substrate Blank 1.0 ml 0 ml 0.1 ml

Extract Blank 0 ml 0.1 ml 1.0 ml

All digest and blank reactions should be incubated at 37 C for 30 min.

Several blank reactions must be run to account for fluorescence which may arise from other

sources other than the enzymic conversion. The extract blank assesses if there is any

contribution to overall flourescence from molecules in the extract while the substrate blank takes

into consideration the fact that there may be some non-enzymic breakdown of the substrate prior

to or during the incubation period. These contributions need to be measured and subtracted from

the total fluorescence so that only fluorescence due to enzyme cleavage is measured.

5. After the 30 min incubation, a 0.2 ml aliquot from each reaction and blank is removed

and added to separate 1.8 ml aliquots of Stop Buffer.

6. Mix and measure fluorescence of blanks and diluted digests with the fluorometer. Read

the display immediately.

To quantitatively measure the amount of product produced by GUS activity, the level of

fluorescence must be measured using a fluorometer (λex=365nm, λem=460nm) and compared to

the amount of fluorescence emitted from a known molar amount of the fluorescent product, 4-

methyl umbelliferone (MU). The fluorometer will be standardized with 0 and 50 nM solutions

of MU.

7. Record your results on the lab whiteboard. You will need to record the results of

everyone in the lab for your report.

43

Lab Report

Students should be familiar with the types of changes to be anticipated at the various stages of

plant regeneration. A weekly log summarizing the OBSERVED changes should be maintained,

recording development as well as specific changes which occur following any change in media.

These changes should be summarized and discussed on a scale of days post-infection.

Calculate the amount of GUS activity (nmoles of MU/g tissue/min) in your tissue for all samples

and show a sample calculation. Remember to take into account the background activity levels of

the plants. Compare the results of all the GUS activities obtained for the various plants analysed

(by all the class) and DISCUSS THE RESULTS.

Questions to be answered with the lab report.

Occasionally shoots arise from calli of the A. tumefaciens-treated explants that are not

transformed even though they have grown on kanamycin-containing media. How can this occur?

What tests other than the GUS activity measurement could be done to determine whether the

plants that you have regenerated from treated explants are in fact transgenic?

In most plant transformation experiments, normally 20 – 40 transformants are recovered. Why

are so many recovered given there is only one construct being inserted?

What factors do you think will influence the number of transformants recovered in this type of

experiment?

References:

R.W. Old & S.B. Primrose, Principles of Gene Manipulation 3rd Ed. Blackwell Scientific

Publications, 1985 p 215-230 (there are later editions as well)

T.M. Murphy & W.F. Thompson, Molecular Plant Development, Prentice Hall, 1988, p 184

R.A. Jefferson et al., J. Molecular Biology 193: 41-46, 1987