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
Lab 10: Computer Lab
--
1
LABORATORY SCHEDULE
(Tuesdays, Rm 342)
Date /2015
Lab Title
Write-up Due Date
Week of
Jan 12th
Sterilization and Aseptic Techniques/Computer Orientation
Jan 19th
Agrobacteria Mediated Plant Transformation (Part I)
Jan 26th
Plant Regeneration
Feb 2nd
Plasmid Construction
Feb 9th
Bacterial Transformation
Feb 23
Feb 23rd
& Mar 2nd
Isolation and Characterization of Plasmids
Mar 10
Mar 9th
Gene Expression and Bioassay
Mar 16
Mar 16th
& 23rd
Isolation of Genomic DNA and PCR
Mar 30
Mar 30th
Agrobacteria Mediated Plant Transformation (Part II) [attached to Part I]
Apr 6th
Computer lab
NOTE:
All lab reports must be handed in on or before April 6, 2014
NOTE:
Most labs will require 2½ - 3 hrs to complete
none
Apr 6th
Feb 24
(Feb 23)
----
2
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 affect 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)
3
USEFUL WEBSITES
National Center for Biotechnology Information:
ExPASy:
http://ca.expasy.org/tools/
Primer 3:
http://frodo.wi.mit.edu/primer3/
http://www.ncbi.nlm.nih.gov
Sequence Manipulation Site: http://www.bioinformatics.org/sms/
pDRAW32:
http://www.acaclone.com/
MultAlin:
http://multalin.toulouse.inra.fr/multalin/multalin.html
MacrogenUSA:
http://orders.macrogenusa.com/
BioEthics
http://www.accessexcellence.org/AB/IE/wholesome.html
http://www.accessexcellence.org/AB/IE/bioethics.html
4
Dilution Calculations
The illustrated two dilution steps were done.
You used the final 40 ul solution to measure
the concentration of solute in that 40 ul and
found a value of 35 ug solute/ml. Determine
i) the concentration of solute in the first tube
and also calculate ii) how much solute was
originally present in tube 1.
Answer
Working in Concentration (mass/volume)
i) Tube 3: 40 ul of solution at a solute
concentration of 35 ug/ml
Recognize that the total solute in the 40 ul
came from the 5 ul added. That means the
solute was diluted from 5 ul to 40 ul in
volume, a dilution factor of 8 x. Therefore
the concentration in the 5 ul transferred was
8 x 35 ug/ml = 280 ug/ml. But remember
that 5 ul came from the 400 ul of solution in
tube 2 -hence the concentration in tube 2 is
also 280 ug/ml.
Tube 2: 400 ul at a solute concentration of
280 ug/ml
Similarly, all the solute in the 400 ul of tube 2 came from the 5 ul delivered from tube 1. That
means that the solute was diluted from 5 ul to 400 ul, a dilution of 80 x. Therefore the
concentration in the 5 ul aliquot transferred from tube 1 was 80 x 280 ug/ml = 22,400 ug/ml.
Therefore this is also the concentration of the solute in tube 1 ie 22,400 ug/ml.
In short, there were two dilutions, 5 to 400 ul (80 x) and 5 to 40 ul (8 x). The total dilution was 8
x 80 = 640 times. Therefore from the concentration determined for tube 3 can be multiplied by
this dilution factor (640) to yield the concentration in tube 1 ie 35 ug/ml x 640 = 22,400 ug/ml
ii) However, we did not have a ml of solution in tube 1, we only had 60 ul (0.060 ml), thus to
calculate the amount of solute originally present in tube 1, you multiple the actual volume (ml)
times the concentration (ug/ml) in the tube.
Thus 0.060 ml x 22,400 ug/ml = 1344 ug solute originally present.
5
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)
75
250
500
1000
1500
2000
Time (min)
25
30
40
45
50
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.
6
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.
7
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.
8
Table 2.
ULTRAVIOLET RADIATION LETHAL DOSES
MICRO-ORGANISM
Lethal Dose For 180μW/cm2
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
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
28-56 min
0.9-1.9 hrs
1.9-3.7 hrs
Spigellla paradysenteriae
Algae, Blue-Green
9
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.
10
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 nontransformed plant cells, the level of expression of the tDNA encoded genes can also be examined
using this enzyme.
11
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 28C
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 109-1010
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
12
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
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.
13
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/l 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.
14
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.
15
Procedure
Culture medium:
A
B
C
D
E
You will be provided with culture medium containing the following
hormone combinations.
0.5 mg/l 2,4-D
2.0 mg/l BAP
2.0 mg/l BAP + 0.5 mg/l 2,4-D
2.0 mg/l 2,4-D + 0.5 mg/l BAP
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?
16
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.
17
PLASMID CONSTRUCTION
Plasmids are small circular pieces of double stranded DNA which occur naturally as extrachromosomal 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 ethidium bromide (final
concentration of 0.5 ug/ml). CAUTION: Take care in handling ethidium bromide and any
solution containing it as this dye is a carcinogen. Use gloves! Ethidium bromide 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. 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.
18
Fragment Separation and Recovery
The demonstrator will demonstrate how to identify and 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 ether 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 150 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.
19
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.
20
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)
ToxA (insert) DNA
Ligase 5x buffer
T4 DNA Ligase
Sterile H2O
X μl
X μl
Y μl
3Y μl
2 μl
2 μl
1 μl (1U)
1 μl
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 -20oC
until next week.
Lab Report -The lab report for this lab should be prepared together with that for Bacterial
Transformation.
21
22
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 nonrecombinant 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.
23
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 1 hour 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, plate out 100 μl aliquots of each 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.
24
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?
25
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 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.
26
27
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.
28
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 4C 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 -20C 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 an ethidium bromide-stained gel by UV light in
the dark. If ethidium bromide 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. To this solution is added ethidium bromide to a final
concentration of 0.5 mg/L. CAUTION Take care in handling ethidium bromide and any
solution containing it as this dye is carcinogenic. Use gloves!
29
2) Add 100 ml of diluted gel buffer-ethidium bromide 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 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 60C, 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, transfer 20 ul 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.
9) When you are finished with the gel, dispose of it into the special container for
ethidium bromide waste.
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!
30
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?
31
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.
32
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 βD-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 20 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.
33
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
Glenlea
Glenlea
Glenlea
Erik
Erik
Erik
Sample
Necrosis
24h
48h
108 h
pSK
pNEC
Ptr toxin
PSK
pNEC
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.
34
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.
Gene A
5'
3’
[EcoRI site]
AATTCCATACATCCTCCGGAGGATGACC-----GGTATGTAGGAGGCCTCCTACTGG------
----------------------------------------------------------------------------------------rest of gene A------G
--------------------------------CTTAA
[EcoRI site]
Gene B
5'
3’
[EcoRI site]
AATTCTGCATAGGCCAAGGGAATGACG------GACGTATCCGGTTCCCTTACTGC-------
---------------------------------------------------------------------------------------rest of gene B-----G
------------------------------CTTAA
[EcoRI site]
35
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)
Weigh 0.5 g of tobacco leaf tissue. Place into a mortar with a “pinch” of fine grinding
sand and grind the tissue to a fine paste - the finer the better!
2)
Immediately add 2.0 ml of hot (65C) CTAB buffer and grind the tissue again.
3)
Transfer the homogenate to two microcentrifuge tubes and incubate the tubes at 65C 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
36
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 500 μl of TE buffer. Add heat-treated RNase A (1.0 μl of 10 mg/ml)
and incubate at 37C for 60 min (RNase is very heat stable while DNases are heat labile
thus contaminating DNases can be destroyed by heat-treatment). Alternatively the
sample plus RNase can be stored overnight at 4C and then frozen until next week to
continue the procedure.
NOTE: Steps 9 - 11 will be carried out for you to allow you to proceed to the digestion step in
week 2.
9)
Extract the DNA with an equal volume phenol (CAUTION phenol is caustic) by gentle
repeated inversion and centrifuge at 5000g for 5 min to separate the aqueous and organic
phases. Transfer the upper aqueous phase to a clean tube and re-extract the aqueous
phase once with an equal volume of phenol:chloroform (1:1) and once with an equal
volume of chloroform (recover the aqueous phase each time and transfer to a clean tube).
10)
Precipitate DNA by addition of 0.1 vol of 3 M sodium acetate and 2.0 vol of ethanol,
allow to stand in ice for 15 min and recover DNA by centrifugation at 10,000g for 15
min. Rinse with 70% ethanol (0.5 ml) and centrifuge briefly (5 min) to repellet. Remove
ethanol as before, invert tube and allow pellet to dry (15 min). It is essential that the
ethanol be removed because its presence will inhibits any enzyme activity.
11)
Redissolve the pellet in sterile water (250 ul/2.5 g of fresh starting material). Heating in a
60C water bath may be necessary to get the DNA into solution. Transfer to a
microcentrifuge tube.
37
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
DNA (your sample)
10x EcoRI Digestion buffer
EcoRI enzyme (10 units/ul)
2)
35-N ul
N ul
4 ul
1 ul
----40 ul
Mix the components carefully and incubate the digests at 37C for 1 hour.
38
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 then 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.
39
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
volume less than 1 μl cannot be measured accurately). A good target concentration would
be 10 ng/μl allowing a 10 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 10 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. Add 40 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 50 ul Reaction
5.0 μl
0.4 μl
0.75 μl
1.0 μl
1.0 μl
0.5 μl
30.6 μl
10x PCR buffer
25 mM dNTP
25 mM MgCl2
primer 1 (20 pmoles/μl)
primer 2 (20 pmoles/μl)
Taq polymerase (5 units/μl)
sterile H2O
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 |
o
58 C
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.
40
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?
41
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
42
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 ßglucuronide 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, 4C) the homogenate to remove cell debris. Recover the
supernatant and store on ice.
43
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.
Digest
Substrate Blank
Extract Blank
MUG substrate
1.0 ml
1.0 ml
0 ml
extract supernatant
0.1 ml
0 ml
0.1 ml
GUS extraction buffer
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, 4methyl 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.
44
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
45
46