Practical 1

DNA, a Gene, an Auxotroph

In this class you will observe the DNA material isolated form bacteria. You will investigate
the phenotype changes caused by a single gene in Arabidopsis thaliana, propose a
genetic hypothesis to explain your observations and test this with statistical analysis.
You will observe the growth, or absence of growth, of Auxotroph bacteria in the presence
of supplements.

After completing this practical you should be able to:

Extract and visualise DNA from E.coli.

Analyse data from a genetic cross involving one autosomal gene locus.
Observe bacterial growth on minimal media to determine which supplements are required
for growth of different Auxotrophs.

Assessment

Pre-practical Test (score 80% = 1 mark)

In-practical assessment (5 marks)
Post-practical Test (4 marks)
This Post-practical Test covers material from the
Introductory Practical.

Password for Post-practical Test:

Test to be completed between 4 pm on the day of

prac and 5 pm the following day.

Your safety in the laboratory is very important:

At all times wear a lab coat and suitable shoes with enclosed heel and toe and safety glasses
when required.

Always work to ensure your safety and the safety of those around you.

Use caution when using sodium perchlorate. It is a strong oxidising agent.

Open Eppendorf tubes with care, do not hold near your face.

Immediately report any injuries or spills to a demonstrator.

Microorganisms will be used in this class, avoid touching things to your mouth and wash hands
carefully after the class.

A risk assessment has been carried out for the practical classes and identified risks minimised. Please
observe the safety signs displayed and ask your demonstrator if you would like to know more, MSDS
( Material Safety Data Sheets) are available in the laboratory. Further information can be found at:

http://safety.unimelb.edu.au.

Time Management
Begin practical with the DNA extraction.
During Steps 1 and 3 incubations (each 15 minutes) start the analysis of the Arabidopsis plates.
DNA extraction should be completed at the end of one hour and shown to your demonstrator for
assessment.
Complete the observations of the Auxotroph.

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Activity 1: DNA Extraction Isolating DNA from E. coli Cells

Escherichia coli, a rod shaped bacterium about 1-2 m in length will be used in this. Some strains
can be found in the human digestive tract, will be used as our source of DNA. The strain we use in
the prac is not harmful. We will extract the DNA from a suspension of cells of E. coli, containing
approximately 1000 million or 109 cells. E. coli has a semi-rigid cell wall of peptidoglycan which
supports the cell and determines its shape. The wall surrounds the plasma membrane. Each cell
contains a single chromosome, which is a DNA molecule tightly coiled inside the cell, forming the
nucleoid (Figure 1). The absence of a nuclear membrane is one of the differences between
prokaryotes and eukaryotes, which was discussed in semester one. The chromosome is circular of
approximately 4.7 million base pairs and contains about 1500 genes. There is also a small volume
of cytoplasm. E. coli commonly contains small circular extrachromosomal pieces of DNA called
plasmids (Knox et al. 2014 (5th edition) p. 319 (Knox et al. 2010 (4th edition) p. 292).
If you are unsure of what a base or base pair in DNA is, consult Knox et al. 2014 (5th edition) p. 35
(Knox et al. 2010 (4th edition) p. 50).

Figure 1. Diagrammatic structure of a cell of E. coli.

The steps involved in extracting and isolating DNA are:

1. Rupturing the cell wall, and liberating the contents of the cell into solution. This is called lysis.
2. Separating the DNA from the protein.
3. Inactivating any enzymes released from the cell, which may break up the DNA when in contact
with it.
Prior to the practical complete a flow chart showing steps 1 to 7 of the DNA extraction.
Flow chart:

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Equipment
A plastic tube containing E. coli suspension
Pipettes

Microfuge tubes containing lysozyme, protease, sodium dodecyl sulphate

Conical flask containing sodium perchlorate
Squeeze bottle containing 100% Ethanol
Procedure
The E. coli cells you will use were grown in nutrient broth and have been resuspended in a lysis
buffer to a volume of 2.5 ml. They are in a screw top tube on your bench.
1. Using a pipette, add 0.5 ml of the enzyme lysozyme (10 mg/ml) to the cell suspension. With
a permanent marker, label the tube with your seat number and loosely apply the cap.
Incubate at 37C for 15 minutes. Lysozyme digests the cell wall.
2. Add 200 L of the 20% ionic detergent named sodium dodecyl sulphate (SDS). Screw the
lid on the tube tightly and gently mix by inverting the tube 5 times. Sodium dodecyl sulphate
causes the plasma membrane of the E. coli to rupture.
3. Loosen the lid of the tube slightly and incubate at 55C for 15 minutes. The solution should
become quite viscous.
4. Add 0.3 ml of protease solution to the cell lysate (20 mg/ml). Screw the lid on loosely and
incubate at 37C for 20 minutes.
5. Cool the tube and its contents on ice. The tube should be cool to the touch before
proceeding.
6. Add 0.8 ml of sodium perchlorate (5 M). Invert gently 5 times. Take care when mixing
because sodium perchlorate is an oxidising agent and is toxic. The sodium perchlorate
will assist in separating the protein from the DNA and stabilising the DNA.
7. Tilt the tube at an angle and using a pipette carefully and slowly add 5 ml of cold ethanol
down the side of the tube so that it forms a layer above the DNA solution. It is at the interface
of the alcohol and lysate that the DNA will precipitate out of solution.
Allow the tube to stand for 2-3 minutes and, if you have been gentle in adding the alcohol,
you will see the DNA suspended at the interface. Take note of the appearance of the DNA at
the interface immediately after the addition of the ethanol.
8. Show your tube to your demonstrator for assessment of the DNA yield.

Demonstrator initials

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Question 1:

Suggest why the solution may become viscous after the cell wall and cell
membrane are ruptured?

Question 2:

Protease is an enzyme. What is an enzyme? Name another enzyme.

Question 3:

What function does protease perform in this experiment?

In a laboratory setting, the DNA you have extracted from E. coli would be purified and used in
other procedures such as gel electrophoresis (see practical 3)

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Activity 2: An Autosomal Gene in Arabidopsis thaliana

In this section of the practical you will investigate the inheritance of
a leaf morphology trait in a monohybrid cross of A. thaliana plants.
One variety of plant has hairs or trichomes on its leaves (called
non glabrous) whereas the other variety has no trichomes (smooth
leaves called glabrous). The hairs are made up of 3 parts shown
in the electron micrograph at right (Figure 2). Leaf trichomes may
protect plants against herbivorous insects, and may increase
tolerance to drought and UV-radiation. Some mutants have an
occasional hair but it is usually misshapen and not in the trichome
shape.

Figure 2. Leaf trichome

Source:http://www.planttrichome.or
g/trichomedb/species.jsp

Two pure breeding (homozygous) varieties of plants have been crossed.

Parents (P1 X P2) = purebreeding F1 = heterozygous F1 X F1 F2

Equipment
One pair of A. thaliana demonstration plates per pair of students

Procedure
1.

Working in pairs, look at the demonstration plates showing

these two varieties in the parents (P1 and P2) and their
offspring (F1). You can use a dissecting microscope to
examine the leaves closely. Make sure you look at the
mature leaves not the first leaves (cotyledons, Figure
3).

Question 4:
P1 =

What is the phenotype of the P1, P2 and F1

plants?
P2 =

F1 =

Figure 3. A. thaliana rosette.

Note the first leaves (cotyledons) at
the base of the plant (see arrow). Do
not look for hairs on these.

Question 5: What does this observation about the F1 plants

indicate?

Plants of the F1 generation were allowed to self-fertilise and set seed (Figure 4). The F2 seed was
germinated.
You have been supplied with a petri dish containing young F2 plants.
2.

Score the glabrous/non-glabrous phenotype of the F2 plants and record your data and the
data from your bench in Table 1.

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Table 1: Data for F2

Phenotype
Number of non glabrous
(hairy) plants

Number of glabrous
(smooth) plants

Total

Own data
Class data

3.

Using the convention for Arabidopsis GL1 = non glabrous = hairy (wild type) and gl = glabrous
= smooth, propose a genetic hypothesis to account for these observations by filling in the
gaps below.
non glabrous

Parental
Genotypes

glabrous

H
H

F1
Genotype

non glabrous

H
h

Meiosis

F2

Figure 4. A. thaliana in fruit.

The plants are staked to
support the fruits for seed
harvesting. The fruit is a
siliqua 520 mm long,
containing 2030 seeds.

Female
gametes
Male
gametes

H
H

H
H

H
h

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Question 6:

The expected F2 phenotypic ratio is

Question 7:

State the genetic hypothesis by completing the following:

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1. There is/ are ______ gene locus/i being considered in this cross with ______ allele/s. The
dominant phenotype is _________________________. The F2 of this _____________ cross will
produce a phenotypic ratio of ____________________.
2. State the null hypothesis:

Rarely do observed results exactly match the predicted results. Therefore we perform a
statistical test to see if the difference between the observed and the expected results is
statistically significant or not.
3. Using the appropriate statistical test (2) compare the predictions of your genetic model with the
observed results. Use the class data.
(Apart from the information in the BioByte, you can also refer to the Appendix 5.)
Calculate
the Chi-square (2) value for this comparison by completing Table 2 (for your
Table
2: 2 Analysis
calculations in genetic problems correct to 2 decimal places).
Phenotype
TOTAL
non glabrous (hairs)
glabrous (non hairy)
Observed
(O)
Expected
(E)
(O-E)2
E
2

(O-E)2
E

=
Use the Chi-square table (below).
Number of degrees of freedom
Probability
4. Conclusion: Circle the appropriate alternatives:
The null hypothesis is rejected / not rejected. Therefore, we accept / reject, the underlying genetic
hypothesis that gave rise to the prediction. That is, the observed results are consistent / not
consistent with the genetic hypothesis.

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Table 3: Chi-square (2) Distribution

PROBABILITY
DEGREES
OF
FREEDOM

NON-SIGNIFICANT
0.95

0.90

0.80

0.70

0.50

SIGNIFICANT
0.30

0.20

0.10

0.05

0.01

0.001

CHI SQUARE VALUES

1

0.004

0.02

0.06

0.125

0.46

1.07

1.64

2.71

3.84

6.44

10.83

0.10

0.21

0.45

0.71

1.39

2.41

3.22

4.60

5.99

9.21

13.82

0.35

0.58

1.012

1.42

2.37

3.66

4.64

6.25

7.82

11.34

16.27

0.71

1.06

1.65

2.20

3.36

4.88

5.99

7.78

9.49

13.28

18.47

1.14

1.61

2.34

3.00

4.35

6.06

7.29

9.24

11.07

15.09

20.52

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Modelling meiosis in the Arabidopsis cross:

The glabrous gene GL1 is located on chromosome 3 in Arabidopsis. On the diagram below track
chromosome 3 with its appropriate alleles through meiosis in the two pure breeding parents to the
F1. Show the chromosome in the parents after DNA replication.

Glabrous (non-hairy) parent

Non glabrous (hairy) parent

X
end of meiosis 1

end of
meiosis
2

F1

You can practice your understanding of meiosis by continuing to the F2.

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Activity 3: Analysis of Auxotrophs in E. coli

Collect your plates from the container.
In the last practical class you prepared these plates each spread with one of the mutant strains of
E. coli (Strain A, B, C or D) onto minimal media.
There is a demonstration plate of the wild type E. coli spread on minimal media.
Work as a group to see all 4 strains.
Hold your plate up to the light and look for a halo of growth around the discs.
Question 8:

Describe what you see on these 5 plates (hold the plate up to the ceiling to see
whether there is growth or not).

a. Wild type E. coli on Minimal Media

___________________________________________________________________________
b. Strain A on Minimal Media
___________________________________________________________________________
c. Strain B on Minimal Media
___________________________________________________________________________
d. Strain C on Minimal Media
____________________________________________________________________________
e. Strain D on Minimal Media
____________________________________________________________________________
Question 9:

What can you conclude from these observations?

Question 10:

Draw the pattern of growth

you see, on the template.

I worked with Strain _________________

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Question 11:

BIOL10005: 2016

Collect data for the 4 strains and enter the results in Table 5. Indicate when
growth occurred around the disc with a + and for no growth

Table 5: Results of auxotroph growth on minimal media

Amino Acid

Strain A

Strain B

Strain C

Strain D

Arg
Leu
Meth
Tyr
Phe
Water
Question 12:
a. Why is one disc impregnated with just water?

b. Had there been growth of E. coli around the disc impregnated with water, how would this
influence the conclusions you could draw from your experiment?

Question 13: Describe in what amino acid pathway there was a mutation for each of the strains.
Strain A
Strain B
Strain C
Strain D

Question 14: What was unusual about strain D? How can you explain this result?