Virology practical report

VRIJE UNIVERSITEIT BRUSSEL
INSTITUTE OF MOLECULAR BIOLOGY AND
BIOTECHNOLOGY

TITLE: VIROLOGY PRACTICALS REPORT

NAMES: KARIUKI SAMUEL MUNDIA

INSTRUCTOR: Prof. Dr. Henri De Greve

ASSISTANCE: Maia De Kerpel and Cine Deboeck

DATE OF SUBMISSION: 27/04/2012

1

LIST OF TABLES
Table 1 Results of plaques on indicator bacteria ............................................................................ 5
Table 2. Number of plaques observed after phage culture in two host bacteria ............................. 7
Table 3 Day 2 phage results showing the plaques after incubation with bacteriophage lambda
overnight. ........................................................................................................................................ 8
Table 4: The day 3 results showing the plaques after culture on the three bacterial strains........... 9
Table 5 showing efficiency of plating ............................................................................................ 9
Table 6: showing phenotypes of the three bacterial strains .......................................................... 10
Table 7: Results of phage immunity test....................................................................................... 10
Table 8: Relative distances and Molecular weight tabulation of bacteria Stx gene ..................... 12
Table 9: Relative distances and molecular weight tabulation of phage Stx gene ......................... 13
Table 10: Number of bacterial colonies after incubation in LB and Mackonkey media .............. 15
Table 11: Results in MinA plates supplemented with different amino acids showing cysteine
positive plates................................................................................................................................ 15

LIST OF FIGURES

Figure 1 showing results of Agarose gel of total bacterial DNA.................................................. 11
Figure 2: Graph of log of molecular weight and relative distance of band migration.................. 12
Figure 3: Results of Agarose gel of phage DNA .......................................................................... 13
Figure 4: Graph of log of molecular weight and relative distance of band migration .................. 14
Figure 5: Agglutination was not observed when the bacteria and yeast were incubated on ice ... 18

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Table of Contents
Spontaneous induction of Bacteriophages................................................................................................... 5
1.1 Introduction ........................................................................................................................................ 5
1.2 Objective: ............................................................................................................................................ 5
1.3 Results and discussion ........................................................................................................................ 5
1.3.1 Results.......................................................................................................................................... 5
Table 1 Results of plaques on indicator bacteria.................................................................................. 5
Suppression of amber mutations.................................................................................................................. 6
2.1 Introduction ........................................................................................................................................ 6
2.2 Objective ............................................................................................................................................. 6
Table 2. Number of plaques observed after phage culture in two host bacteria......................................... 7
Restriction and modification of bacteriophage λ in different E.coli strains. ................................................ 8
3.1 Introduction ........................................................................................................................................ 8
3.2 Objective ............................................................................................................................................. 8
Table 3 Day 2 phage results showing the plaques after incubation with bacteriophage lambda overnight.
...................................................................................................................................................................... 8
Table 4: The day 3 results showing the plaques after culture on the three bacterial strains ...................... 9
Table 5 showing efficiency of plating............................................................................................................ 9
Table 6: showing phenotypes of the three bacterial strains ...................................................................... 10
Immunity of lysogenic bacteria...................................................................................................................10
4.1 Introduction ...................................................................................................................................... 10
4.2 Objective ........................................................................................................................................... 10
4.3 Results and discussion ......................................................................................................................10
Table 7: Results of phage immunity test. ................................................................................................... 10
Isolation of total bacterial DNA and phage DNA and PCR amplification of shiga toxin gene.....................11
5.1 Objective ........................................................................................................................................... 11
5.2.1 Results of total bacterial DNA.................................................................................................... 11
Table 8: Relative distances and Molecular weight tabulation of bacteria Stx gene ................................... 12
Figure 2: Graph of log of molecular weight and relative distance of band migration................................12
5.2.2 Results of phage DNA.................................................................................................................13

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Figure 3: Results of Agarose gel of phage DNA...........................................................................................13
Table 9: Relative distances and molecular weight tabulation of phage Stx gene ...................................... 13
Figure 4: Graph of log of molecular weight and relative distance of band migration................................14
Transposition of TN10 from phage λ vehicles.............................................................................................14
6.1 Introduction ...................................................................................................................................... 14
6.2 Objective ........................................................................................................................................... 15
6.3.1 Results and Discussion ...............................................................................................................15
Table 10: Number of bacterial colonies after incubation in LB and Mackonkey media.............................15
6.3.2 Results isolation and identification of amino acid biosynthesis mutants.................................. 15
Table 11: Results in MinA plates supplemented with different amino acids showing cysteine positive
plates...........................................................................................................................................................15
Generalized Transduction: P1 Transduction...............................................................................................16
7.1 Introduction ...................................................................................................................................... 16
7.2 Objective ........................................................................................................................................... 16
7.3 Results and Discussion ......................................................................................................................16
Yeast agglutination ..................................................................................................................................... 17
8.1 Introduction ...................................................................................................................................... 17
8.2 Objective ........................................................................................................................................... 17
8.3 Results and Discussion ......................................................................................................................17
Figure 5: Agglutination was not observed when the bacteria and yeast were incubated on ice .............. 18
References ..................................................................................................................................................18

4

Spontaneous induction of Bacteriophages

1.1 Introduction
Bacteriophages follow either a lytic or lysogenic life cycles when they infect a bacteria cell.
Lysogeny is obtained when a bacteriophage DNA is integrated in the host genome, a condition
that arises during adverse conditions of the environment, rise in virus population or physiological
changes of the bacteria cell. Phage induction occur when host DNA is damaged or heat treatment
of temperature sensitive repressor protein. However, one million out of one billion bacterial cells
containing prophage can undergo spontaneous induction and revert to lytic type of lifestyle. This
is due to activation of the genes that enhances the lytic life cycle.

1.2 Objective:
To isolate viable phages from lysogenic bacterial strains and illustrating it by using indicator
Escherichia coli strains.

1.3 Results and discussion
1.3.1 Results

Table 1 Results of plaques on indicator bacteria
100 101
λ plaque plaque
Ψ933 plaque No plaque

Lysis of the indicator E.coli C was observed in both the 100 and 101 dilutions of bacteriophage λ
and in 100 of ψ933 but not in 101 dilution of ψ933 phage. However, more complete confluent
plaque was observed in 100 dilution as compared to 10-1 dilution of λ phage. In addition λ had
more complete confluent plaque compared to ψ933.

Since spontaneous induction is a radomn chance event, we expected to observe plaques if it
really happened. The observation of plaques shows that the phage DNA was excised from the
bacterial genome and followed the lytic lifestyle.

5

Bacteriophage λ and its relatives are temperate phages, this means that they can show both
lysogenic and lytic lifestyles. In lysogeny, the phage DNA integrates into a very specific region
of the bacteria genome called attB using its attP, a process catalyzed by λ Int protein and the
Integration host factor (IHF) from E.coli host. This idea of integration of the λ genome into the
host chromosome by a process of reciprocal crossing over was first proposed by Allan Campell
in 1962. The reversal of this process, excision of the prophage, requires besides Int and IHF also
the λ Xis protein and occurs by recombination between the attL and attR sites.

The results showed that spontaneous induction was possible without use of any inducing
substance such as UV light or chemicals.

Suppression of amber mutations

2.1 Introduction
Amber mutation is a change in nucleotide sequence from a codon that codes an amino acid to
UAG stop codon leading to production of non-functional protein since UAG when on a
messenger RNA is a signal for termination of translation. Most codons on messenger RNA code
for amino acids which are gradually added to the growing polypeptide chain which eventually
leads to production of functional proteins. Stop codons on the other hand bind release factor
leading to the dissociation of the two ribosomal subunits and consequence termination of
translation. Other stop codons in RNA are opal (TGA) and ochre (TAA).

Bacteriophages with amber mutations are able to grow in bacteria cells that have a mutant tRNA
called amber suppressors. These bacteria’s tRNA is able to read through UAG and produce a
functional protein. This replaces the original functional protein of the wild type virus hence these
bacteria are called amber suppressor mutants.

2.2 Objective
To observe the results of culturing λ1098 bacteriophage carrying Tn10tet transposon in
suppressor minus (Su-) and suppressor plus (Su+) host.

6


Table 2. Number of plaques observed after phage culture in two host bacteria
107 DILUTION FACTOR
CSH 110 25 PLAQUES
C600 NO PLAQUES

There were no plaques observed in the plate with. E.coli C while 25 plaques were observed on
107 dilution and more confluent plaques on other dilutions in the petri dish with E.coli CSH110
strain.

These results reveal that the E.coli CSH110 strain is suppressing the amber mutations of the
derivative phage. In contrast, E.coli C600 is a wild type isolate which does not suppress the
amber mutations of the λ1098 phage.

The presence of amber suppressor in CSH110 strain enabled the protein synthesis to occur
normally despite the presence of amber mutations. This allowed the replication of the phage
genome with eventual lysis of bacteria which produced visible colonies on the plates. On the
other hand, strain C did not display the phenotype under permissive conditions.

Amber Suppressors are mutant tRNA genes that code for tRNA whose anticodons have been
altered so that they respond to UAG (amber) codons and insert the amino acid of the wild type
tRNA instead of release factors.

The titre of the phage stock is calculated as follows:

As indicated in the above table, 25 plaques were observed on Su+CSH110 petri dish in the 10-7
dilution. Therefore, the 10-7 dilution tube contains 25PFU per 0.02ml, and the titre of the virus in
the stock is 25/0.02x107 PFU/ml or 1.25x1010 PFU/ml.

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Restriction and modification of bacteriophage λ in different E.coli strains.

3.1 Introduction
Restriction modification system is used to protect bacteria from incoming foreign DNA from the
environment including bacteriophages at the same time modify its DNA to prevent restriction of
its chromosome from this nuclease activity. This system has been observed to prevent growth of
bacteriophages from other bacterial host. Restriction is realized by use of sequence specific
endonucleases while modification it majorly by methylation of these specific sequences.

Restriction modification systems are classified as type I, type II and type III depending on the
order of their discovery. Type II restriction modification systems are the major one used in the
labs in genetic engineering because they are site-specific. Type I system is not site-specific and is
the major system in E.coli K12.

3.2 Objective
To illustrate the phenomenon of restriction and modification of DNA using bacteriophage λ as a
model.


Table 3 Day 2 phage results showing the plaques after incubation with bacteriophage
lambda overnight.
Plaques at 107 dilution Stock titre
K514 20 1x1010
C600 48 2.4x1010
HB101 21 1.05x1010

The 10-7 dilution factor showed clearly countable plaques in all the three E.coli K12 strains as
indicated in the table above. Each mini-lysate was titrated after dilutions on the three E.coli
strains again and the results were as follows.

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Table 4: The day 3 results showing the plaques after culture on the three bacterial strains
λK514 λC600 λHB101
K514 27 = 1.35x109pfu/ml confluent confluent
C600 confluent 34 = 1.7x109pfu/ml 17 = 8.5x104pfu/ml
HB101 confluent confluent 24 = 1.2x109pfu/ml

Table 5 showing efficiency of plating
Original strain titre (pfu/ml) New strain titre (pfu/ml) Efficiency of plating (no units)
K514 = 1.0x1010 K514 = 1
C600 = 1
HB101 = 1
C600 = 2.4x1010 K514 = 1
C600 = 1
HB101 = 8.5x104 3.5x10-6
HB101 = 1.05x1010 K514 = 1
C600 = 1
HB101 = 1

The efficiency of plating reduced by 10-6 when λHB101 was grown in C600 bacterial strain plate
and only a small number of bacteria produced progeny phages. This clearly confirms that C600
has a restriction and modification system since every bacterium with a restriction system must
have a modification system to protect its DNA from degradation by nucleases. The titre reduced
after introduction of λHB101 in C600 bacterial strain due to degradation of the phage DNA by
the restriction enzymes of the bacterial host. It is clear here that bacterial strain HB101 does not
have a modification system otherwise it could have given the progeny phage the modification to
survive C600 restriction. Consequently, it cannot have a restriction system as well, otherwise it
would destroy its own DNA.However, some of the phage DNA was able to survive due to
acquiring the modification by methylation of the restriction site by C600 as it modified its DNA.

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When λHB101 was introduced in K514 it showed an efficiency of plating of 1 which shows that
it does not have the restriction capability as shown by C600 strain against the same phage.

Finally, when λK514 was introduced in C600 it showed an EOP of 1. This implies that it had a
modification system which prevented degradation of its incoming phage unlike the case of
λHB101. The conclusion is summarized in the table below.

Table 6: showing phenotypes of the three bacterial strains
Restriction Modification
K514 - +
C600 + +
HB101 - -

Immunity of lysogenic bacteria

4.1 Introduction
Bacterial phages in symbiosis or lysogeny with host bacteria give immunity to that host against
lysis by consequence infection by homologous free phages. This implies that lambda phage
participates in its own interference. It does so by the prophage coding for a diffusible protein that
is involved in this interference by blocking the expression of the incoming bacteriophage
genome.

4.2 Objective
To illustrate the phenomenon of bacteriophage immunity, using bacteriophage lambda as a
model.


Table 7: Results of phage immunity test.
Bacteriophage Observation
C600 Plaques
C600(λ) No Plaques
C600(λimm34) Plaques

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Plagues were observed in petri dishes with bacteria strains C600 and C600(λimm34) but not in
C600λ. This is because the lambda prophage already in C600(λ) prevented expression of the
genome of incoming homologous phage. It does so by producing a repressor protein that blocks
the expression. C600 is a wild type and therefore does not contain the lambda prophage while
C600(λimm34) has a mutant lambda phage giving both of the ability to lyse the bacteria since the
repressor protein works on a homologous phage.

The resident lambda phage produces a cI repressor protein that binds to the O L and OR sites of
the super infecting lambda phage as soon as it enters the cell turning off the expression of the
incoming phage gene and hence no plaques result.

Isolation of total bacterial DNA and phage DNA and PCR amplification of shiga toxin gene

5.1 Objective
To isolate bacterial total DNA, perform PCR and calculate its molecular weight of the shiga
toxin genes.

5.2.1 Results of total bacterial DNA
Total bacterial DNA was obtained and PCR amplification of shiga toxin performed and results
were as below.
1λ 2ψ 3c 4 5 6 7 8 9 10 11 12 13 14 15 Pst

Figure 1 showing results of Agarose gel of total bacterial DNA

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Table 8: Relative distances and Molecular weight tabulation of bacteria Stx gene
Migration Distance Relative Distance Molecular weight (bp) Log molecular weight
(cm)
0.9 0.24 11501 4.06
1.1 0.29 5077 3.71
1.6 0.42 2838 3.45
1.9 0.50 2140 3.33
2.1 0.55 1986 3.30
2.4 0.63 1700 3.23

Dye front 3.8cm

4.5 y = -1.8146x + 4.3344
4 R² = 0.8695

3.5
Log of molecular weight

3
2.5
2
1.5
1
0.5
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Relative distance of band migration

Figure 2: Graph of log of molecular weight and relative distance of band migration
Sample relative distance = 0.47

Equation of line y = -1.8146x+4.3344

Substitution = -1.8146(0.47)+4.3624

Antilog = 3.4

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Molecular weight = 2818bp

5.2.2 Results of phage DNA
1λ 2ψ 3c 4 5 6 7 8 9 10 11 12 Pst

Figure 3: Results of Agarose gel of phage DNA

Table 9: Relative distances and molecular weight tabulation of phage Stx gene
Migration Distance Relative Distance Molecular weight (bp) Log molecular weight
(cm)
1.2 0.30 11501 4.06
1.6 0.40 5077 3.71
2.2 0.58 2838 3.45
2.5 0.63 2140 3.33
2.6 0.65 1986 3.30
3.1 0.78 1700 3.23

Dye front = 4cm

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Sample Relative Distance = 0.575

4.5
y = -1.7377x + 4.4807
4 R² = 0.934
3.5
log of molecular weight

3
2.5
2
1.5
1
0.5
0
0 0.2 0.4 0.6 0.8 1
Relative distance of band migration

Figure 4: Graph of log of molecular weight and relative distance of band migration
Equation = y= -1.7377x + 4.4807

Substitution = x = 0.575

Y=-1.7377 (0.575) + 4.4807

Molecular weight = 2960bp.

The PCR analysis showed a visible band at the 933W phage and not at the λ or control well. This
confirmed that the shiga toxin was gene was present in the 933W phage and after integreation
into the bacteria its presence could be located as well by use of forward and reverse PCR primers
specific for it.

In addition using Pst marker with known molecular weight it was possible to confirm the
molecular weight of our shiga toxin gene which was approximately 2930 base pairs.

Transposition of TN10 from phage λ vehicles

6.1 Introduction
Transposable elements are discrete DNA segments that can repeatedly be inserted in the host
genome. The process occurs without DNA sequence homology as contrasted to DNA
recombination. Kleckner, 1981 has classified transposable elements into the classes depending
on DNA sequence homology, structural properties and mechanism of transposition. TN10

14

belongs to class II of transposable element with an accessory determinant of antibiotic resistance
in addition to transposition functions.

6.2 Objective
To show the power of using the transposons to isolate mutants and to subsequently characterize
the mutants specifically in amino acid biosynthesis.

6.3.1 Results and Discussion

Table 10: Number of bacterial colonies after incubation in LB and Mackonkey media
100 10-1 10-2
LB Tet10 222 11 3
Mackonkey 101 9 2

Pale white colonies representing lactose mutants were not observed, however, white and pink
colonies were observed in both LB and Mackonkey media respectively.

Mackonkey as a selective media for enteric bacteria is supplemented with bile salts and high
concentration of sodium chloride. It is suitable for detecting lactose catabolism by noticing the
color change from pink to red. If the lactose catabolism gene is interrupted by a transposon for
example as we anticipated, defective beta-galactosidase enzyme is produced which ferment
lactose and therefore pale color cannot be observed.

6.3.2 Results isolation and identification of amino acid biosynthesis mutants

Table 11: Results in MinA plates supplemented with different amino acids showing
cysteine positive plates.
P1 P2 P3 P4 P5
P6 Cys+ Met- Ala- Lys- Gly-
1,2,3,4,5
P7 His- Leu- Ile- Pro- Val-
P8 Phe- Tyr- Trp- Thr- Gln-
P9 Glu- Asp- Asn- Arg- Ser-

15

After incubating 100 colonies overnight in MinA plates supplemented with different amino acid
mixtures and rich LB media replica , it was observed that some plates had colonies on LB and
not at replica spots in Min A media. Specifically, plate 1 and plate 6 had had five mutants which
correspond to cysteine deficient plates from the table provided. This implies that the transposon
insertion was in the cysteine biosynthetic pathway.

Generalized Transduction: P1 Transduction

7.1 Introduction
Generalized transduction is a process where a bacterial DNA is transferred from one bacterium
to another using a bacteriophage, typically carrying bacteria DNA and not phage DNA. It is the
packaging of the bacterial DNA into the viral capsid and occurs either through headful packaging
or via recombination.

P1 phage moves genetic elements from one E.coli bacteria to another, a technique pioneered by
Nat Sternberg among others. P1vir is a mutant that ensures lysis of bacteria upon infection. Since
P1 packages approximately 90kb DNA, it is possible to include a selectable marker to enhance
recovery of your gene of interest. Once the phage population has been grown on donor bacteria,
it is then then infected to recipient one. Through homologous recombination, the donor DNA can
be incorporated in the recipient genome. To prevent the subsequent phage from lysing the
infected recipient bacteria control of infectivity is necessary and this is achieved by regulating
the calcium in the media since the phage requires calcium for adsorption. A calcium chelator-
citrate is used to lower the amount of calcium to prevent phage adsorption but low enough to
staff the cells of calcium.

7.2 Objective
To use P1 to transduce an antibiotic resistance gene and confirm its location by PCR.

7.3 Results and Discussion
The following tubes were prepared and 0.1ml of each mixture cultured on LB agar supplemented
with chloramphenicol and Na-citrate.

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a) 0.1 ml MG1655 + 0.2 ml of P1 vir lysate

b) 0.1 ml MG1655 + 0.2 ml of P1 vir lysate

c) 0.1 ml MG1655 + 100µl LB with 100mM MgSO4 and 50mM CaCl2

d) 0.2 ml of P1 vir lysates + 100µl LB with 100mM MgSO4 and 50mM CaCl2 (control)

Colonies were observed in both plate A and B each containing 0.1ml of recipient E.coli MG1655
strain and 0.2ml P1vir lysate from donor strain. No growth was observed in plate mixture C and
D. this shows that the chloramphenicol resistance gene was transduced into the recipient E.coli
MG1655 strain.

Yeast agglutination

8.1 Introduction
Escherichia coli have been known to agglutinate erythrocytes for a long time. In addition, they
can as well agglutinate yeast cells, Saccharomyces cerevisiae that are known to contain mannans
on their cell surface and this activity can be inhibited by using D-mannose. Yeast cells has high
mannose chains on their surface which make it possible for the fimbriae of bacteria to bind and
crosslink them.

8.2 Objective
To test the P1 transductants for their capability to agglutinate yeast cells

8.3 Results and Discussion
Agglutination was not observed. However, wild type colonies formed agglutination with the
yeast cells. This is attributed to the fact that they have type 1 fimbriae which bind to mannose
sugar on the surface of yeast cells.

The mutants on the other hand has the clustering gene interrupted by the cat cassette and
therefore cannot produce the type 1 fimbriae important for this agglutination. This explains the
reason why agglutination could not be observed in the P1 transductants MG1655 with yeast
cells.

17

Figure 5: Agglutination was not observed when the bacteria and yeast were incubated on
ice

References
Atsumi S. and JW Little (2006) Role of the lytic repressor in prophage induction of phage
lambda as analyzed by a module-replacement approach. University of Arizona, USA.

Gary E. Kaiser (2011) Doc Kaiser’s Microbiology, The Community College of Baltimore,
Catonsville Campus

Henri De Greve (2012) Virology Manual. Vrije Universiteit Brussel, Belgium.

Rokney A. et all (2008) Host Responses Influence on the Induction of Lamda Prophage, Hebrew
University, Hadassal Medical School, Jerusalem, Israel.

Svenningsen SL et all (2005) On the role of Cro in Lambda prophage induction. National Cancer
Institute, Bethesda, USA.

Yuval E. et all (1981) Participation of Pili and Cell Wall adhesion in the Yeast Agglutination
activity of Escherichia coli, Max-Plank Institute of Immunology, Germany.

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