Construction of a Bacteriophage Genomic Library

Matthieu Kratz
Group 3, Finn Werner
Partner: Muhammad Mohd Nasir

Abstract
In this research project we aimed to construct a DNA genomic library of the
bacteriophage. This was done via the use of the chromosome, the pUC19 plasmid
and a number of enzymes, particularly PSt1, DNA ligase and Alkaline Phosphatase.
These materials, combined with techniques like PAGE and Southern Blotting,
allowed us to digest and visualize both and pUC19 DNA and eventually ligate
these two fragments to create multiple recombinant plasmids which were then
transformed into E.coli and grown on agar-ampicillin plates. The E.coli colonies that
grew contained the pUC19 plasmids, with a certain portion of them containing
pUC19 with a insert. We were able to identify these colonies via blue-white
selection, where a successful insertion produces a white colony due to Lac-Z
inactivation. In the plates 7 to 8, we were able to obtain a relatively high rate of Lac-Z
inactivation (57.5% and 74.7% respectively), insinuating that these two plates
contained a high proportion of colonies with a pUC19 plasmid with a insert.
The inserts in the pUC19 plasmids of select white colonies were ran on a gel and the
WC7 colony was also isolated to produce DIG-labelled gene probes for our Southern
blot. This Southern blot and gel showed that the inserts obtained were 4749 bp or
5077 bp (WC7, 28th fragment or 26th fragment) and 805 bp (WC9, 3rd fragment). The
former contained genes involved in general recombination, as well regulation of lysis
and lysogeny and the latter contained exclusively structural genes.

Introduction
The bacteriophage is part of a group of viruses, known as bacteriophages that
have adapted to infecting bacteria. These phages will enter bacterial cells via a
number of routes, such as by interactions with surface receptors or creation of pores
within the cell wall/membrane. At this point, phages will usually hijack the host cells
machinery, multiply and burst out of the host cell (lysis) or integrate their genome into
the hosts chromosome (lysogeny). From a structural point of view, the virion
particle has an icosahedral head of about 50-60 nm and a tail of around 150 nm [1].
The genome, as with almost all phages, is contained within this head structure.
The icosahedral bacteriophage is a particularly interesting member of this group
due to the fact its target species is the heavily employed E.coli. Additionally, it is a
temperate phage, one capable of both growth strategies, the choice of which, will be
made as function of the phages metabolic/nutritional environment. As a result of its
great importance to research, the control and components of s genome is very well
characterized and hence perfect for our experiment as there is no doubt as to the
number of genes, as well as their positioning and grouping (Figure 1).

All of these gene groups were digested via the use of the aforementioned restriction
enzyme, Pst1 which should produce 29 distinct DNA fragments. These fragments
were then ligated to our pUC19 vector, who prior to this, was treated with Pst1 and
alkaline phosphatase (avoids self-ligation). As a result, all these fragments of the
DNA were inserted into our pUC19 plasmids, which were then transformed to our
host cells, E.coli DH5, which were cultured on ampicillin agar plates.

Figure 1 bacteriophage genetic map [2]. A circular (linear if in virion) genome consisting of 48,514 base pairs,
divided into 27 genes. The genome can be divided by function, creating 7 groups, among them, Recombination
and Integration + Excision. These 2 groups are particularly important in the lysogenic pathway. The remaining
groups, bar Control Region (controls choice of pathway), are mostly important for the lytic pathway.

However, at that final point, we do not know whether or not the E.coli DH5 that
managed to survive contain a pUC19 plasmid with an inserted fragment or merely a
pUC19 plasmid which somehow self-ligated. This was remedied via the use of bluewhite selection, made possible by our pUC19 plasmid containing an insertion site in
the middle of the peptide of the Lac-Z gene. The -peptide produced by pUC19 will
combine with the peptide produced by the E.coli cell to produce the active form of
-galactosidase. Our agar plate contained X-gal, an analogue of lactose, which is
converted to a blue product by -galactosidase (production stimulated by IPTG). As
a result, if an insertion occurs, the Lac-Z peptide loci will be inactivated and hence
X-gal will not be converted into its blue product, producing a white colony. If the
insertion fails or does not occur, the Lac-Z loci on pUC19 will remain intact and
hence active -galactosidase will be produced and a blue product will be formed.

Finally, we had to verify the identity of these insert fragments. The inserts were
identified in a preliminary fashion via isolation of the insert plasmid, digestion with
Pst I and the subsequent running of the sample on a gel. However, the more robust
technique of Southern blotting was also used to identify the insert on one of our two
colonies. In parallel to treating our nucleic acids with Pst1, another batch of the DNA
was treated with Eco RI and Hind III. This parallel batch of DNA was then run on
agarose gel which was subsequently transferred to a nylon membrane which served
as the basis for the Southern blot. The plasmids insert of a select white colony
(WC7) was then used to construct a DNA probe which would hybridize to the
digested DNA on the nylon membrane. As a result, if a gene sequence was
successfully integrated into the pUC19 plasmid and subsequently transformed in
E.coli DH5 cells, we would observe a purple band at the corresponding band on the
membrane. From that, we deduced which Eco R1/Hind III fragment our probe
hybridized to and hence deduce the identity of our Pst 1 fragment.

References
[1]
Hendrix RW, Roberts JW, Stahl FW, and Weisberg RA. Lambda II. Cold Spring
Harbour Laboratory. CSHL press. 1983. 3-12
[2]

Cain A (2015) Bacteriophage Lambda. 9

Results and Discussion

11509 bp
5077 bp
4749 bp
4507 bp
2838 bp
2556 bp
2459 bp
2443 bp
2140 bp
1986 bp
1700 bp
1159 bp
1093 bp
805 bp

Figure 2. Agarose (0.8%) gel electrophoresis of and pUC19 (Week 1 Gel). AGE of both cut and uncut and
pUC19 DNA. Lanes 1 and 2 contain controls, uncut and uncut pUC19 DNA (relaxed and supercoiled) respectively.
Lane 3 contains a DNA marker where the relevant bands are annotated with their corresponding base pairs. Lanes 4,
7, 10, 13 and 15 contain pUC19 DNA that was treated with the restriction endonuclease Pst1 and lanes 5, 8, 11, 14
and 17 contain DNA that was also treated with Pst1.

The purpose of this agarose gel was to verify the proper digestion of the DNA and pUC19
DNA samples by the Pst 1 restriction endonuclease. This was done by comparing the band
patterns and sizes with their respective undigested versions (Lanes 1 and 2).
There is only one Pst 1 restriction site on the pUC19 plasmid, hence proper digestion would
produce a linear fragment. Lane 4 (cut pUC19), contains a fragment of around 2600 base
pairs, which travelled a further distance than the shortest band in Lane 2 (uncut pUC19) but
not as far as the other fragment in Lane 2. This insinuates that we do indeed have the linear
form of pUC19 in lane 4, as the fragment travelled between the relaxed form (shortest band)
and the supercoiled form of pUC19. Thus, Pst 1 has properly digested the pUC19 DNA
sample.
If Pst 1 digested the DNA properly, it would produce 28 individual fragments of DNA.
Unfortunately, we are unable to identify each of these fragments in the gel photo for two
reasons, first of all, some of the fragments produced have very few base pairs (fragment 4,
15bp) and hence run out of the frame. Finally, there are a number of fragments that overlap

due to having similar base pair counts, making it hard to discern individual fragments within
a band. However, it is evident that the sample was digested with a type II endonuclease, as
there are a larger number of discrete bands. Hence, if we assume we used the proper
restriction endonuclease, we can say that Pst 1 functioned properly on the DNA sample.
A quantitative analysis of the lanes containing DNA is possible via the production of a
standard curve (Figure 3) using the DNA marker in the photo. From the line of best fit, we
can construct an equation that can be used to estimate the sizes of each of these fragments
(Equation 1). The calculated sizes using this equation and the expected sizes of these
fragments were quite similar, with all calculated values being within 15% of the expected
values, and the majority being within 10%. A full comparison of the results can be seen
below, in Table 1.

Figure 3. Standard Curve for the AGE containing and pUC19 DNA. Standard Curve created using the DNA
marker on the first gel photo, Figure 2, and is used to calculate base pair number of bands of interest.

Equation 1. Base pair estimation equation. An equation constructed using the data from Figure 2 which allows to
estimate the number of base pairs for a particular band given the migration distance.
Nbp= base pair number
d=migration distance

Table 1. Table comparing the expected fragment size vs calculated fragment size of Pst 1 digested
Genome. Expected size data was found in the appendix of research project booklet [1], and calculated size data
was produced using Equation 1.

Expected Size of
Fragment
(bp)
5077
4749
4507
2838
2556
2459
2443
2140
1986
1700
1159
1093
805

Calculated Size of
Fragment
(bp)
5550
5182
4838
3206
2795
2795
2795
2395
2198
1852
1248
1145
813

Percentage Error
(|sizeex- sizecalc/ sizeex|)
9.32%
9.12%
7.34%
12.97%
9.35%
13.66%
14.41%
11.92%
10.67%
8.94%
7.68%
4.76%
0.99%

Figure 4. Photo of agarose gel (0.8%), containing digested , later transferred to nylon membrane (Week 2
gel). Lane 2 contains DNA that was digested with Hind III and was used as a molecular weight marker
(unusable due to gel red dye causing a malfunction). Lanes 4, 7, 10, 13, 16 contain DNA that was digested with
Eco R1 and lanes 5, 8, 11, 14 and 17 were digested with Mlu 1.

This gel was ran and transferred to a nylon membrane (precursor for Southern blot) and can
be used to verify the proper digestion of the DNA sample by the restriction endonucleases
Eco R1 and Mlu 1. The appearance of the relevant gel lanes is consistent with type II
endonuclease digestion, i.e. multiple, discrete bands of DNA. At an individual level, Mlu 1
should produce quite a few very small fragments (Figure 5) when digesting the genome,
due to having a few cleaving site in close proximity to one and other. As a result, the lanes
digested by Mlu 1 will have a number bands very far from the wells, and close to the edge of
the frame. Eco R1, on the other hand, should produce fragments of similar size, bar the first,
due to having evenly spaced cleavage sites. As a result, the lanes treated with Eco R1 will
contain some medium size fragments that are closely stacked one upon another. These key
characteristics are present in the appropriate lanes, and hence Eco R1 and Mlu 1 properly
digested the DNA.
A more definitive conclusion cannot be reached using the DNA marker, ( DNA digested with
Hind III) as it did not run properly on the gel. As a result, a reliable standard curve cannot be
produced and thus a comparison of the expected base pair values and calculated base pair
values is not possible.

Figure 5. Restriction/Cleavage sites for Eco R1 and Mlu I on the chromosome [2]. Due to lack of space,
the 1st, 6th and 7th fragments when using Mlu 1 are denoted as F1, F6, and F7. F1 = 458 bp, F6 = 956 bp and F7 =
1268 bp. This diagram helps to explain the patterns found in Figure 4 and verify the proper action of the
restriction endonucleases.

Table 2. Results of transformed E.coli DH5 on ampicillin agar plate equipped for blue-white assay. All
samples had been heat shock treated prior to plating. Plates 1-3 had the E.coli cells transformed with undigested
pUC19. Plates 4-6 had E.coli cells transformed with pUC19 that was digested with restriction endonuclease Pst
1, phosphatased and religated. Plates 7-9 contained E.coli cells transformed with pUC19 that was digested by
Pst 1, phosphatased and ligated to Pst 1 treated DNA. Each set of plates had a decreasing concentration of

pUC19 DNA added. The final column is meant to correlate directly with the number of successful fragment
insertion events and subsequent transformations for plate 7-9.
Plate
Number
1

pUC19
amount
(ng)
10

White Colony
Count
(CFU)
30.0

Blue Colony
Count
(CFU)
1200

Colony per mass of

DNA
(CFU per ug of DNA)
123000

Lac-Z Inactivation
Rate
(CFUwhite/ CFUtotal)
2.45%

1.0

0.00

24.00

24000.0

0.00%

0.1

0.00

11.00

111000

0.00%

90

112

112.0

1578.00

21.1%

45

68.0

68.00

1555.00

2.86%

4.5

0.00

0.000

0.00000

0.00%

90

487

360.0

9411.00

57.5%

45

218

74.00

6489.00

74.7%

4.5

0.00

0.000

0.00000

0.00%

This table is supposed to allow us to gauge how many of the E.coli DH5 contained pUC19
plasmid with a insert and how efficient our cells were at taking up pUC19 DNA.
The first 3 plates are meant to act as controls, essentially showing us what the natural value
of Lac-Z inactivation should be for untreated pUC19. Theoretically, it should be 0%, but in
practice (Plate 1), Lac-Z inactivation may not always happen via gene insertion. This means
that just because a white colony is produced (Lac-Z inactivation), that does not necessarily
mean that it is a colony with a insert i.e. Lac-Z inactivation n insertion. These plates also
show that our E.coli cells are taking up this uncut supercoiled pUC19 plasmid, and hence
that the heat shock treatment has functioned properly.
Plate 4 shows a very high rate of Lac-Z inactivation, something which may be due to the
increase treatment and manipulation of the plasmid. However, the value is just too high,
particularly when compared to plate 5, and thus this is potentially the result of a major
counting error or contamination. Plate 5 shows a Lac-Z inactivation rate very similar to plate
1, reinforcing the theory that there is indeed a natural rate of Lac-Z inactivation in this
process. Using the data, from Plate 1, 2 and 5 we can tentatively say that natural rate of LacZ inactivation (NRLI) varies between 0 and 3%. Plates 3, 4 and 6 are disregarded in this
calculation due to lack of overall growth or lack of reliability. These plates have a notably
lower colony per unit mass of DNA value than the previous plates. This is most likely the
result of the pUC19 being in an open circular form, as opposed to a supercoiled form,
making it harder for the pUC19 to diffuse through pores created by the divalent cations.
Plates 6-9 are the samples of real interest, as they contain E.Coli DH5 that were
transformed with pUC19 that potentially contained inserts. We can see a very high rate of
Lac-Z inactivation (Table 2), insinuating that a very large amount of the colonies on these
plates (bar Plate 9) contain a pUC19 plasmid with a insert. Considering, the previously
calculated NRLI, we can tentatively estimate the rate of successful insertion to be 53.557.5% (plate 8) and 71.7-74.7% (plate 9). As a whole, the expected results were obtained
(bar plate 4), i.e. very low rates of Lac-Z inactivation for plates 1-6 and much higher rates for
plates 7-9. The relatively low colony per unit mass of DNA is present for the very same
reasons as in plates 4, 5 and 6.

Figure 6. Agarose gel of isolated plasmids digested with Pst 1 (Week 3 gel). Lane 1 contains the DNA
marker, lane 2 contains pUC19 digested with Pst 1 (no insert). Lanes 9, 10 and 11 are the lanes that will be
analysed, lane 9 contains digested pUC19 isolated from a blue colony. Lanes 10 and 11 contain digested
pUC19 from the WC7 sample and WC9 sample, respectively. (Group 6 data was used as our data was not
viable for analysis)

The purpose of this gel was to have a preliminary estimation of the size of the insert in
pUC19 plasmids of the selected white colonies. Unlike our Week 2 gel, this gels marker ran
perfectly and as a result, a standard curve (Figure 7) has been produced from the marker
bands.
Using Equation 2, the insert is estimated to be around 4730 bp for lane 10 (WC7) and 806
bp for lane 11 (WC9). By cross-referencing these sizes with a list of all fragments produced
during Pst 1 digestion, we can predict which of the fragments correspond to our inserts.
The lane 10 insert most likely corresponds to the 28th fragment (32252 bp-37001 bp, 4749
bp), however it is also possible that it corresponds to the 26th fragment (26928 bp- 32005
bp, 5077 bp), due to the smearing of the band. The lane 11 insert seems to correspond to
the 3rd fragment (2820 bp-3625 bp, 805 bp).

Figure 7. Standard Curve for Week 3 Gel. Standard curve created using the DNA marker (lane 1) in Figure 6,
the line of best fit was used to derive the Equation 2.

Equation 2. Base pair estimation equation. An equation constructed using the data from Figure 7 and allows to
estimate the number of base pairs for a particular band, given the migration distance.

Figure 8. Southern Blot using WC7 insert as probe (Week 4 Blot). Lane 1 contains the DNA marker, which
did not run correctly, lanes 3 and 6 contain probes that hybridized to Eco R1 digested genome. Lanes 4 and 7
contain probes that hybridized to Mlu 1 digested genome. One must keep in mind that the insert, and hence the
probe, used in Lane 3 and 6 as well as in lane 4 and 7 are (most likely) not the same sequence. Lanes 6 and 7
will be analysed below.

This Southern blot was done to verify the identity of our insert fragment in our WC7 colony
sample. Unfortunately, the marker did not run correctly, so estimation of sizes will be done by
comparing migration distance with the week 2 gel. This is possible as we assume that the
genome was correctly digested by Eco R1 and Mlu 1, and hence we can tell which
hybridized bands correspond to which fragments using page 48 in our handbook.
Using this, we are able to see that our WC7 insert has hybridized to the 4th fragment of the
Eco R1 digest (31747 bp-39168 bp) and to the 1st fragment of the Mlu 1 digest (22220 bp48510 bp). Hence, the zone from which the insert in WC7 comes from must be between
31747 bp and 39168 bp regions of the genome. There are 4 possible fragments in that
zone, fragments of size 5077 bp, 247 bp, 4749 bp or 11509 bp. The analysis from the week
3 gel revealed that our WC7 insert should be around 4730 bp, immediately eliminating 247
bp and 11509 bp fragments as inserts. Thus, the WC7 insert is either the 5077 bp (26th
fragment) or the 4749 bp (28th fragment) with the latter seeming more likely.

The only colony that was used to create the probe for the Southern blot was the WC7
colony, however discussion about the WC9 insert is still possible due to the Week 3 Gel.
From the information in the week 3 gel and week 4 Southern blot, we can look at what genes
of the genome are present in the insert fragments of both white colonies via the use of a
detailed genome map (Figure 9)

Figure 9. A) Detailed genome map of bacteriophage [3]. The genome map has been cropped to only include
the regions of the genome which our WC7 (32.252 kbp-37.001 kbp or 26.928 kbp-32.005 kbp) and WC9 (2.820
kbp-3.625 kbp) inserts span.
B) Structure of head. A diagram of bacteriophage , showing which structural genes in the map corresponds to
which head/tail structure.

The WC7 colony possibly contained a fragment spanning a region from 32252 bp-37001 bp
in the genome, this large region most notably includes: the PL promoter, the N-anti
termination factor gene as well as a number of smaller genes involved in general
recombination. The N-anti-terminator gene is undoubtedly one of the most important genes
contained in the region, considering its major role in allowing to extend the transcript from
both PL and PR, thus causing expression secondary regulatory factors as well as genes
involved in replication. Additionally, our region is just a few 100 base pairs away from the CI
gene which codes for the very important repressor that plays a key role in maintaining
lysogeny and repressing lysis [4].
However, it also possible that our WC7 insert contained a fragment spanning a region from
26928 bp-32005 bp. This region includes all the genes involved in site specific
recombination, such as the int (integrase endonuclease system) and xis (recognizes hybrid
excision site) loci, both which code for gene products important for viral genome integration
and excision. Additionally, this region encompasses the Ea region of the genome, which
codes for proteins that seem to be involved in either binding directly to nucleic acids or
interacting with other viral and host proteins [5].
The WC9 contained a fragment spanning from 2820 bp-3625 bp region of the genome, a
comparatively much smaller region than the possible WC7 inserts. This region contains a

very small amount the W gene and about half of the B gene, both of which are part of the
icosahedral head structure (Figure 9.B). More precisely, the protein encoded by the W gene
is heavily involved in the morphogenesis of the head, and seems to be part of the interface
between the head and tail structure [6]. The protein encoded on the B gene is directly
connected to the W gene in the head structure, and it is involved in the packaging of the
genome (binds to subunits of the terminase enzyme system), head assembly and
connecting the head and tail proteins [7] [8].
Although we did experience quite a few malfunctions with the Gel Red dye and our week 3
gel, we were generally able to work around these obstacles, and produce viable data. This
data allowed us to determine the identity of the fragments within our selected clones (WC7
and WC9) from our transformation experiment. The WC7 colony contained two possible
fragments (26th and 28th), unfortunately indistinguishable without further testing due to similar
base pair counts. The WC9 colony was not southern blotted but via use of the week 3 gel,
we were able to determine the insert to be the 3rd fragment of Pst 1 digested genome.
These 3 inserts had an interesting variety of genes, with some coding for very important
proteins like the N-anti termination factor, xis, and others coding for somewhat obscure
proteins such as Ea 8.5. All in all, this research project was a great success as we are able
to successfully create a genomic library, and identify one or more fragments from selected
clones.

References
(1)

BIOC2001 Lab Practical Manual. Appendix. 48

(2)

BIOC2001 Lab Practical Manual. Appendix. 46

(3)
Rajagopala SV, Casjens S, Uetz P (2011) The protein interaction map of
bacteriophage lambda. BMC Microbiology.11:213
(4)

Cain A (2015) Bacteriophage Lambda. 16-21

(5)
Kwan JJ, Smirnova E, Khazai S, Evanics F, Maxwell KL, and Donaldson LW (2013)
The Solution Structures of Two Prophage Homologues of the Bacteriophage Ea8.5 Protein
Reveal a Newly Discovered Hybrid Homeodomain/Zinc-Finger Fold. Biochemistry. 52: 36123614
(6)
Murialdo H, Xing X, Tzamtzis D, Haddad A and Gold M (2003) The product of the
bacteriophage lambda W gene: purification and properties. Biochemistry and Cell Biology.
81: 307-315
(7)
Kochan J, Carrascosa JL and Murialdo H (1984) Bacteriophage lambda
preconnectors. Purification and structure. J. of Molecular Biology. 174: 433-437
(8)
Kochan J and Murialdo H (1983) Early intermediates in bacteriophage lambda
prohead assembly. II. Identification of biologically active intermediates. Virology. 131: 100115