This was a research paper I wrote for my Integrated Laboratory Techniques in Biological Sciences II course. Evaluation of the lacZ gene in Escherichia coli mutagenesis using pBluescript and pTn5: Km vectors

1. 1
Evaluation of the lacZ gene in Escherichia coli mutagenesis using pBluescript and pTn5:
Km vectors
Emilio Solomon
5580132
Abstract
The lacZ gene in Escherichia coli was used for mutagenesis using pBluescript and pTn5:
Km vectors to evaluate its functionality and the activity of β-D-galactosidase, its encoded
enzyme. Microbiological and molecular lab protocols were performed, including medium
preparation, competent cell preparation, transposition, transformation, screening, alkaline lysis,
restriction endonuclease digestion, and gel electrophoresis. Medium preparation, competent cell
preparation, transposition, transformation, and screening were conducted to evaluate the lacZ
gene’s functionality and the activity of β-D-galactosidase. Total loss of lacZ gene function and a
non-functional β-D-galactosidase were observed. Alkaline lysis, restriction endonuclease
digestion, and gel electrophoresis were used to determine the size of the DNA fragments in
recombinant plasmid; pBluescript and pTn5: Km and its non-recombinant counterpart;
pBluescript, as well as to map restriction sites in both recombinant and non-recombinant
plasmids. Proposed restriction maps corresponded with the DNA fragment sizes from gel
electrophoresis and were validated by the concentration of agarose used.
1. Introduction
Transposons are transposable elements that are capable of undergoing translocation
within chromosomal, phage, or plasmid DNA. There are three classes of transposons, including
Class I elements, Class II elements, and Helitrons. Class I elements are known as
retrotransposons. These transposons transpose via a copy and paste mechanism. In this copy and
paste mechanism, the mRNA transcribed from RNA polymerase II is converted into cDNA by
reverse transcription and then integrated at a new position in the genome. Class I elements can
further be sub-divided into long terminal repeats (LTR) and non-LTR elements. Both differ in
the mechanism of integration. Long terminal repeats encode all of the necessary proteins for

2. 2
transposition. Non-LTR elements require enzymes, encoded by LTR elements. Class II elements
transpose via a cut and paste mechanism. In the cut and paste mechanism, the element excised
from the chromosome is reintegrated at a new location. This process involves a transposase
enzyme encoded by the transposon. Helitrons are transposons that transpose via a rolling circle
mechanism. This process involves nicking at the Helitron terminus, followed by strand invasion,
DNA synthesis, strand displacement, and the resolution of a heteroduplex by DNA replication.
Its rolling circle mechanism is regulated through flanking. Flanking occurs when DNA synthesis
and strand displacement proceeds farther than the end of the Helitron.
The lacZ gene is a gene that encodes for the enzyme, β-D-galactosidase. This enzyme is
responsible for hydrolyzing lactose into galactose and glucose. The lacZ gene is regulated in the
lac operon. For example, when the lac operon is turned on, the lacZ gene will facilitate the
hydrolysis of lactose into galactose and glucose. When the lac operon is turned off, the lacZ gene
will not be activated. This will only occur when glucose is present in high concentrations in cells.
The lacZ gene is used for a variety of purposes, according to various journals. According to a
journal from FEMS Microbiology Letters, the lacZ gene can be used for the characterization and
expression of genes from Yersinia pestis and Escherichia coli (Bobrov & Perry, 2006). The lacZ
gene can also be used for cloning PCR-amplified gene promoters on antibiotic resistant plasmids
in the lac operon (Datsenko & Wanner, 2000). According to a journal from Gene Expression
Patterns, the lacZ gene can be used for investigating the functions of Dapper antagonist of
catenin-1 (Dact1) in Wnt-mediated organogenesis and tissue homeostasis in mice (Suzuki, Leu,
Brice, & Senoo, 2014). In humans, the lacZ gene can be used to study Tem1 ontogeny (Huang et
al., 2011). Researchers from Mutation Research/Genetic Toxicology and Environmental
Mutagenesis have used the lacZ gene in order to detect mutagens and clastogens in mice
(Mahabir et al., 2008).
X-gal, or 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside is an analogue of lactose. β-
D-galactosidase is also capable of digesting X-gal, however the product after hydrolysis is
different. X-gal is digested into galactose and 5-bromo-4-chloro-3-hydroxyindole. The
byproducts can further be dimerized and oxidized into 5, 5’ –dibromo- 4, 4’-dichloroindigo. 5,
5’-dibromo- 4, 4’- dichloroindigo would appear as an insoluble, blue product. Escherichia coli
(E. coli) is a gram-negative, facultative anaerobic bacterium commonly found in the intestine of

3. 3
warm-blooded organisms, including humans. It is capable of growing rapidly and is able to grow
with or without the presence of oxygen. Its genome is well understood by scientists and is often
used as a host for cell culturing and molecular cloning. E. coli contains genes, including the lacZ
gene in the form of plasmid DNA. Plasmid DNA in E. coli contains restriction sites, where
restriction enzyme digestion can occur. Restriction enzyme digestion is facilitated through
restriction enzymes, restriction endonucleases. Many restriction endonucleases, including EcoRI,
HinDIII, and BamHI are capable of digesting the plasmid DNA at various restriction sites. These
restriction sites contain specific segments of nucleotide bases.
This research investigation aims to evaluate the lacZ gene in Escherichia coli
mutagenesis using vectors pBluescript (pGEM), containing the lacZ gene and pTn5: Km
(pMOD). Parameters such as the functionality of the lacZ gene and β-D-galactosidase activity
will be investigated. The research investigation also aims to determine the restriction sites in
pBluescript with or without pTn5: Km. The experiment will involve the use of Luria-Bertani
(LB), a medium present in complex and chemically-defined forms. The complex medium
appears as a liquid, composing of Peptone, yeast extract, and sodium chloride, NaCl. Peptone
acts as a protein source, which will provide amino acids and peptides to E. coli. Yeast extract
contains vitamins, minerals, and other nutrients, acting as a carbon source. NaCl in LB helps in
providing sodium and chlorine ions. The chemically-defined medium appears as a solid. This
medium is composed of the broth, with the addition of agar. The agar helps in solidifying the
medium. Both complex (broth) and chemically defined (agar) media will be prepared. The
prepared agar and broth will then be used for making competent cells. Competent cells will be
made using techniques such as cell suspension, centrifugation, and incubation. Cell suspension
will help increase cell competence. Centrifugation will help separate the pellet (protein) from the
supernatant (DNA) in the competent cells. Incubation will help maintain the competent cells
under optimal conditions. Once the competent cells are made, the cells will be transferred into
agar plates containing ampicillin only. These competent cells will be spread through the agar to
distribute cells evenly. The spreading of competent cells will be done aseptically to avoid
contamination. Ampicillin agar plates, containing competent E. coli cells will be treated with X-
gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and reaction mixture containing
reaction buffer, vectors pBluescript (pGEM) and pTn5: Km (pMOD), enzyme transposase, and
sterile water for the transposition reaction, followed by transformation. Transformation will

4. 4
result in either the formation of blue colonies, white colonies, or both. Blue colonies will
demonstrate β-D-galactosidase activity, while white colonies will not show any β-D-
galactosidase activity. After transformation, only the white colonies will be selected for
screening. The screening process will help determine the functionality of the lacZ gene.
Ampicillin and ampicillin/kanamycin agar plates will be used for screening. Blue and white
colonies will be observed following the screening process. Blue colonies will indicate a
functional lacZ gene whereas white colonies will indicate a non-functional lacZ gene. The
frequency of transposition, which is defined as the number of colonies after transformation over
the number of colonies before transformation will help determine the extent of lacZ gene
functionality. Techniques such as alkaline lysis and restriction enzyme digestion, will be
employed subsequently in order to extract and purify the plasmid DNA of E. coli and cut the
plasmid DNA/determine the site of insertion of pTn5: Km vector, respectively. Alkaline lysis
will be performed using suspension, lysis, neutralizing, and alcohol solutions. Finally, the
plasmid DNA will be separated through gel electrophoresis using 1% agarose, Tris-base EDTA
buffer, purple tracking dye, and fluorescein staining solution. Tris-base EDTA buffer will help
maintain the ionization of the DNA. Purple tracking-dye will help make the DNA denser. This
will help in visualizing the separated DNA. Fluorescein staining solution will help in visualizing
the DNA under UV light. The sizes of the DNA fragments will be determined and used to map
restriction sites in pBluescript, with or without pTn5: Km insert.
2. Materials and Methods
2.1 Chemicals/solutions
 0.2 M NaOH
 1% (w/v) SDS
 100x BSA
 10x buffer
 15% sucrose
 70% ethanol
 Acetic acid
 Agar
 Agarose powder
 Ampicillin
 Calcium chloride
 Distilled water
 EDTA
 Ethanol
 EZ- Tn5 reaction buffer
 Fluorescein solution

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 Glycerol
 Ice
 Isopropanol
 Kanamycin
 Manganese (II) chloride
 Milli-Q water
 pBluescript
 Peptone
 Potassium acetate
 pTn5: Km
 Restriction enzyme (EcoRI,
BamHI)
 Sodium acetate
 Sodium chloride (NaCl)
 Stop solution
 SYBR®
 Transposase
 Tris-base EDTA (TBE)
buffer
 Tris-HCl
 X-gal (5-bromo-4-chloro-3-
indolyl-β-D-galacto-pyranoside)
 Yeast extract
2.2 Devices/machines and miscellaneous materials
 Aliquot
 Autoclave
 Beaker
 Burner
 Centrifuge
 Eppendorf tube
 Erlenmeyer flask
 Horizontal electrophoresis
chamber
 Incubator
 Petri dish
 Pipettes
 Refrigerator
 Spreader
 Tip
 Toothpick
 UV trans illuminator
2.3.1 Identification of an unknown sequence
Using the partial DNA sequence and partial amino acid sequence of an unknown gene, a
BLAST search was performed. Through the BLAST search, the unknown gene of the partial
DNA sequence and partial amino acid sequence was identified. Information on the name of the
unknown gene, host organism, protein encoded by the gene, metabolic reaction, and the use of
the gene in molecular cloning were obtained.

6. 6
2.3.2 Medium preparation
Luria-Bertani (LB) broth and agar were prepared. 100 ml of LB broth was prepared using
1 gm of Peptone, 0.5 gm of Yeast extract, 0.5 gm of sodium chloride (NaCl), and 100 ml of
distilled water. 30 ml and 3 ml of LB broth were then aliquoted into a flask and plastic tubes,
respectively. The medium was then autoclaved. LB agar was made using LB broth and agar and
transferred to bottles. Antibiotics, ampicillin and kanamycin were added into the bottles,
containing LB agar. Ampicillin was added to one bottle to a final concentration of 100 μg/ml.
Both ampicillin and kanamycin were added to another bottle. Ampicillin was added to a
concentration of 100 μg/ml, followed by the addition of kanamycin to a final concentration of 50
μg/ml. LB agar with Amp and LB agar with Amp/Kan were then poured into their respective agar plates
and were left to solidify.
2.3.3 Competent cell preparation
Starter cultures were grown in 5 ml of LB broth until the stationary phase was achieved.
0.3 ml of starter cultures were then transferred into pre-warmed (37° C) 30-ml LB broths. Broths
were then grown at 37° C, followed by shaking for 2.5 hrs. Shaking was done until OD600 of 0.3-
0.4 was achieved. After the growth and shaking of culture, culture was poured carefully into a 50
ml centrifuge tube. The tube was chilled on ice for 15 minutes and cells were centrifuged for 10
min at 7520 ×g and 4° C for harvest. The supernatant was discarded into a special disposal bin
and the pellet was resuspended into culture medium containing 10 mM of sodium acetate,
CH3COONa (pH= 5.6), 50 mM of manganese (II) chloride MnCl2, and 5 mM of sodium
chloride, NaCl. The mixture was then incubated on ice for 20 min and centrifuged at 7520 ×g, 4°
C for 10 min. The supernatant was discarded and the pellet was resuspended in culture medium
containing 10 mM of CH3COONa (pH= 5.0), 70 mM of CaCl2, 5 mM of MnCl2, and 5%
glycerol. The mixture was then incubated on ice for at least 30 minutes, but no more than 1 hour.
Meanwhile, five Eppendorf tubes were prepared and labelled. Once cold incubation was done,
100 μl of the mixture, containing the competent cells were transferred into 1.5 ml Eppendorf
tubes (with labels). This was done aseptically. The mixture was then stored at -80° C.

7. 7
2.3.4 Transposition
10 µl of reaction mixtures were prepared for the transposition reaction. One reaction
mixture using 1 μl of 10x EZ- Tn5 reaction buffer, 1 μl of pBluescript (pGEM), 1 µl of pTn5:
Km (pMOD), 1 μl of transposase, and 6 µl of distilled water. One reaction mixture (without
pMOD) containing 1 μl of 10x EZ- Tn5 reaction buffer, 1 μl of pGEM, 1 μl of transposase, and
7 μl of distilled water. One reaction mixture (without transposase) containing 1 μl of 10x EZ-
Tn5 reaction buffer, 1 μl of pGEM, 1 μl of pMOD, and 7 µl of distilled water. Reaction mixtures
were then incubated at 37° C for 2 hrs. Stop solution was then added into each mixture and
further incubated at 70° C for 10 min. Reaction mixtures were then transferred into the
Eppendorf tubes (containing competent cells) and incubated on ice for 30 min. Tubes containing
competent cells and transposition reaction mixture were transferred into new, empty Eppendorf
tubes. Tubes were then placed in a water bath at 42° C and incubated for exactly 45 seconds.
Tubes were transferred and placed in a cold bath, for 30 min. After incubation, 1 ml of LB broth
was added into each tube and resuspended. 300 µl, 100 µl, 50 µl, and 5 µl of suspension were
added into 4 ampicillin-LB agar plates. 300 µl of reaction mixture without pMOD and 300 µl of
reaction mixture without transposase were added into their respective ampicillin-LB agar plates.
All agar plates were incubated for 24 hrs for the transposition reaction to occur. Transposition
was repeated using two reaction mixtures; one with 1 µl of reaction buffer, 1 µl of pGEM, 1 µl
of pMOD, 1 µl of transposase, and 6 µl of distilled water and another one with 1 µl of reaction
buffer, 1 µl of pMOD, 1 µl of transposase, and 7 µl of distilled water, if no growth was observed
at all during the transformation process. For the protocol repeat, only two amp agar plates with
100 µl of reaction mixture were used.
2.3.5 Transformation/Screening
After transposition, white colonies were selected for transformation. Prior to
transformation, ampicillin agar plates were gridded into 50 regions. Selected white colonies were
inoculated/transferred to one region in the ampicillin-LB agar plates (repeat protocol) one by one
using toothpicks aseptically. Ampicillin-LB agar plates containing the colonies were then,
incubated for 24 hrs for growth. Following incubation, only white colonies were selected and
transferred to two gridded LB agar plates (1 ampicillin-LB, 1 ampicillin/kanamycin- LB) using

8. 8
toothpicks aseptically. Both agar plates were incubated for 24 hrs for growth. Clones from only
amp/kan agar plates were then inoculated in 3 ml LB broth using a pipette aseptically. The tip
was left inside the tube with LB broth containing ampicillin and kanamycin and incubated for 24
hrs for growth. Cells were then, harvested by centrifugation for alkaline lysis.
2.3.6 Alkaline lysis
The supernatant was discarded and the pellet was resuspended in 250 µl of suspension
solution containing 25 mM Tris-HCl (pH= 8.0), 10 mM EDTA (pH= 8.0), and 15% to create
pores in the cell membrane, as well as maintain the cell’s osmolarity. 250 µl of lysis solution
containing 0.2 M sodium hydroxide (NaOH) and 1% (w/v) sodium dodecyl sulfate (SDS) was
then applied. The tube was then inverted for cell lysis. This helped in separating the DNA from
protein. Once the cells have been lysed, 350 µl of neutralizing solution containing 2 M potassium
acetate (CH3COOK) and 1 M acetic acid (CH3COOH) was added to prevent further lysis. This
was followed by tube inverting and centrifugation to reanneal the lysed DNA. The supernatant
was then transferred into a new tube and added with 500 µl of isopropanol. Tube was inverted
and centrifuged for 5 minutes. The supernatant was discarded and the pellet was resuspended in
500 µl of 70% ethanol and centrifuged for 2 minutes. The addition of isopropanol and 70%
ethanol helped in precipitating the DNA. The supernatant was discarded and the pellet was air
dried over paper towels at room temperature for approximately 30 min. The pellet was then
resuspended in 50 µl of sterile Milli-Q water for purification.
2.3.7 Restriction enzyme digestion
10 to 15 µl of purified plasmid DNA was mixed with 5 µl of 10x buffer, 0.5 µl of 100x
BSA, 1 µl of restriction enzyme in a new, empty Eppendorf tube. Milli-Q water was added to a
total volume of 50 µl. Mixture was then incubated for 24 hrs for restriction enzyme digestion to
occur.

9. 9
2.3.8 Gel electrophoresis
5 µl of digested DNA was aliquoted into 5 new, empty Eppendorf tubes each. 1 µl of 6x
purple loading dye was added into all 5 tubes. Meanwhile, 1% agarose with Tris-base EDTA
(TBE) buffer was prepared, using 0.2 gm of agarose powder and 10 ml of TBE buffer. The
mixture was then poured into a well to solidify into a gel-like form. Once solidified, the gel was
transferred to a horizontal electrophoresis chamber containing TBE buffer. The horizontal
electrophoresis chamber was then powered with electricity and left for 30 min for the DNA to
migrate/separate. Gel electrophoresis results were analyzed and restriction maps were proposed.
Proposed restriction maps were checked for validation using the concentration of agarose used.
3. Results
3.1 Unknown sequence
After the BLAST search was performed using the given partial DNA and partial amino
acid sequences, the name of the unknown gene, as well as information about its host organism,
protein encoded, metabolic reaction, and uses in molecular cloning were obtained. The name of
the unknown gene is the lacZ gene. This gene encodes for the enzyme, β-D-galactosidase, an
enzyme that catalyzes the hydrolysis of lactose into galactose and glucose (Formula 3.1.1). The
lacZ gene is found in a variety of warm-blooded organisms, including humans. The gene is
commonly used in the molecular cloning of E. coli.
Formula 3.1.1 Enzymatic reaction
lactose
β-D-galactosidase
→ galactose + glucose
3.2 Transformation/screening
White colonies were transformed using Amp agar and screened using both Amp and
Amp/Kan agar to determine the functionality of the lacZ gene.

10. 10
Table 3.2.1 Number of colonies in transposition reaction mixtures
Colony
1 2 3
300 µl 100 µl 50 µl 5
µl
300
µl
300
µl
White 0 0 0 0 0 0
Blue 0 0 0 0 0 0
Table 3.2.1 presents the number of colonies observed after transposition. Colonies,
including white and blue colonies were not observed in any of the agar plates, including 1 (EZ-
Tn5 reaction buffer, pGEM, pMOD: Km, transposase) and controls 2 (EZ- Tn5 reaction buffer,
pGEM, transposase), and 3 (EZ- Tn5 reaction buffer, pGEM, pMOD: Km). Results
demonstrated that transformation did not occur with, nor without transposition. This indicated
poor execution of the transposition reaction protocol. The protocol was repeated using two amp
agar plates (Table 3.2.2).
Table 3.2.2 Number of white and blue colonies in ampicillin agar plates
Agar (Amp) Number of white
colonies
Number of blue
colonies
1 807 0
2 807 0
Table 3.2.2 presents the number of white and blue colonies, observed in ampicillin agar
plates. Only white colonies were observed in both samples, with 807 colonies present in both.
Blue colonies were not observed. The sole presence of white colonies in both ampicillin agar
plates denoted the total loss of function of the lacZ gene, following transformation. The sole
presence of white colonies also indicated a non-functional β-D-galactosidase.

11. 11
Table 3.2.3 Number of colonies in Amp and Amp/Kan agar plates (n= 200)
Agar Plate Number of white
colonies
Number of
blue colonies
Amp 1 50 0
2 50 0
Amp/Kan 1 0 0
2 0 0
Table 3.2.3 shows the number of colonies in amp and amp/kan agar plates after
screening. Only white colonies were observed in amp plates. There were 100 white colonies in
both ampicillin plates, with 50 colonies in both. Blue colonies were not observed in both
ampicillin plates, however. In other words, the 50 white colonies observed in both amp plates
were able to grow in the presence of ampicillin only (ampicillin resistance), but demonstrated
indigestion of X-gal. On the other hand, both white and blue colonies were not observed in
amp/kan plates. Both plates had 0 white colonies and 0 blue colonies. This indicated that
colonies were not able to grow in the presence of amp/kan, making the digestion of X-gal non-
existent.
Table 3.2.4 Frequency of transposition between Amp and Amp/Kan agars
Agar (Amp + Amp/Kan) Frequency of Transposition
1 0
2 0
Using the results from Table 3.2.4, the frequency of transposition was calculated for both
sets of amp and amp/kan agar plates using Formula 3.2.1. Both sets had a frequency of
transposition of 0, indicating that all of the colonies grown in the presence of ampicillin were not
able to grow in the presence of ampicillin with kanamycin. In other words, transformation did
not occur.
Formula 3.2.1 Frequency of transposition

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Frequency of transposition =
# of colonies on Amp, Kan agar
# of colonies on Amp
3.3 Gel electrophoresis
Gel electrophoresis was employed in order to separate the DNA into fragments for size
determination. Prior to gel electrophoresis, DNA was extracted/purified and cut during alkaline
lysis and restriction enzyme digestion protocols, respectively.
Figure 3.3.1 DNA fragments after gel electrophoresis
Figure 3.3.2 1 kb DNA ladder (GeneRuler™)
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
M= DNA size marker
Well
Size of
marker
(bp)
4000
2000
1500
1000

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Figure 3.3.1 displays the DNA fragments after gel electrophoresis. The sizes of the DNA
fragments (bp) in wells 14 (pBluescript), 15 (pBluescript + pTn5: Km), and 16 (control) (Figure
3.3.1) were determined using the sizes from the DNA marker (bp); 1 kb DNA ladder (Figure
3.3.2), the distance of migration (cm), and the logarithm of fragment size from DNA marker
(Table 3.3.1). The distance of migration was plotted with the log of the DNA marker fragment
size and the linear equation was obtained from the line of best fit of the graph (Figure 3.3.2).
The linear equation was then used to estimate the size of the DNA fragments, observed after gel
electrophoresis (Table 3.3.2).
Figure 3.3.3 Migration distance vs Log (fragment size)
f(x) = -0.2824x + 3.9404
R² = 0.9039
2.50
2.70
2.90
3.10
3.30
3.50
3.70
1.00 1.50 2.00 2.50 3.00 3.50 4.00
Log(fragmentsize)
Distance(cm)
Migration distance vs Log(fragmentsize)

14. 14
Table 3.3.1 Estimated sizes of the DNA fragments (kb)
DNA
ladder
fragment
sizes (bp)
Distance of
migration
(cm)
Log(DNA
ladder fragment
sizes)
Size of
fragments (bp)
Estimated size
of fragments
(kb)
4000
1.50
3.60
3287
3.0
2000
2.00
3.30
2374
2.0
1500
2.50
3.18
1716
2.0
1000
3.50
3.00
895
1.0
Table 3.3.2 Estimated sizes of plasmids
Plasmid Size (bp)
Estimated size
(kb)
pBluescript +
pTn5: Km 5661
5.0
pBluescript 2611 3.0
The sizes of fragments (Table 3.3.3) were used to determine the size of plasmids
pBluescript with and without pTn5: Km insert (Table 3.3.4). Using the sizes of pBluescript with
or without insert, restriction maps were deduced.
1.0 kb
pBluescript + pTn5:
Km (5.0 kb)
BamHI
EcoRI
EcoRI
pTn5: Km (2.0 kb)
pBluescript (3.0 kb)
pBluescript (3.0 kb)
BamHIEcoRI
2.0 kb

15. 15
Figure 3.3.4 Restriction maps of pBluescript + pTn5: Km and pBluescript
Figure 3.3.4 shows the restriction maps of pBluescript with insert (pBluescript + pTn5:
Km) and without insert (pBluescript). In first restriction map, pBluescript and pTn5: Km were
present. It is known that the size of the recombinant plasmid is approximately 5.0 kb (Table
3.3.2). It is also known that the size of pBluescript is approximately 3.0 kb. This means that the
size of pTn5: Km is approximately 2.0 kb. Since the restriction map included pBluescript and
pTn5: Km, this suggested that two restriction enzymes were involved in digesting the plasmid.
Those enzymes included EcoRI and BamHI. EcoRI digested the plasmid at two restriction sites,
whereas BamHI digested the plasmid at only one restriction site. In the second restriction map,
pBluescript was present only. The plasmid without insert is approximately 3.0 kb, suggesting
that EcoRI and BamHI cut at one restriction site only. The restriction maps in Figure 3.3.4
corresponded with the results from Tables 3.3.1 and 3.3.2, as well as Figure 3.3.1.
Figure 3.3.5 Recognition sites of various restriction enzymes in E. coli
Figure 3.3.5 shows the recognition sites of various restriction enzymes capable of
digesting E. coli. Since only EcoRI and BamHI were used, the recognition sites of these
restriction enzymes were sought. According to Figure 3.3.5, EcoRI digested between A and G
(5’ to 3’) and BamHI digested between G and G (5’ to 3’) in E. coli, resulting in a sticky end
cleavage patterns.

16. 16
Table 3.3.3 Percent agarose (w/v) and their DNA resolution size
Percent agarose (w/v) DNA resolution size (1 kb = 1000 bp)
0.5 1 – 30 kb
0.7 800 bp – 12 kb
1.0 500 bp – 10 kb
1.2 400 bp – 7 kb
1.5 200 bp – 3 kb
2.0 50 bp – 2 kb
Table 3.3.3 displays the concentration of agarose used and their uses in specific DNA
sizes. Since 1.0% agarose was used, the DNA should have ranged from 500 bp- 10 kb. The
estimated sizes of both plasmids corresponded with the DNA resolution size of 1.0% agarose.
5.0 kb (pBluescript + pTn5: Km) and 3.0 kb (pBluescript) lie within the range, indicating a
successful gel electrophoresis.
4. Discussion
It was initially hypothesized that the function of the lacZ gene would be lost following
the mutagenesis of Escherichia coli using vectors pBluescript and pTn5: Km. Mutagenesis
involved techniques, including transposition, transformation, and screening, alkaline lysis,
restriction endonuclease digestion, and gel electrophoresis. Transposition, transformation, and
screening were used for evaluate the functionality of the lacZ gene and the activity of β-D-
galactosidase. Alkaline lysis, restriction endonuclease digestion, and gel electrophoresis were
used to determine the restriction sites in pBluescript with or without pTn5: Km insert. After all
processes, it was confirmed that the function lacZ gene was lost.
The loss of function in the lacZ gene was expected following transposition,
transformation, and screening. However, during the transposition, transformation, and screening
processes, the lacZ gene demonstrated a total loss of gene function. According to Table 3.2.2,
white colonies were observed in ampicillin agar plates only, with a total of 807 colonies in both.

17. 17
Blue colonies however, were not observed in the agar plates. Results from the screening process
(Tables 3.2.3 and 3.2.4) helped confirm the total loss of lacZ gene function observed during the
transformation process. In Table 3.2.3, white colonies were observed in ampicillin agar plates
following the screening process only. Both ampicillin agar plates had 50 white colonies. Blue
colonies were not observed in the ampicillin agar plates. On the other hand, both white and blue
colonies were not present in the ampicillin/kanamycin agar plates. There were 0 white colonies
and 0 blue colonies in the ampicillin/kanamycin agar plates. The sole presence of white colonies
in ampicillin plates following the screening process, indicated that colonies were able to grow in
the presence of ampicillin only, but were not able to grow in the presence of ampicillin with
kanamycin. The total absence of white and blue colonies in both ampicillin/kanamycin agar
plates demonstrated that growth did not occur, making the digestion of X-gal non-existent. In
Table 3.2.4, both sets of ampicillin and ampicillin/kanamycin agar plates had a frequency of
transposition of 0. These frequencies supported the results from Table 3.2.3. Results from the
transformation and screening process did not support the literatures. According to Analytical
Biochemistry and the Journal of Virological Methods, blue and white colonies were expected to
be present (Wessels et al., 2015), (Winnard Jr, Challa, Bhujwalla, & Raman, 2014). The total
loss of function of the lacZ gene may have been caused by poor execution of the lab protocols,
including media preparation, competent cell preparation, and transposition. Perhaps, not enough
LB broth nutrients such as Peptone, yeast extract, and sodium chloride or antibiotics such as
ampicillin and kanamycin were supplied when preparing the LB broth and agar. Also, the
competent cells may have been incubated for too long during competent cell preparation, causing
the cells to reach the death phase. Perhaps, the competent cells were not distributed evenly
enough in the agar plates or that the alcohol spreader was excessively hot during transposition.
The total loss of function of the lacZ gene may have also been caused by non-technical errors,
such as the cell’s innate competence.
Following alkaline lysis, restriction endonuclease digestion, and gel electrophoresis, the
sizes of the DNA fragments and the size of plasmids were determined and the restriction sites in
pBluescript with or without pTn5: Km were mapped. Using the sizes of the DNA fragments (bp)
in wells 14, 15, and 16, the sizes from the DNA marker (bp); 1 kb DNA ladder (Figure 3.3.2),
the distance of migration (cm), and the logarithm of fragment size from DNA marker (Table
3.3.3) four DNA fragments with sizes 1.0, 2.0, 3.0, and 5.0 kb were observed. The sizes of the

18. 18
DNA fragments and prior knowledge of vector sizes helped map the restriction sites in
pBluescript with or without pTn5: Km insert (Figure 3.3.4). According to Figure 3.3.4, the
recombinant plasmid (pBluescript + pTn5: Km) has three restriction sites, including two EcoRI
restriction sites and one BamHI restriction site. The non-recombinant plasmid (pBluescript) has
two restriction sites, including one EcoRI and one BamHI restriction site. In other words, EcoRI
is capable of digesting the recombinant plasmid twice and once in the non-recombinant plasmid,
whereas BamHI is capable of digesting in both the recombinant and non-recombinant plasmids
once. EcoRI and BamHI restriction/recognition sites were specifically determined in E. coli
(Figure 3.3.5), with EcoRI digesting between A and G (5’ to 3’) and BamHI digesting between G
and G (5’ to 3’). Overall, the restriction maps in Figure 3.3.4 corresponded with results from
Figures 3.3.1, 3.3.3, 3.3.4 and Tables 3.3.1 and 3.3.2. Proposed restriction maps were validated
by the concentration of agarose used (Table 3.3.3). 1.0% agarose was used for the separation of
DNA, with sizes ranging from 500 bp to 10 kb. Sizes of plasmids ranged from 500 bp to 10 kb
indicating a successful gel electrophoresis.
5. Conclusion
This research investigation primarily aimed to determine the restriction sites in plasmid
vector pBluescript with or without pTn5: Km. The investigation also aimed to evaluate the
functionality of the lacZ gene, as well as the activity of β-D-galactosidase, the enzyme encoded
by the lacZ gene. Escherichia coli mutagenesis was performed in order to evaluate such
parameters. The mutagenesis of E. coli involved the use standard microbiological lab procedures
such as transformation and screening and molecular lab procedures such as transposition,
alkaline lysis, restriction endonuclease digestion, and gel electrophoresis. Transposition,
transformation, and screening helped determine the functionality of the lacZ gene, as well as
evaluate the activity of its encoded enzyme, β-D-galactosidase. Following these three processes,
both the lacZ gene and β-D-galactosidase appeared to be non-functional at all, indicating a total
loss of function of the lacZ gene. White colonies were present in ampicillin plates during the
transformation and screening processes only. Blue colonies were absent in both ampicillin and
ampicillin/kanamycin agar plates during these processes. This was shown in Tables 3.2.1. 3.2.2.
3.2.3 and 3.2.4. Alkaline lysis, restriction endonuclease digestion, and gel electrophoresis on the

19. 19
other hand, helped determine the sizes of the DNA fragments, as well as helped map the
restriction sites of EcoRI and BamHI in pBluescript with or without pTn5: Km insert. The
proposed restriction maps (Figure 3.3.4) corresponded with the results obtained from gel
electrophoresis (Figures 3.3.1-3.3.4, Tables 3.3.1-3.3.2) and were validated by the concentration
of agarose used. For further research, other genes involved in the lac operon, such as lacY and
lacA should be used to determine their functionalities and their encoded enzymes’ properties and
activities. Using lacY and lacA, the activities and properties of galactoside permease and
galactoside transacetylase can be observed. Sufficient knowledge on these genes can help
researchers understand the lac operon better in terms of its regulation and roles in various
organisms.
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