Health and Life Sciences Faculty
Course Title: Biological and Forensic Science
Module code:
216 BMS
Module Title:
Molecular Genetics
Overview of the Recombinant DNA technologythe process of subcloning a foreign gene into
the plasmid vector pUC19
Ciobanu Maria Alice
SID: 3395606
Word count:
1617
Overview of the Recombinant DNA technology- the process of
subcloning a foreign gene into the plasmid vector pUC19
Introduction
The term ‘’gene cloning’’ refers to a wide variety of techniques that makes it possible
to manipulate DNA in order to return it to living organisms where it can function
normally. Essentially, it involves isolating a piece of DNA from an organism and
introducing it into a cloning host, for example bacterium Escherichia Coli which
grows and divides rapidly. It is therefore possible to study the cloned DNA or
produce the protein encoded by the gene .The cDNA may be inserted into vectors
and then cloned. The choice of vector represents the most important consideration in
molecular cloning experiments. (Krebs et all, 2010)
Many bacteria contain an extra chromosomal element of DNA known as a plasmid.
This is a relatively small, covalently closed circular molecule, which carries genes for
antibiotic resistance, conjugation or the metabolism of unusual substrates. One of
the most notable plasmids, termed pUC19 was widely adopted for cloning of small
DNA fragments in E.coli. It is an antibiotic resistance gene for ampicillin and contains
an origin of replication enabling the vector to replicate in E.coli. (Lodge et all; 2007)
Plasmid vectors permit the identification of recombinant clones by looking for
insertional inactivation of either antibiotic resistance genes or lacZ. However,
potential recombinant clones still need to be analyzed. This involves purifying the
plasmid DNA from individual clones, cutting it with restriction enzymes in order to
analyze the size of the fragments produced. (Krebs et all, 2010)
Restriction
endonucleases recognize certain DNA sequences and cleave them in a defined
pattern. Covalent joining of ends on each of the two strands may be brought about
by the enzyme DNA ligase, which enables the construction of recombinant DNA
fragments. However, it is often convenient to cut a vector with two restriction
endonucleases which do not produce complementary overhanging ends (described
as sticky ends and are generated by cleavage). EcoRI is one of the most commonly
used restriction enzyme and it is produced by the bacterium E. coli strain RY13.
(Walker, J.M., Rapley, R. ;2000)
The next step in cloning a gene is to find a way of joining the DNA molecules
together in a new combination (termed as recombinant). The most common used
DNA ligase is a protein produced by a bacteriophage known as T4. In the energy
consuming process of ligation, DNA ligase catalyzes the formation of a covalent
phosphodiester bond between the 5’ phosphate on one DNA strand and a 3’hydroxyl
on another. (Krebs et all, 2010)
The final step is described as the transformation which involves the introduction of
the new recombinant plasmid into E. coli. For the DNA to get into the bacterial cell, a
selection of the cells containing the plasmid is necessary. Considering that E.coli is
not naturally capable for transformation, its cells need to be treated by chemical
treatment and electroporation in order to enable them to take up DNA. (Lodge et all;
2007)
This laboratory report is based on three practical sessions and its aim is to transfer a
fungal gene (termed CIH-1)from a plasmid vector (called pBK-CMV) into a different
plasmid vector (known as pUC19) through the use of a number of techniques and
procedures as restriction endonuclease digestion of DNA, analysis of DNA
fragments by agarose gel electrophoresis, ligation of DNA fragments into a vector,
introduction of the ligated DNA into host bacteria by transformation, selection of
colonies containing recombinant vector molecules and isolation and analysis of
recombinant plasmids. (Coventry University; 2011)
Methods:
The experiments were carried out as described in the schedule. (Laboratory
Schedule, Coventry, 2011)
Results
In order to clone DNA, it needs to be cut up in a precise and repeatable way by using
enzymes. Therefore, the foreign gene (CIH-1) needs to be cut out of the pBK-CMV
with the restriction endonucleases EcoR1 and Xbal, same as the pUC19. (Coventry
University; 2011)
To check if the restriction digestion has been successful, gel electrophoresis is used
to measure the size of the fragments generated as it can been seen in Figure 1
below:
Figure 1. Agarose gel electrophoresis of the restriction digest plasmids pBKCMV and pUC19
It can be observed that the nucleic acids migrated in gel describing a linear
movement of the DNA fragments. The DNA is visualised by staining with ethidium
bromide, which fluoresces under ultraviolet light. From this image, the measurement
of the distance moved by each DNA size markers from the well can be determined
using a ruler. The results concluded have been recorded in Table 1 below.
Table 1. Distance moved by DNA fragments from the well
DNA fragments size (base pairs)
Distance moved from well (mm)
5000
10
3000
12
2000
14
1000
19
500
23
It can be observed that the distance that DNA fragments have migrated from the
well is proportional to the size of the DNA fragment, with small fragments moving
faster than large ones, describing a variation from 10 to 23 mm distance travelled
from well.
Furthermore, the size of fragments needs to be determined. A very useful tool is to
plot a graph of log marker size against distance travelled in the gell. A calibration
curve will be produced and used afterwards to calculate the size of pUC19 and pBKCMV DNA fragments. A representation of it can be observed in Figure 2
Figure 2. Representation of the measurement of the distance moved by DNA
size markers
The results established from the plotted graph have been recorded in the table
below. Taking into consideration that the recombinant plasmids have been cut with
EcoR1, two fragments have been generated: pUC19 which varies in size between
600 bp and 3800. On the other hand, when Xbal is used to cut the plasmid, only one
band occurs: pBK-CMV which has a length of 3100 bp. (Table 2)
Table 2. Distance moved by restriction plasmids from the well
Distance moved
Size (bp)
(mm)
pUC19
pBK-CMV
9
3800
20
600
12
3100
Following the ligation process, DNA needs to be introduced into the host cell.
Therefore, the antibiotic ampicillin is added to the L-agar plates in the experiment in
order to select for bacteria which have taken up the plasmid pUC19. Also, a mixture
of X-gal and IPTG are added. As it is an artificial substrate for β-galactosidase, X-gal
produce a blue product. On the other hand, IPTG is an artificial inducer of the lac
operon, stimulating its transcription (Coventry University; 2011). As a result, bacterial
colonies which express β-galactosidase will appear blue, while the non-producing
ones will be white. After counting the colonies, the results were recorded in Table 3
below.
Table 3. Results determined from the transformation plates
Sample
Dilution
No. blue colonies
No. white colonies
Plate 1
Plate 2
Mean
Plate 1
Plate 2
Mean
10-6
0
0
0
257
190
223.5
10-7
0
0
0
63
51
57
10-8
0
0
0
2
6
4
None
0
0
0
0
0
0
None
162
94
128
0
0
0
10-1
22
41
31.5
0
0
0
2)
10-2
1
2
1.5
0
0
0
Tranformation
None
15
18
15.5
23
23
23
10-1
1
0
0.5
0
0
0
Compotent
cells
Tranformation
negative
control (tube
3)
Tranformation
positive
control (tube
Ligation (tube
1)
The Figure 3 represents the agarose gel electrophoresis of B1, W1 and W2
restricted plasmids. As it can be observed, W1 and W2 contain recombinant DNA,
therefore they form 2 DNA fragments in the gel. Conversely, pUC19 is nonrecombinant DNA, so just a single line is shown.
Figure 3. Agarose gel electrophoresis of B1, W1 and W2 restricted plasmids
Measurements of distance travelled by the DNA fragments in the marker track are
documented below. (Table 4)
Table 4. Distance travelled by DNA fragments in the marker track
DNA fragment size (base pairs)
Distance moved from well (mm)
5000
6
3000
8
2000
9
1000
13
500
17
In order to determine the DNA fragments size, a calibration curve was plotted. It can
be visualised in Figure 4 below.
Figure 4. Representation of the measurement of the distance travelled by the
DNA fragments
The estimation of the size of bands in the restriction digest is recorded in Table 5
and its based on measurements made on the calibration curve above.
Table 5. Distance travelled by B1, W1 and W2 in the marker track
Sample
Distance moved
Size (base pairs)
B1
7
3300
W1
7
3300
14
550
7
3300
14
550
W2
It can be observed that the plasmids moved proportional to their length, resulting in
the same number of base pairs. Due to the fact that W1 and W2 contain cDNA, they
produced 2 DNA fragments with a size of approximately 500 bp.
Discussion
In the first experiment the fungal gene, CIH-1 which is isolated from the fungus
Colletrotrichum lindemuthianum needs to be inserted into pCU19.The CIH-1 cDNA
have been cloned in a plasmid vector called pBK-CMV. In order to clone DNA, it
needs to be cut up in a precise and repeatable way by using enzymes. Therefore,
the foreign gene needs to be cut out of the pBK-CMV with the restriction
endonucleases EcoR1 and Xbal, same as the pUC19. Restriction endonucleases
recognize certain DNA sequences which are polindromic, usually 4-6 base-pairs (bp)
in length, and cleave them in a defined pattern. This means that the nucleotide
sequence reading is the same in both directions on each strand. Usually they leave a
flush (blunt –ended) or staggered fragment when cleaved, depending on the
enzyme. (Krebs et all, 2010)
After inactivating the restriction enzymes, the plasmid and restriction enzyme
fragments are mixed in the presence of T4 DNA ligase. In this experiment,
throughout the ligation reaction the digested pBK-CMV and pUC19 were mixed
together. As a result, the foreign gene (CIH-1) from pBK-CMV is ligated into the MCS
of pCU19. However, the desired outcome from the cloning experiment is that one
vector molecule to be joined to one of the genomic DNA fragments in order to
circularize and form a new recombinant molecule. The last step in gene cloning is
the introduction of the recombinant plasmid into E. coli. During transformation, the
DNA associated with the lipopolysaccharide on the outer surface of the competent
cells in order to uptake the DNA. (Lodge et all; 2007)
The most popular restriction sites are concentrated into a region called the multiple
cloning site (MCS) which is located within the gene lac Z’. Nevertheless, the MCS is
part of a gene in its own right and codes for a portion of polypeptide called βgalactosidase which is caused by adding an inducer known as IPTG (isopropyl-β-Dthiogalactopyranoside). The functional enzyme is able to hydrolyse a colourless
substance named X-gal (5-bromo-4-chloroindol-3-γl-β-galactopyranoside) into a blue
insoluble material. When a disruption in the gene occurs through the insertion of a
foreign fragment of DNA, a non-functional enzyme results which is incapable to
perform hydrolysis of X-gal (Krebs et all, 2010). Moreover, X-gal is the artificial
substrate used in this experiment and IPTG is the artificial inducer which takes care
of the repressor gene and stops it from working. Hence, it is easy to detect the
recombinant pUC19 plasmid since it is white in the presence of X-gal, whereas a
non-recombinant pUC19 plasmid will be blue as the gene is not disrupted, therefore
fully functional and expressing β-galactosidase activity. This impressing system,
termed blue/white selection permits the initial identification of recombinants to be
undertaken very rapidly. It is based on the lac Z’ gene and requires the use of
special E.coli host strains which are naturally lac + . In fact, this represents one of the
biochemical characteristics routinely used in the identification if E.coli. From Table 3
it can be observed that the number of white colonies overcomes the number of the
blue ones. The white colonies are formed as a result of the insertion of DNA
fragments into the multiple cloning sites of pUC19 which interferes with lac Z. If the
bacterial colonies have taken up the plasmid pUC19 they are coloured in blue.
(Walker, J.M., Rapley, R.; 2000)
The final step is to prove that the inserted DNA fragment in pUC19 generated in this
experiment is in fact the fungal cDNA molecule, CIH-1. To start with, parts of DNA
molecules from two chromosomes differ from each other by a single base pair, which
results in the absence of an EcoR1 site in one of the chromosomes. Upon digestion
with EcoR1, the chromosome without the extra EcoR1 site produces a larger
fragment than the other one. This difference is recognised using a probe that
hybridises within the region encompassed by two flanking EcoR1 sites present in
both molecules. A probe represents a molecule able to bind very specifically to other
molecules, therefore it is used to identify the relevant clone among the undesired
ones. Two different kinds of probes are recognized: antibodies and polynucleotides .
(Sudbery, P. & Sudbery, I.; 2009)
To conclude, significant improvements have been made at the molecular level. Many
new and powerful ways for isolation, analysis and manipulation of nucleic acids have
been discovered. The recently developed cloning strategies heralded a new and
exciting era in the exploitation of DNA molecules. Gene cloning especially enabled
numerous discoveries to be made and provided precious insights into gene
structure, function and regulation, becoming not only an extremely useful tool but
also an absolute requirement in the area of bioscience. (Strachan, T., Read, A.;
1999)
List of references
Coventry University. (2011) Laboratory schedule for 216BMS Molecular Genetics –
DNA cloning Labs 1-3. Coventry: Coventry University
Krebs, J.E., Goldstein, E.S., Kilpatrick, S.T. (2010) Lewin`s essential genes. 2nd
edition. London: Jones and Bartlett Publishers
Lodge, J., Lund, P., Minchin, S. (2007) Gene cloning Principles and Applications. 1st
edition. Abingdon: Taylor & Francis
Strachan, T., Read, A. (1999) Human molecular genetics. 2nd
edition. Oxford:
Garland Science
Sudbery, P., Sudbery, I. (2009) Human molecular genetics. 3rd
edition. Essex:
Benjamin Cummings
Walker, J.M., Rapley, R. (2000) Molecular biology and biotechnology. 4th edition.
Cambridge: The Royal Society of Chemistry