DNA Cloning of the α -amylase gene and

gDNA of B.licheniformis

Fatimah Edwards
215826977

TA: Hassan Arif

Abstract

The fundamentals of cloning were explored involving essential molecular biology techniques
such as DNA extraction, PCR, recombinant DNA, and transformation. The gene of interest, the α
-amylase gene which was isolated from B. licheniformis, was recombined with pET15b plasmid
and was made to be transformed into E. coli. Bacterial Plates with E.coli were analyzed for
successful transformation. Plate A containing ampicillin was treated with E.coli potentially
transformed with recombinant plasmid. No growth occurred however, growth occurred on the
control, plate B which had no insert. While cloning of the α-amylase gene was being done,
simultaneously, the cloning of the B.licheniformis genome was being done as well. It was to be
transformed into E.coli using pRL498 as its vector. Plate D, which was the control, had no
growth and neither did the experimental plate C. Possible explanation as to why no growth
occurred can be related to a problem in the ligation and/or transformation method. A Bradford
assay of maltose was conducted using purified α-amylase gene from E.coli to measure the
activity, thus efficacy, of the cloned α-amylase enzyme which catalyzes the hydrolysis of starch
into maltose and glucose.

Introduction

DNA cloning is a method used to quantify genes and larger fragments of DNA sequences.
Molecular biology uses DNA cloning to determine functions of transcribed proteins from the
resulting genes[ CITATION Alb02 \l 3084 ]. Cloning has a similar function to PCR however, cloning
can accommodate much larger sequences of nucleic acids. The purpose of this experiment was
to preform gene cloning, effectively using a variety of essential molecular biologic techniques
while understanding their positive and negative results. A simplistic view of gene cloning using
bacteria entails isolating genomic DNA containing the gene of interest, quantify the gene by
PCR, using restriction enzymes to insert the gene into a plasmid, transform the plasmid into a
different bacteria, and then analyze bacterial cultures to see whether the plasmid with the gene
of interest was absorbed. The bacteria used for DNA extraction was B.licheniformis and the
bacteria that was to take up the recombinant plasmid was E.coli. The gene of interest is the α-
amylase gene which transcribes the enzyme α-amylase. This digestive enzyme catalyzes the
hydrolysis of starch into maltose and glucose[ CITATION Smi10 \l 3084 ].

Materials and Methods

Refer to BIOL 3140 Lab manual, pages 24-40.

Discussion

The point of this experiment was to attempt to successfully clone the α-amylase gene from B.
licheniformis into E. coli. Essential molecular biological techniques such as DNA extraction, PCR,
recombinant DNA, and bacterial transformation, were used for cloning and their individual
results were analyzed with agarose gels and used as checkpoints to monitor progress. An
unsuccessful result from any of these techniques will result in an unsuccessful cloning.

To make sure PCR of the α-amylase gene was successful, an agarose gel electrophoresis was
carried out to analyze the products. The negative control was not treated with template DNA
therefore there would be a low concentration of DNA which can explain the low weight and
faint band [ CITATION Lee12 \l 3084 ]. The positive control was predicted to have a band that
approximately matched the molecular weight of α-amylase which is 1.5kb[ CITATION Ras09 \l
3084 ]. In that case, Lane 3 which contained PCR products from B. licheniformis, was supposed

to have a similar band which would confirm that the PCR product was the α-amylase gene. The
PCR products from B, licheniformis was congruent with the prediction of the α-amylase gene
have a weight of 1.5kb however the positive control could not support this. The abnormality of
the positive control may be due to contamination or a disruption in the balanced concertation
of the PCR reagents. Both defects can allow for nonspecific binding thereby preventing the
desired product[ CITATION Lor12 \l 3084 ].

The unpurified PCR products from figure 2 were digested by restriction enzymes for preparation
for ligation. These clean and cleaved digests provided confirmation that the α-amylase gene
was still intact and present (figure 3, lane 2) since the band was 1.5kb. No band for the pRL498
was present. It is possible for ssDNA to form hairpin pin loops thus inhibiting the PCR process
from continuing [ CITATION Lor12 \l 3084 ]. This can be an explanation as to why no band
occurred.

To know whether the α-amylase gene and the DNA library was successfully cloned in E.coli, 4
plates with the appropriate growth medium that exploits the fact that plasmids contain
antibody resistance, was set up. The plasmid pET15b had resistance to ampicillin therefore, the
growth medium used for it contained ampicillin. The plasmid pRL498 had resistance to
kanamycin therefore, it was grown in kanamycin. Only E.coli successfully transformed with
functional plasmids, meaning the ligation was successfully as well, will grow cultures. No
growth suggests problems with the transformation or ligation method.

Plate B was treated with E.coli, assumed to be transformed with pET15b plasmid only. Our
results showed that there was growth which means the transformation was successful. Our
plate A, however, which was treated with E.coli assumed to be transformed with recombinant
pET15b + cleaved PCR product, had no growth. Since Plate B was positive, that could mean that
the transformation for plate A was successful but the ligation.

Plate D was treated with E.coli and the vector pRL498. There being no growth was at first odd
because that would mean there was a problem with the transformation method however, It
appears that there is an existence of a special group of plasmids, including pRL498, who contain
long palindromic sequences which are highly unstable and nonviable in the majority of E.coli
strains[CITATION Elh88 \l 3084 ]. This supports the reason why no growth occurred with the
vector alone. Plate C was treated with E.coli and the recombinant pRL498 plasmid with the
genomic DNA library. The addition of the genomic DNA is supposed to disrupt the long
palindromic sequence of the pRL498 plasmid, allowing for the transformed E.coli to replicate as
per normal[CITATION Elh88 \l 3084 ]. There being no growth despite the DNA insertion, could be
due to a problem with the the ligation method as well as the transformation since there is no
positive control to compare it to.

If a bacteria culture grew, the transformation must have worked being that the vector plasmids
contain the antibody resistance needed for the E.coli to survive. Further analysis would be
needed however, to know for certain if the α-amylase gene is in fact present. The PCR colony
products from figure 4 was supposed to help conclude the presence of the α-amylase gene by
targeting the gene with primers. Using the results from figure 2 and 3 of which the weight of
the α-amylase gene was 1.5kb, it was expected to see bands of 1.5kb to confirm its presence
within the colonies. No bands occurred from any of the colonies selected. The positive control
should have had a band of 1.5 to correspond to the α-amylase gene but instead the and is very
light. Non-specific bands around 100 bp, such as the positive control, are likely due to primers
forming dimers resulting in a faulty PCR product [ CITATION Lor12 \l 3084 ].

Due to the plates that contained plasmid with insert not growing, a Lugol’s iodine staining to
view α-amylase activity could not be conducted.

Results
A spectrophotometric quantification was preformed to determine the DNA concentration and
of the extracted B.licheniformis. DNA concentration = 8691 µg/µl.

Lane 7 Lambda
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6
digested by HindIII
1kb 10-1 10-2 10-3 10-4 10-5

(kb)
10
8.0
6.0
5.0
4.0
3.0

2.0
1.5

1.0

Figure 1 shows a 0.7% agarose gel electrophoresis of genomic DNA from B. licheniformis. Lane 1
contains the 1 kb DNA ladder. Lanes 2-6 contain diluted Genomic DNA of concentrations 10 -1, 10-2, 10-3 ,
10-4, and 10-5 respectively. Lane 7 contains bacteriophage Lambda DNA digested by Hind III.
 Lane 4 α
Lane 2 Lane 3 amylase gene
from
Lane 1 1negative Positive
kb control control B.licheniformis

(kb)

3.0
2.0
1.5
1.0
0.5

Figure 2 shows a 1% agarose gel of unpurified PCR products. The target gene was the α -amylase gene.
Lane 2 contains the negative control, lane 3 contains the PCR products from pAMY8 positive control, and
lane 4 contains PCR products from B.licheniformis.
 Lane 2 α
amylase Lane 3 Lane 4 Lane 5
Lane 1
gene PCR gDNA pET15b pRL498
1kb
product
(kb)

3.0
2.0
1.5
1.0
0.5

Figure 3 shows a 1% agarose gel of various DNA digests. Lane 1 contains 1kb DNA ladder. Lane 2
contains clean PCR product double cleaved by Ndel and BamHI. It shows a band of approximately 1.5kb.
lane 3 contains clean genomic DNA from B. licheniformis single cleaved by HindIII, lane 4 contains clean
pET15b double cleaved by Ndel and BamHI, and lane 5 contains clean pRL498 single cleaved by HindIII.
 Lane 2 Lane 3 Lanes 4-7 PCR colony
negative Positive 100 bp
Lane 1
control control
1kb

kb
bp
3.0
2.0
1.5 1000
1.0 500
0.5
100

Figure 4 shows a 1% agarose gel of colony PCR products. Lane 1 contains 1kb DNA ladder and lane 8
contains 100 bp DNA ladder. Lanes 2 and 3 contain the negative and positive control respectively. Lanes
4-7 all contain colony PCR products however, no bands were present.

Table 1 shows Bradford assay results as well as enzyme activity of α -amylase. The Bradford assay
measured the concentration of maltose produced by the enzymatic activity of α -amylase. The value was
then compared to the actual concentration of the α -amylase enzyme. This new value was when
compared to the time (min) the enzymatic process took to convert the starch into maltose.

Amy E Negative B.licheniformi Amy E Negative E. B.licheniformis

E.coli E.coli s medium E.coli coli lysate
medium medium lysate
A540 nm 0.077 0.00819 0.072 1.049 0.004 1.524
Mg of maltose 0.509 0.0668 0.477 6.756 0.0398 9.8084
produced during
assay
Calculated mg of 5.09 0.0668 0.477 67.56 0.0398 9.8084
maltose produced
by 1ml of test
solution (mg/ml)
Enzyme activity 0.424 0.0557 0.039 5.63 3.316x10-3 0.8173
(mg/ml/incubation
time (min))
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