Biotechnology 2010 Project:
Isolation of the β-glucosidase gene (bglA) from B. halodurans and its introduction into vector
pLATE for protein expression in BL21 E. coli
The Biotechnology 2010 lab project implemented principles of recombinant DNA technology,
such as molecular cloning and gene expression, to isolate a β-glucosidase gene (bglA) from
Bacillus halodurans and introduce it into another host species, a BL21 (DE3) strain of E. coli, via
the vector plasmid pLATE11. To achieve this, both the gene insert of interest and the vector
plasmid were subject to an alternative method of cloning known as LIC (ligation-independent
cloning) that eliminated the need for enzyme restriction and ligation and possessed a much
higher efficiency/success rate than traditional cloning. In this procedure, LIC-ready (linearized)
pLATE11 and previously purified bglA were treated with T4 DNA polymerase, which in the
presence of only dGTP switches to its 3’5’ exonuclease activity, removing the nucleotides and
using its 5’3’ polymerase role to restore a dGTP when it encountered cytosine. As a result,
the polymerase created 14-21 nucleotide single-stranded overhangs that were complementary
to the ends of the pLATE11 vector. The bglA-pLATE11 sample was used to transform competent
BL21 cells, which successfully grew on an LB/Amp plate. Five colonies were extracted and then
their genome was screened for successful annealing of plasmid and gene insert using
Polymerase Chain Reaction (PCR). PCR confirmed that the annealing process was indeed
successful. A new batch of cells were transformed with the verified gene-containing plasmid,
and an expression culture was readied. This BL21 culture was induced for expression of the βglucosidase enzyme with Isopropyl β-D-1-thiogalactopyranoside, or IPTG. IPTG is an inducer
that mimics allolactose but cannot be hydrolyzed by β-galactosidase in the cell due to the
presence of a sulfur bond. Consequently, the concentration of this allolactose variant did not
decrease and the lac operon remained active over the entire course of the experiment. Analysis
of gene expression via SDS-PAGE confirmed presence of the desired enzyme, as presented by a
band on the gel that matched the expected size of the protein, about 50kD.
1. Introduction:
What is an enzyme?
An enzyme is a protein or RNA molecule that acts as a biological catalyst. Enzymes, regardless
of the type, have a very unique function that is specific to their structure. They facilitate
thousands of metabolic processes that are essential in the lives of all organisms on Earth. Like
any other protein, enzymes are synthesized by the expression of their respective genes, when
they are activated by various cascade reactions that involve transcription factors.
For example, hydrolases are enzymes that catalyze reactions that decompose a substrate by
reacting with H2O. Cellobiase is a hydrolase that breaks down a partially-decomposed cellulose
molecule, known as cellobiose, into glucose in the following order:
β -1,4 glucanase
cellobiase (β -glucosidase)
Cellulose ---------------> Cellobiose -------------> Glucose
β-glucosidase is a cellobiase that is ubiquitous in nature. It can be found in many species, both
prokaryotic and eukaryotic. Due to its abundance and role in the degradation of cellulose into
glucose, which can be fermented to ethanol and then processed as fuel, this enzyme offers a
very promising path to the quest for complete fossil fuel replacement.
Ligation-Independent Cloning
Photo courtesy of Thermo Fisher Scientific, Inc.
Ligation-Independent Cloning (LIC) is a simple and highly efficient method of molecular cloning
that completely eliminates the need for restriction enzymes and ligase, two factors that affect
the success rate of conventional cloning methods. The procedure consists of: (1) amplification
of gene insert using special gene insert primers that contain vector-specific sequences; (2) The
creation of overhangs or sticky ends on gene insert that are complementary to the LIC-ready
plasmid (provided with the kit). This step involves the 3’5’ exonuclease and 5’3’
polymerase functions of DNA polymerase. These two functions are balanced by the addition of
only one nucleotide, usually dGTP, but any nucleotide can be used if the vector-specific
sequences on the primers are adjusted accordingly. DNA polymerase performs its
‘proofreading’ function by removing nucleotides until it reaches a guanine. The enzyme
proceeds to remove this nucleotide, but in the presence of dGTP, it immediately reverts to its
polymerase function and restores the chain using the cytosine on the complementary strand as
template; (3) the annealing step, in which the LIC-ready plasmid and gene insert are mixed.
Within an hour, the mixture is ready to be used in the transformation of competent E. coli cells.
This project focused on the cloning of a potentially useful cellobiase for transforming cellulose
to glucose. A β-glucosidase gene, bglA, was isolated from B. halodurans and transferred to a
BL21 strain of E. coli via pLATE11 vector plasmid. The transformed bacterial cells were induced
with IPTG (Isopropyl β-D-1-thiogalactopyranoside). IPTG is very similar to allolactose, but a
slight difference in its structure allows this molecule to trigger the activation of the lac operon
without ever being exhausted in the process. If bglA was successfully inserted into the
cloning/expression region of pLATE11, then the induction of the lac operon by IPTG would also
trigger the expression of the gene of interest.
2. Materials and Methods
Materials:
Materials used in each experiment are listed in the following table:


Amplification of bglA Using B. halodurans
Genome as a Template and Purification
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


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LIC
Bacterial Transformation with bglA-pLATE11
and Colony Screening
Thermocycler
PCR 2X Master Mix (contains Taq
polymerase, MgCl2, and dNTP’s)
1.0ng/μl FWD LIC primer
1.0ng/μl REV LIC primer
Template DNA: genomic DNA
dH2O
1% agarose gel (previously poured)
Gel electrophoresis equipment
6X blue loading dye
QIAquick PCR Purification Kit


aLICator LIC Cloning and Expression Kit 1
(untagged), #K1241
5X LIC buffer
0.5 M EDTA
pLATE11, LIC-ready vector (60 ng, 0.02 pmol
DNA)
purified PCR product from above, 78.7 ng/μl
T4 DNA Polymerase








5.0 μl of annealing reaction from above
Competent BL21 cells
42°C heat block
2X Master Mix
1.0ng/μl FWD LIC primer
1.0ng/μl REV LIC primer
dH2O
QIAprep Spin Miniprep
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


Induction of lac Operon by IPTG and SDSPAGE to analyze Protein Expression
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Expression cultures, both induced and
uninduced
3X SDS sample buffer
1M DTT
10% SDS gel (previously poured)
SDS-PAGE equipment
220kDa Protein ladder
6X blue loading dye
Methods:
Amplification of bglA Using B. halodurans Genome as a Template and Purification:
A previously isolated and verified B. halodurans genome sample was amplified with Polymerase
Chain Reaction (PCR), using LIC primers that consisted of vector-specific sequences on the 5’
and 3’ ends overlapped with the sequence of bglA:
pLATE11 forward primer:
5'-AGAAGGAGATATAACTATG–bglA
sequence 3'
pLATE11 reverse primer:
5'-GGAGATGGGAAGTCATTA–blgA
sequence 31
This experiment created the gene insert that was later used in the LIC and annealing processes.
One 50 μl PCR reaction per group was prepared using the reagents listed below in the following
order:
50 μl PCR reaction: 22 μl dH2O; 25 μl Master Mix; 1.0 μl FWD LIC primer; 1.0 μl REV LIC primer;
1 μl genomic DNA.
The samples were placed in the thermocycler overnight. 30 reactions occurred: 95°C for 5
minutes; 95°C for 30 seconds; 50°C for 30 seconds; 72°C for one minute; 72°C for 5 minutes;
storage at 4°C until removed.
6 μl of 6X blue loading dye was added to the sample and then the sample was loaded into a
previously poured 1% agarose gel. The gel was run at 150 V for about 45-50 minutes.
Purification: The PCR product from above was purified in a bind-wash-elute prodecure to
completely remove unincorporated dNTPs and primers, to inactivate DNA polymerase, and to
1
Primer sequences obtained from manual included in aLICator LIC Cloning and Expression kit #K1241
remove contaminating DNA. The QIAquick PCR Purification Kit uilitized to achieve this provided
all necessary materials for purification and collection of bglA.
Ligation-Independent Cloning: The purpose of this experiment was to clone the gene fragment
into the vector plasmid using the 3’-5’ exonuclease and 5’-3’ polymerase activities of T4 DNA
polymerase to eliminate the need for restriction digestion and ligation. The procedure
consisted of two sections:
a. LIC cloning: To create the necessary 5’ and 3’ overhangs on the purified PCR
template, the following reaction was set up: 2 μl 5X LIC buffer; 0.1 pmol purified PCR
product (~1.3 μl); 5.7 nuclease-free H2O. The mixture was vortexed briefly and
incubated at room temperature. The reaction was immediately stopped after 5
minutes by adding 0.6 μl EDTA.
b. Annealing: 1 μl of LIC-ready pLATE11 (60ng, 0.02 pmol DNA) was added to the
reaction, vortexed briefly for about 3-5 seconds, and annealing proceeded for 5
minutes at room temperature.
Bacterial Transformation with bglA-pLATE11: In this section of the project, competent BL21
(DE3) cells were transformed with the annealing reaction created above. The steps were as
follows:
a.
b.
c.
d.
e.
5.0 μl of the purified plasmid was transferred to tube containing BL21 cells.
Tube was incubated on ice for 5 minutes.
Cells were then heat shocked at 42°C for 30 seconds.
Heat shocked cells were placed back on ice for 5 minutes.
Finally, the mixture was poured onto an LB-Agar-amp plate and spread evenly
throughout the plate with a sterile L-shaped rod.
f. Plate was incubated at 37°C overnight.
Colony Screening: Five colonies were selected from the LB-Agar-Amp plate for screening. Five
PCR reactions were set up with these colonies, in which the target sequence was that of bglA.
This step was performed to assess the results of LIC reaction. The following PCR recipe was
followed:
5 30-μl PCR reactions: 75 μl 2X Master Mix; 1.0 μl FWD LIC primer; 1.0 μl REV LIC primer; 73.0 μl
dH2O; colony sample (1-5).
Tubes were placed in the thermocycler overnight.
6 μl of 6X blue loading dye was added to the samples and then the samples were loaded into a
previously poured 1% agarose gel. The gel was run at 150 V for 60 minutes.
Second Purification and Bacterial Transformation: Colony #3 was selected and transferred to LB
medium and incubated at 37°C overnight. The pLATE11 vector containing bglA was extracted
from the cells and purified using a QIAprep Spin Miniprep kit. The purified plasmid was
introduced into another vial of BL21 cells following the transformation procedure outlined
above.
Induction of lac Operon by IPTG and SDS-PAGE to analyze Protein Expression:
Two separate colonies were extracted from the plate by the instructor. These colonies were
used to inoculate two separate tubes containing 2 ml of LB. The liquid cultures were incubated
at 37°C in a shaking incubator for one hour. Next, one of the cultures was induced for
expression of proteins in the lac operon by addition of 2 μl 0.5 M IPTG. Both cultures were
incubated at room temperature in a shaker overnight.
SDS-PAGE to Analyze Protein Expression: The OD600 of each culture created above was
measured in the spectrophotometer using a 1-ml cuvette. 900 μl of each culture was
transferred to a microcentrifuge tube and then the samples were spun down for one minute.
The supernatant resulting from the centrifugation was discarded and the pellet of cells was
resuspended in 80 μl of 3X SDS sample buffer and 10 μl of 1M DTT. The cells were then placed
in a 95°C heat block for exactly 5 minutes.
The amount of resuspended cells to load onto the gel was calculated using the equation
(0.9/measured OD600) x 10. The OD600 was measured to be 0.941 for uninduced cells, and 1.004
for induced cells. Therefore, the calculated volume of cells was ~10 μl uninduced and ~9 μl
induced.
The calculated volumes were loaded onto a previously poured 15% SDS gel. Invitrogen’s
Benchmark protein ladder was also loaded. After filling the chamber with TRIS-Glycine SDS
buffer, the gel was run at 180V for about 35 minutes. Finally, the gel was stained using the
Quick Stain method so a photograph could be taken.
3. Results
Thermo Fisher Scientific Inc.
Lane Organization
1 – 1Kb DNA ladder
2 – Group #1
3 – Group #2
4 – Group #3
5 – Group #4
6 – 1Kb DNA ladder
Figure 1. Gene insert amplified by LIC primers.
Figure 1 depicts the results from the amplification of bglA using primers that contain vectorspecific sequences in addition to the gene sequence. The total size of the PCR fragment came to
be 1449 bp long. The circled band, as well as the bands of the other groups, falls just above the
1.5k-bp band (see GenerulerTM1kb DNA Ladder diagram).
Lane Organization
1 – 1Kb DNA ladder
2 – Colony #1
3 – Colony #2
4 – Colony #3
5 – Colony #4
6 – Colony #5
Figure 2. Analysis of plasmid in selected colonies via PCR.
Figure 2 displays the results of the LIC experiment. Through LIC, the gene fragment correctly
annealed to pLATE11. BglA is approximately 1430 bp long, and the vector-specific sequences
are 19bp, for a total of 1449 bp. Colonies 1-3 confirm this result as represented by the sample
bands that lie just under the 1.5k-bp ladder band. This indicates that the identity of the PCR
product is indeed bglA. Colonies 4 and 5 show no appreciable bands.
Lane Organization
1 – Uninduced culture (control)
2 – Induced culture
3 – Protein ladder
Figure 3. SDS-PAGE of the IPTG-induced and uninduced cultures.
The gel photograph above shows the results of the SDS-PAGE for the analysis of protein
expression. As can be seen, both the induced and uninduced cultures expressed proteins coded
by the lac genes lacY, lacZ, and lacA, even though only one of them was exposed to IPTG. In
addition to these there was a protein represented by a strong band in the induced culture and a
very faint band in the uninduced culture. The fragment is approximately 50kDa.
4. Discussion
The ultimate goal of the project was to clone a potentially useful cellobiase found in B.
halodurans, an enzyme of particular interest in the biofuel industry. A β-glucosidase gene, bglA,
was isolated from this species and transferred to a BL21 strain of E. coli via pLATE11 vector
plasmid. The transformed bacterial cells were induced with IPTG (Isopropyl β-D-1thiogalactopyranoside). Once pLATE11 is in the cells, protein expression would reveal the
product of the gene insert cloned into the plasmid.
Ligase-Independent Cloning (LIC) was successful as expected. By eliminating the need for ligase
and restriction enzymes, the success rate of this method was increased dramatically, and the
cloning procedure itself is significantly simplified. It makes sense that the success rate is higher
because there is only one product that can be obtained. DNA polymerase was able to create
overhangs on the gene insert that were complementary with the ends of linearized plasmid
pLATE11. In the absence of 3 of the dNTP’s, DNA polymerase activated its “proofreading”
mode, and began removing nucleotides from the 3’ end of the DNA until it reached guanine. At
that point, the enzyme still removed the nucleotide, but due to the presence of dGTP in the
solution, it switched back to its polymerase function and restored the chain with a new dGTP
using the complementary DNA strand as a template. Thus, the two functions of the DNA
polymerase are equilibrated at the first occurrence of cytosine in the complementary strand.
The results were sticky ends that were 14-21 bp long. If a restriction enzyme had been used to
create the sticky ends, the length of the overhangs would have been much shorter, and
therefore much more unstable. In addition, ligase would have had to be used to hold together
the gene insert and the plasmid, and not very strongly. This is one of the leading factors that
cause conventional cloning to fail. In contrast, LIC created purposely long overhangs to
eliminate the need for both restriction enzymes and ligase. When mixed, bglA and pLATE11
annealed tightly to each other almost spontaneously.
60% of the colonies screened were successfully transformed by a gene-containing plasmid. This
can be seen in the second gel picture. The primers used only targeted the sequence of the gene
insert specifically, and the presence of bands in colonies 1-3 of the correct size indicated that
the correct product was amplified. Colonies 4 and 5 showed fainter bands that were of smaller
size. The faint bands likely represented the primers that did not find the target sequence. Since
the primers did not find a complementary DNA strand to attach to, they travelled through the
gels as any other fragment. This result indicates that colonies 4 and 5 were “false positives,”
meaning that they were transformed by a plasmid that de-linearized before the gene fragment
could insert itself. This perfectly explains why the primers were seen in the picture as very faint
bands.
It is important to note that the bands shown in the two gels, though they obviously represent
very similar sizes, are located at slightly different distances compared to the ladder band. For
example, the band in gel 1 is located just above the 1.5k-bp band and in the second picture the
bands representing the same exact product are located just below it. This is mostly likely due to
the fact that the first gel was run for a shorter period of time, a difference of approximately 1015 minutes. Therefore, it can be concluded that the band was higher up than it was expected to
be because the gel was not allowed to run until it was completely ready. The time discrepancy
did not alter the outcome of the project.
The presence of bglA in pLATE11 was further confirmed by expression of the lac operon. Both
the induced and uninduced cells expressed the expected proteins coded by the lac operon,
such as β-galactosidase (LacZ), β-galactoside permease (LacY), and β-galactoside
transacetylase (LacA). The cells in both samples also expressed β-glucosidase, encoded by bglA.
The cells in the uninduced sample must have been exposed to a source of lactose other than
IPTG that triggered activation of the operon in the plasmid, and in their main genome.
However, some of the bands are fainter than in the induced sample, which can possibly be
attributed to the fact that the lactose source was being depleted fast at the time the cells were
split. This would help to explain why the band representing β-glucosidase was significantly
stronger in the induced culture, which was exposed to the unhydrolizable IPTG.
The size of β-glucosidase is approximately 50kDa. The band that represents the target enzyme
matches the band on the protein ladder of roughly that size. This confirms the overall success
of the Biotechnology 2010 project.
LIC allowed for an efficient insertion of bglA into pLATE11. Transformation of E. coli cells and
subsequent expression of β-glucosidase has demonstrated that the production of the target
enzyme via molecular cloning can be used as a means for more efficient biofuel production.
This is definitely a sound strategy that should be investigated further.