MOLECULAR AND CELLULAR BIOLOGY

LAB REPORT SESSION 10-11

BM2102

Bacterial Transformation

Pamela Wijaya
Bio Medicine
19010203

4th March 2021

i3L - Indonesia International Institute of Life Sciences
Jakarta
Introduction
Bacterial transformation or cloning is the process of inserting exogenous DNA into a bacteria cell
in order to produce new recombinants without conjugation steps (Griffiths et al., 2000). The term of
transformation by infection (transfection) is introduced when the cells used are mammalian cells. It is
said by infection as mammalian cells need an infective delivery fecal like virus to inject the genetic
material into the cells (Kim & Eberwine, 2010). The purpose of both techniques are to produce proteins
in vitro by enabling the replication of exogenous DNA within the cell lining, in which the purified proteins
could then be obtained by lysing the cell. Nevertheless, both techniques are used in different
applications. Bacterial transformation produces massive genes of interest very rapidly that can be used
for drug production (Griffiths et al., 2000). Meanwhile, for targeting specific cells, transfection is
preferable which could be used for the applications of gene therapy (Kim & Eberwine, 2010). Only the
bacterial transformation technique is covered in this study.
Bacterial plasmid/vector used for genetic modification or massive protein production should
have several features, including origin of replication (ORI), selection marker, restriction sites, and
promoter (Solar et al., 1998). The origin of replication instructs the plasmid to begin the DNA replication
on that region and also instructs the cell to reproduce the plasmid itself. The selection marker consisted
of antibacterial resistance gene which indicates if the bacteria has taken up the plasmid or not by
growing the bacteria in the antibacterial drug. Those surviving bacterias indicated that they have taken
up the antibacterial resistance gene within the plasmid. Furthermore, the exogenous DNA mostly is
inserted in the form of circular DNA as it contains only exon, not intron, and this cDNA is introduced to
the plasmid within the restriction site. Restriction enzymes are also involved to cut this restriction site so
that the plasmid becomes an open region and the cDNA can enter the plasmid. The promoter is placed
near and before the cDNA sequences in order to signal the beginning of the transcription process
(Griffiths, 1999).
This present study aimed to learn the process of replicating red fluorescent protein (rfp gene)
through the pKAN-R and pARA plasmids in bacteria cells. These plasmids are suitable for the bacterial
transformation technique as they consist of all four required features. The process is done by cutting the
restriction site with the help of a double digest mechanism, followed by confirmation of restriction
digest through the gel electrophoresis, and the proper plasmid could then be transformed into the
bacteria cell. In order to be replicated, the bacteria has to be grown in proper condition which is called as
the plating process. This report also covered the use of other two commonly used cloning systems,
including pUC18 vector and gateway cloning system.
Materials & Methods (Noted that personal protective equipment and sterilization process were done
before doing any experiments in the lab.)
Sample Preparation
Two plasmids were used, including pKAN-R and pARA plasmids. The pKAN-R plasmid (K solution)
consisted of the gene of interest (rfp), pBAD promoter, and antibacterial resistance gene known as
kanamycin (kanR). In contrast, the pARA plasmid (A solution) consisted of the activator gene known as
arabinose activator (araC) that would activate the pBAD promoter when the bacteria was grown in the
presence of arabinose. The absence of araC did not allow the pBAD promoter to bind with the RNA
polymerase, so that the transcription process could not begin. Moreover, pARA vectors also contain the
origin of replication and antibacterial resistance gene known as ampicillin (ampR). All these materials,
also the restriction buffer, restriction enzymes, and distilled water were stored in the ice box before
being used to keep the plasmid stable and avoid enzyme denaturation (Nguyen et al., 2018).
Restriction Enzyme Digest
This process aimed to cut the fragments of interest on both plasmids, resulting in the same
sticky ends that could then be combined together. Water bath was set to 37℃ prior to the experiment.
Four tubes labeled with (K+, K-, A+, A-) were prepared and a restriction buffer (2.5XB) was added to the
all four tubes. The next step was followed by the addition of pKAN-R to both K+ and K- tubes, while pARA
was added to both A+ and A- tubes. Restriction enzymes, including BamHI and HindIII, were involved in
this step to cut the restriction site of both plasmids so that the recombinant plasmid could be obtained.
As two different enzymes were used in each end of the plasmids, this term was called a double digest
mechanism which functioned to ensure proper insertion orientation (5’ end located in the 5’ site near
the promoter). These restriction enzymes were added to both positive tubes, while the negative tubes
were only added with the distilled water as both were set as the controls. Centrifugation and incubation
processes were initiated afterwards to optimize the digestion by the restriction enzymes.
Verifying Recombinant Plasmid
Samples were divided into the undigested plasmids (gK- and gA-), the digested plasmids (gK+ and
gA+), and the ligation of the digested pKAN-R and pARA plasmids (gLIG). The restriction enzymes from
the previous procedure would result in an open region plasmid which allowed it to be inserted with the
exogenous gene of interest and they would be combined with ligase enzyme, forming recombinant
plasmid. This recombinant plasmid could be separated into linear fragments based on the molecular size
through the electric current conducted by the agarose gel electrophoresis. This process was performed
in order to determine if the desired plasmid (pARA-R) had carried the rfp gene during the restriction and
ligation processes.
Addition of dH2O and loading dye prior to doing the gel electrophoresis were necessary to
initiate the electricity, also enabling the visualization of the DNA bands. The step was continued by the
centrifugation in order to mix all the solutions. A 1X sodium borate buffer was poured into the gel
electrophoresis box and a DNA ladder was pipetted into the first well. This DNA ladder consisted of a
mixture of DNA fragments of known size that could be used to estimate the unknown size of the
fragments of interest. The following wells were then filled by the gK-, gK+, gA-, gA+, and gLIG solution
respectively. The electricity conducted through the gel electrophoresis then began at 130V for 40
minutes.
Bacterial Transformation
Bacterial transformation process was aimed to facilitate the uptake of the plasmid of interest by
the bacteria cell. This study used Escherichia coli to uptake the pARA-R plasmid which contained all the
essential features. Nevertheless, only the competent bacteria cell that could take up the plasmid DNA.
Therefore, the E.coli should be chemically treated to induce the cell competency and this was done
through several processes. Samples were divided into control group (P-) and experimental group (P+).
The control group contained E.coli cells without the recombinant plasmid, while the experimental group
contained E.coli with the recombinant plasmids. These samples were incubated/heat shocked at 42℃ in
order to make the cell membrane more porous so that the recombinant plasmid could enter the cells.
This was done only in 45 seconds to prevent bacteria cell death and samples were immediately placed
back into the ice bucket so that the cell membrane became intact again. Electroporation or electric shock
could be used as the alternative method to enlarge the pores of the cell membrane with the use of
electric current. These both processes needed a pre-treatment of calcium chloride that would help to
neutralize the negatively charged ion of cell membrane and DNA. After all processes were done, samples
were incubated at Luria Bertani (LB) broth and were stored in the refrigerator.
Plating Transformed Bacteria
Plating transformed bacteria was performed to confirm that E.coli had acquired recombinant
DNA plasmids through the bacterial transformation process. This experiment performed three different
conditions to plate the transformed (P+) and non-transformed (P-) bacteria. The three different
conditions performed were LB, LB/amp, and LB/amp/ara plates. The LB and LB/amp plates were divided
into two sections, in which P- samples were put on the left side of the plates and the other side was
filled with the P+ samples. On the other hand, the LB/amp/ara plate was used only to grow the P+
sample. Cell spreader was necessary in this experiment to spread the bacteria equally inside the plates.
All tubes were inverted to prevent condensation dropped into the gel then were incubated for 24 hours
in the incubator.

Result
Restriction Enzyme Digest
Two DNA fragments were produced by BamHI and HindIII, including the gene of interest and the
plasmid backbone. These fragments would appear as linear fragments (A+ and K+ tubes) which animated
as shown in figure 1. However, the fragments were difficult to see with the naked eye in real life and,
therefore, gel electrophoresis was necessary to predict the presence of the fragments corresponding to
their molecular size. Both A+ and K+ samples showed two bands, while K- and A- showed only one band
at the top of the well. This indicated that pKAN-R plasmid (K+ sample) had been cut into rfp and pBAD
linear fragments, while pARA plasmid (A+ sample) had been cut into ampR and araC linear fragments.
The result also showed the importance of restriction enzymes in cutting the plasmid into linear
fragments as the negative tubes, which were not added by these enzymes, only showed one large uncut
circular fragment.

Figure 1. Restriction enzyme in positive tubes resulted in linear fragments

Figure 2. Clear fragmented bands displayed by the gel electrophoresis

Verifying Recombinant Plasmid
The ligase enzyme would join all the fragments of interest into one recombinant plasmid. A lot of
variation might appear and the one with all important features, including ORI, rfp, araC, ampR, and
pBAD, was the expected result (pARA-R) as shown in figure 3. The gel electrophoresis was conducted to
identify the presence and the size of the fragments of interest in the sample. The positive samples (gK+
and gA+) showed two fragments of interest which indicated successful restriction digest. Different with
the positive samples, the negative samples (gK- and gA-) appeared in three different conformations,
including multimer, nicked and supercoiled as it consisted of circular plasmid. The nicked plasmid was
damaged plasmid while the farthest fragment travelled was the supercoiled plasmid as it was more
dense compared to the others. The fragmented circular plasmid did not really correspond to the size of
the DNA ladder as the DNA ladder consisted of linear fragments. Furthermore, the gLIG sample showed
multiple bands near each other which indicated a lot of recombinant plasmids possibilities occurred and
they had their own sizes. All the results also showed partial or incomplete cuts which might be due to
non optimal conditions performed during the experiment.

Figure 3. The recombinant plasmid should have all the essential features

Actual Result Ideal Result

gK-
gK+

gA-

gA+

gLIG

Table 1. Actual vs ideal results of gel electrophoresis

Bacterial Transformation & Plating Transformed Bacteria
The ideal result should yield plasmid within the E.coli cells in the P+ tubes as animated in figure
4. This indicated that the process to induce the E.coli cells was successful as it was able to uptake the
plasmids of interest. This result was confirmed through the plating transformed bacteria process by
observing their growth. Three different growth mediums were used and the result was shown in figure
5. The Luria Bertani (LB) broth was competent for most bacteria cells to grow and therefore the result
showed abundant bacteria in the plate. The second medium consisted of LB and ampicillin which aimed
to detect the presence of ampR in the plasmids. AmpR codes for the enzyme B-lactamase which
inactivated ampicillin and allowed the bacteria to grow in its presence. Those bacteria that still survived
in the P+ section indicated that the bacteria had taken up the ampR antibacterial resistance gene in the
plasmids. Moreover, arabinose was also added to the growth medium which would bind to araC then
promote the RNA polymerase to bind with pBAD promoter and initiate the transcription of rfp gene. This
rfp gene was derived from a sea anemone that produced a red fluorescent protein and thus the red color
bacteria in LB/amp/ara plate indicated that they had successfully produced the genes of interest.

Figure 4. Animation of transformed bacteria vs non-transformed bacteria

Figure 5. Growth of bacteria in three different conditions

Discussion
There were still several pitfalls that might occur during the bacterial transformation process. The
incomplete cleavage of the restriction enzymes due to being blocked by salt buffer and PCR components,
as well as too short incubation period might lead to few or absence of fragments. This could be
overcomed by involving DNA purification steps prior to doing the restriction digest. Sterile materials,
equipment, and proper incubation period were also necessary to prevent those pitfalls (Wigand, 1987).
Pitfalls might also come from the error during the gel electrophoresis. Extended running time
would cause the samples to fall off the gel, while insufficient running time could lead to unclear
separation of the fragments (Sanderson et al., 2015). Non optimum temperature and restrict buffer used
also resulted in incomplete fragments as shown in the result. Appropriate materials and equipment
used, as well as technique to load the samples into the well were needed to prevent all the pitfalls. The
insufficient heat shock step could also have an impact on the bacterial transformation efficiency. Bacteria
faced a heating process for too long could cause denaturation of protein even lead to cell death, while
too short heating process could not result in complete uptake of plasmids (Rahimzadeh et al., 2016). A
problem with the transformed bacteria might result in absence of bacteria growth in the mediums.
Sterile equipment was a must in handling the bacteria to prevent any contamination either to the body
or the result itself.
Two different methods could be used as the alternative of bacterial transformation, such as
using the pUC18 vector or gateway cloning system. The pUC18 vector contained ORI, ampR and lacZ
selection marker, multiple restriction site, and promoter. Double digest mechanism could be performed
with the use of EcoRI and NotI restriction enzymes in both ends of the restriction site (Adam et al.,
1999). Bacterial transformation with the pUC18 vector mainly had the same procedures with the pKAN-R
and pARA plasmids. The differences spotted include: buffer used was NEBuffer, samples were heat
shocked at 65℃, and the bacteria would be grown in the addition of ampicillin also x-gal within the LB
broth (Nagamani et al., 2019). The presence of ampR enabled the bacteria to continue growing in the
presence of ampicillin and the lacZ (B-galactosidase) would digest the x-gal, resulting in the blue
coloration (Adam et al., 1999).
Gateway cloning system, on the other hand, was the innovative technique to do the bacterial
transformation. It allowed multiple DNA fragments to be inserted into the plasmid which then could be
transferred between plasmids (Khan, 2016). This technique involved two enzymes, including BP and LR
clonase enzymes. The BP clonase enzyme recombined attB and attP sites that would result in attL and
attR sites, while the reverse reaction was done by the LR clonase enzyme (Reece-Hoyes & Wallhout,
2018). This method could be used for certain functions, such as developing a synthetic biological circuit
to treat cancer. Expression of miRNA in cancer cells would bind to the mRNA which resulted in the
inhibition of the translation process. In this case, manipulating the biological circuit by performing
promoter, coding sequence (BAX gene), and complementary region of miRNA that had low expression in
cancer cells, but high expression in healthy cells were expected to cure patients with cancer. This
synthetic biological circuit then needed to be copied multiple times in bacteria cells through the gateway
cloning system to be set as the gene therapy.
All of the three different mechanisms generally aimed for the same function that was genetic
manipulation. They were used to make multiple copies of DNA or known as DNA cloning. These artificial
transformations could be used for several applications in various fields. Bacterial transformation allowed
the scientists to develop effective drugs regarding a certain disease. For instance, bacteria could be
manipulated to produce large amounts of insulin proteins to cure diabetes patients (Khan, 2016).
Moreover, it could also be used for generation of genetically-modified crops and set as advanced
diagnostic tools (Good, 2018). In the agricultural field, bacterial transformation could be used to produce
nitrogen-fixing crops in order to stimulate food production rapidly and reduce the use of fertilizers (Khan,
2016).

Conclusion
Bacterial transformation can be used to generate a massive production of modified genes with
the help of bacteria or eukaryotic cells. The process is done by first cutting the genes of interest then
recombining them into plasmid. Gel electrophoresis is performed to check these genes of interest and
the proper variants of plasmid will be inserted into the competent bacteria cells. Some processes must
be done in case the bacteria can not naturally occur as competent cells, such as heat shock and pre
treatment of CaCl2. The purpose of inserting this plasmid inside the bacteria cell is to allow the rapid
replication of the plasmid by the bacteria machinery. These whole processes can be the future hope in
improving various fields, including medical fields, agricultural, etc. They can be applied for genetic
modification, drugs development, certain disease treatment, as well as gene therapy. Nevertheless,
there are still some pitfalls that might occur during this process which can be optimized by proper
materials, equipment, and technique to perform the bacterial transformation.
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