Emirhan İnan 10.10.

2023
2495372 – Section 1

BACTERIAL TRANSFORMATION

Introduction:
Horizontal gene transfer is surely one of the most important features of bacteria.
It plays a crucial role in natural bacterial evolution and development of new
biotechnologies. These gene transfer mechanisms allow bacteria to adapt to
environmental changes by acquiring new genes without mutation (Yu et al., 2023).
Bacteria can transfer genes to each other in 3 ways. These are conjugation,
transduction and transformation which is the main point of the experiment. The
process that a bacterium absorb genetic material from the environment is called
transformation. Competency which refers to the ability of a bacterium to take DNA
from environment is required for transformation to take place. Since naturally
competent bacteria are very rare in nature, they must be made artificially competent
beforehand. Heat-shock with CaCl2 treatment and electroporation are two common
methods for artificial transformation (Pandey et al., 2021). In this experiment,
NEBstable strain of E. coli were made Ampicillin resistant with artificial
transformation by using heat-shock with CaCl2 treatment method. There are 3
negative controls including no addition of plasmid, addition of plasmid but no heat-
shock and addition of plasmid but no ice incubation after heat-shock.
Materials and Methods:
Materials that are used in this experiment are NEBstable strain of E. coli,
ampicillin resistant plasmids, LB medium, agar plates with ampicillin, micropipettes,
L-shaped spreader, Bunsen burner, centrifuge, water bath, incubator.
Competent E. coli and plasmid solution are placed in ice. 2 μL of plasmid
solution (50 ng/μL) are added to the tube and mixed carefully. Mixture is incubated on
ice for 30 minutes to decrease the temperature of the cells because the difference
between room temperature and 42°C is not enough for heat-shock. After incubation,
the tube is put in water bath whose temperature is 42°C for 50 seconds. Heat-shock
opens pores in the membrane of the cells so that plasmids can enter inside. Then
positive control tube is placed in ice for 10 minutes. The cells are recovered by closing
the pores in the membrane while in ice. 500 μL LB medium is added to the tube.
Sample is incubated at 37°C for 1 hour to allow cells to express the resistance gene.
After incubation, cells are centrifuged at 3500 rpm. 450 μL of supernatant is removed
from the tube and the pellet is resuspended with LB medium. The concentrated
bacteria solution is spread on agar plate including ampicillin near Bunsen burner so
that transformants can be differentiated from nonresistant cells. The sample is
incubated overnight at 37°C.
Results:

Figure 1. Group 1A positive control Figure 2. Group 1A negative control with

no plasmid

Figure 3. Group 1B positive control

Figure 4. Group 1B negative control with no
plasmid

Figure 5. Group 2A positive control Figure 6. Group 2A negative control with

no heat-shock
Figure 7. Group 2B positive control Figure 8. Group 2B negative control with
no heat-shock

Figure 9. Group 3A positive control Figure 10. Group 3A negative control with
no ice incubation

Figure 11. Group 3B positive control Figure 12. Group 3B negative control with
no ice incubation
 Number of colonies
Positive control Negative control
Group Name
Group 1A (no plasmid) 905 0
Group 1B (no plasmid) 2360 0
Group 2A (no heat-shock) 1620 10
Group 2B (no heat-shock) 1848 436
Group 3A (no ice incubation) 1984 1937
Group 3B (no ice incubation) 240 2660

Table 1. Colony numbers in controls of each group

It can be seen from Table 1. that colony numbers of positive controls are high in
Group 1 and the negative controls are both 0. In Group 2, positive controls are again
high but there is a significant difference in negative controls. Group 2B has greater
number of colonies in the negative control. Group 3A has a balanced number of
colonies in both controls while Group 3B has an extreme difference. They have the
lowest number among positives and the highest number among negatives.

Discussion:
For Group 1, positive controls are expected to be high and negative controls are
expected to be 0 because the negative control cells are not transformed with antibiotic
resistant gene in the beginning of the experiment. As it can be seen from Figure 2 and
Figure 4, observed results are matched with expected. The positive control of Group
1A has relatively low number of colonies compared to the other groups, and this might
be due to couple of reasons. The first is that the cells might not have been given
enough time for growth before plating. Improper sterilization of spreader with ethanol
might be another reason causing death of some of bacteria. It is reported that ethanol
can kill bacteria by disrupting their cell membrane and proteins (Chatterjee et al.,
2006).
For Group 2, negative controls are expected to be 0 like Group 1 because pores
need to be formed in membrane by heat-shock so that exogenous DNA can pass but
observed numbers are different than the expected according to Table 1. We can see
colonies in the negative controls of both groups. Especially 2B has significantly more
colonies than 2A. This implies that heat-shock procedure is not the only factor that is
responsible for increasing membrane permeability. It is suggested that CaCl2
treatment also play an important role in pore formation in cell membrane (Rahimzadeh
et al., 2016). However, if we compare the number of colonies in negative controls of
Group 2 and Group 3, it can be said that heat-shock step significantly increases
transformation efficiency.
For Group 3, the negative controls are expected to be lower than the positive
controls because the cells are not recovered in ice after heat-shock. It is reported that
recovery of cells in ice provide higher transformation efficiency than in room
temperature (Singh et al., 2010). However, we see that the number of colonies in
negative controls are almost same or more than positive controls. That can arise from
several reasons. One can be that a lot more cells are plated in negatives. Therefore,
more colonies are formed. Other can be that there is no significant difference between
ice and room temperature incubation. 3B has the lowest number of colonies among
positive controls. This implies a major error during experiment procedure. Reasons for
that can be insufficient time for growth or improper sterilization of spreader similar to
Group 1A. In addition, the antibiotic concentration of the agar plate might be higher
than others and result in death of most of the colonies.
Most of the samples look well distributed except couple plates. It can be seen
from Figures 1, 5, 7, 12; there are some regions that contain a smaller number of
colonies compared to the rest of the plate. Constant rotation of plate and wide range of
motion with spreader while spreading can be done to fix it. No satellite colonies are
observed in any of the plates. Satellite colonies are nonresistant cell aggregations near
resistant colonies. They can form due to delayed incubation time or plating very high
numbers of cells (Medaney et al., 2015). These produce so many colonies and result in
breakdown of antibiotic in environment. Therefore, satellite colonies can grow.
Most of the results fit to the expectations but still there are some parts that are
different than the expected. To correct these errors, procedure steps should be followed
more precisely.

References

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