CHEMICAL AND BIOMOLECULAR ENGINEERING LAB /

BIOENGINEERING LAB

CH1801
BG1801

Gene Transformation

Location: N1.2 B2-41

Name: MATTHEW KEVIN HARDJOPRANOTO

Matric Number: U2220991G

Group: CBE04

Date of experiment: 17-AUG-2022

 LOG REPORT (GENE TRANSFORMATION)
Name: MATTHEW KEVIN HARDJOPRANOTO (U2220991G)
Group: CB204
Date: 17-AUG-2022

1. Results

(1) Schematically draw what you observed on the agar plates

(2) Count the colonies and calculate the transformation efficiency.

Total transformation volume = 250 µl (SOC) + 50 µl (E.coli sample) = 300 µl

Volume plated: 100 µl, Dilution factor = 1
Total colonies: ± 40 colonies
 6
40 10 𝑝𝑔 300 µ𝑙 7
10 𝑝𝑔
× μ𝑔
× 100 µ𝑙
×1 = 1. 2 × 10 𝑐𝑓𝑢/μ𝑔

2. Discussion

(1) Define the vocabulary used in this experiment: transformation, heat-shock

transformation, host, plasmid, and competent.

Transformation A procedure that modifies bacteria's DNA by introducing

external genetic material—such as DNA fragments,
plasmids, etc.—through the membrane cell.

Heat-shock A method of creating membrane holes by subjecting the

Transformation cell to a rapid temperature shift, which will enable the
insertion of external genetic material into the cell.

Host A cell that is introduced with external genetic materials.

Plasmid A tiny, circular DNA molecule that can replicate on its

own and is distinct from chromosomal DNA. Plasmid,
which is often present in bacterial cells, has the potential
to convey advantageous genetic traits like antibiotic
resistance.

Competent A cell that can absorb external genetic material from its
environment.

(2) State why E. coli is used in many genetic engineering experiments.

There are several reasons why E.coli is used for scientific and engineering experiments
a. E.coli is easy to modify and also competent because DNA molecules are introduced
into cells with a high degree of efficiency in E. coli, this organism is a favorite host
for gene cloning.
b. Due to its quick growth and capacity for extremely high protein expression, E. coli
is a favoured host for the manufacture of proteins. It can produce millions of E. coli
in only a few hours since it just takes 20 minutes for it to double.
c. E. coli grows well at a temperature of 37°C, which is simple to maintain and manage
for an extended period of time. As a result, the culture of E. coli requires very
inexpensive culture medium.
d. Large DNA pieces may be transferred from one bacterium to another through
bacterial conjugation.
e. Due to the extensive understanding of its nucleic acid and protein biosynthesis
pathways, E. coli is a favoured host for the study of phage biology.
f. E.coli is also safe to use as it has non-pathogenic strains which result in little or no
harm to the environment in case of poor sterile technique is conducted.
(3) Explain why competent cells, ampicillin, and S.O.C. medium were used for the
transformation.

Competent cells are utilized because they can absorb exogenous genetic material
more efficiently than regular cells. In order for the E.Coli used in this experiment to
be able to take up the pUC19 plasmid DNA across the cell membrane, it underwent
some transformation by adding various chemical substances.

The bacteria that successfully accepted the pUC19 plasmid may be distinguished
from those that did not using Ampicillin. Bacteria cannot survive on Agar media if
the gene transformation was not successful. Successfully converted bacteria will also
be resistant to penicillin, enabling them to survive on Agar media.

S.O.C. medium is utilized as a source of nutrients and other vital components for
the growth of E. coli. Following a heat shock, it enables the bacteria to develop and
recuperate. Super Optimal Broth with Catabolite Repression, or S.O.C., is the
abbreviation for this broth, which is often used to increase the effectiveness of
bacterial plasmid transformations.

(4) Explain the purpose of the controls in this experiment.

There are two controls in this experiment: positive control and negative control.
While the negative control is not treated with any external genetic material, the
positive control will experience gene transformation as a result of the pUC19
plasmid treatment. Positive controls are those that are predicted to be able to resist
the antibiotic ampicillin and hence proliferate on the agar plate. In the meantime,
negative control is being carried out to make sure that the addition of the pUC19
plasmid, which is the independent variable of the experiment, is what produced the
colonies discovered in positive control. If there is no colony identified in negative
control, the experiment is successful.

(5) Explain how the colony growth relates to gene transformation.

Antibiotics are used to treat or prevent some types of bacterial infection. They work
by killing bacteria or preventing them from spreading. E. coli bacteria is being
cultivated on agar plate containing ampicillin antibiotic. The antibacterial ampicillin
is used to distinguish between cells that successfully acquired the pUC19 plasmid
and those that did not. Cells that have successfully undergone transformation will be
able to express the genetic code for antibiotic resistance. The colonies that these
cells eventually create through self-division. The number of colonies that can be
seen in the positive sample corresponds to the number of cells that underwent the
heat shock transformation effectively. A colony of cells represents one modified cell.
As none of the cells in the negative sample have undergone any genetic alteration,
no colony is seen there.
(6) Describe how ionic strength of DNA solution affects electroporation.

By suddenly applying a high electric voltage to the cell, a process known as

electroporation, holes are created in the cell, allowing the cell to absorb external
genetic material. The DNA solution's ionic strength must be kept as low as feasible
because electroporation depends on it. High ionic strength solutions will create
arcing, which could kill a cell because the cell membrane and DNA are both
negatively charged, which will generate considerable repulsion, arcing occurred
during electroporation.

(7) If your transformation efficiency is lower than 1 × 109 cfu/g, conjecture and explain
potential reasons for the low efficiency.

The transformation efficiency in the experiment was 1.2 × 107 cfu/g, which is two
order less than the predicted efficiency. The following are a few mistakes that could
happen in the experiment:
a. Cells may die from heat shock. In addition, failing to return the vials to the ice
bath might result in E. coli death.
b. Since there are hundreds of colonies, each with a different size that might be
difficult to distinguish with the human eye, it is impossible to count them all
precisely. This might lead to a lower count, which would ultimately result in a
poorer efficiency.
c. Some of the pUC19 plasmid may not mix thoroughly and end up on the vial's
wall after being added to the container with the cells. As a result, less cells
may be transformed as a result of the effective volume of plasmid injected
being smaller than the prescribed volume, which would affect efficiency.
d. Competent cells are extremely fragile. There are several reasons technical
mistake in the laboratory that might kill the E.coli, for example exposed heat
and UV sunrays.

(8) Discuss current and potential applications of gene transformation techniques in

biotechnology.

Gene modification is being used extensively in agriculture and medicine. For

instance, human insulin is produced outside of the human body through gene
transformation. Patients with Type I diabetes often utilize insulin. These
individuals' insufficient insulin hormone production results in elevated blood
glucose levels. In addition, gene engineering is employed to create certain
proteins or enzymes for use in medicine or research. Gene modification is
employed in agriculture to improve the productivity of the crops. The
introduction of several desired qualities, such as insect resistance, low
temperature tolerance, and increased yield, to the seeds occurs through gene
transformation. As a result, it will increase land productivity and could be able
to feed more people on Earth.

The use of gene transformation has numerous potential applications as well. One
of these involves giving human cells a gene change. It may be feasible to create
 a person with the desired characteristics, which would lower the danger of
hereditary disorders and unwanted qualities. However, it is seen to be morally
wrong and its development is unknown. Additionally, replacing the infected
cells with healthy cells or maybe altering healthy cells to be able to destroy the
bad cells could enable gene therapy to treat chronic disorders. These studies are
still ongoing since human cells are far more complex than bacterial cells. There
is actually limitless potential for genetic engineering to help humans in the
future.

Experiment Conclusion:

In conclusion, this experiment provides the accepted procedure for carrying out
gene transformation. In this experiment, two controls were obtained: a positive
control with transformed cells and a negative control with normal cells. Colonies
of bacteria were seen at the positive control after an incubation, demonstrating
the effectiveness of the gene transformation while there is no bacterial colony
seen in the negative control. The experiment's result in the transformation
efficiency is 1.2 × 107 , which is much lower than the predicted efficiency.