CHEMICAL AND BIOMOLECULAR ENGINEERING LAB /

BIOENGINEERING LAB

CH1801
BG1801

Gene Transformation

Location: N1.2 B2-41

Name : Felix Ivander Salim

Matric Number : U2320432K

Group : CB04

Date of experiment : 23-AUG-2023

INTRODUCTION Gene Transformation

Probably the greatest strength of molecular biology is the ability to manipulate genetic
material, commonly called genetic engineering. Specific fragments of DNA may be
isolated, cut into discrete pieces by the action of restriction endonucleases, and rejoined
by the action of DNA ligase to create novel genes and other genetic constructs. This
technology allows scientists to study the activity of genes to understand their function.
One of the applications of this technology is the potential to treat genetic diseases, such as
cancers, by gene replacement.

The genetic manipulations described above require large quantities of DNA. One of the
easiest ways to get large amounts of DNA is to place the desired DNA into bacteria, grow
the bacteria, then harvest the bacteria, and isolate the DNA. Bacteria can maintain DNA
as plasmids: circles of DNA that usually contain a gene that allows the bacterium to grow
in the presence of an antibiotic. In this experiment, students will introduce a plasmid into
bacteria. This process is called transformation. Bacteria are treated so they will take the
plasmid up into their cells. These are called competent cells. Transformation involves
mixing competent bacteria with plasmid DNA and then selecting bacteria containing the
plasmid using agar plates that contain an antibiotic.

Purpose

This laboratory protocol will allow students to demonstrate the phenomenon of

transformation in a relatively simple procedure. Competent Escherichia coli (E. coli) cells
which have been kept in freezer storage will be thawed on ice, introduced to a transforming
plasmid by electroporation, and then plated out on a nutrient agar which contains the
antibiotic ampicillin that selects for transformants. Using E. coli without the plasmid as a
negative control (-) and E. coli with the plasmid as a treated or positive (+) sample,
students will be able to directly observe the transformation of bacteria to ampicillin
resistance. The (+) strain will survive on the antibiotic containing agar plate due to the
laboratory procedures conducted by the student. The procedure and observation made by
the student brings home the concept of genotype and phenotype being directly controlled
by the genes which are made of DNA.

Genetic Map of pUC19 plasmid

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[Modified from Sciences Education Foundation]
Sterile Technique

Bacteria are classified into different categories based on whether they present a hazard
under shipping and handling conditions. Nonpathogenic strains, such as the E. coli that
you will be working with, present no or minimal hazards, whereas some other bacteria,
including Salmonella, do, and should be treated with caution. Whenever you are working
with bacteria it is a good idea to treat the bacterial strain as though it were pathogenic and
thus, you will always have good sterile technique regardless of the pathogenicity of the
strain. It is also important to use good sterile technique in order to avoid contaminating
your sample with outside bacteria or contaminating the working area with your sample.
Several rules should be followed when working with bacteria: the benchtop should be
cleared of any unneeded materials; you should wash your hands thoroughly before and
after the experiment; flame containers and instruments when appropriate; and dispose of
all bacterial waste in the biohazard trash.

When bacteria are plated onto a solid media at the proper dilution, a single cell will divide
forming daughter cells that remain together in a clump. This clump is called a colony and
contains more than 107 cells that were derived from one original cell and thus have
identical genomes. Proper sterile technique is important to be able to generate single
colonies. Single colonies are used to confirm the genetic makeup of the bacteria with
which you are working. Single colonies also confirm that your population is not
contaminated with other unwanted bacteria.
[Modified from "Teaching and Learning in the Biological Sciences"]

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PREPARATION

Materials Needed For Hot Shock Transformation You

will need the following items for transformation:

• 50 µl E.coli competent cells (Kept in -80°C)

• 10 pg/1µl pUC-19 plasmid DNA (Kept in -80°C)
• Ultrapure water (Kept in 4°C)
• LB agar plates containing 100 g/mL ampicillin (Kept in 4°C)
LB medium: A medium that consists of tryptone, yeast extract, and NaCl.

• S.O.C medium (Kept in 4°C)

S.O.C medium: A rich medium that consists of tryptone (pancreatic digest of
casein) 2% (w/v), yeast extract 0.5% (w/v), NaCl 8.6 mM, KCl 2.5 mM, MgSO4
20 mM, and Glucose 20 mM

• Ice bucket with ice

• 42°C dry bath
• 37°C rotary shaking incubator
• 37°C incubator
• Digital timer
• 50 ml centrifuge tubes
• Micropipette and micropipette tips (1000 L, 200 L, 2.5/10 L)
• L-shaped spreader

Preparing for Hot Shock Transformation

It is crucial for high efficiency transformation that the cells, plasmid DNA, and
ultrapure water remain cold until after the heat is applied. After the heat is applied,
SOC must be added to the cells immediately to ensure good results. Follow the steps
below to prepare for transformation.

1) Fill ice bucket with ice.

2) Warm LB agar plates containing ampicillin and SOC medium in the 37°C
incubator.

3) Switch on the dry bath, rotary shaking incubator, and incubator to desired
temperatures stated.

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PROCEDURES

1. Place and thaw E.coli competent cells, pUC19 plasmid DNA, and ultrapure water on
ice.
Caution: Use these cells immediately. Do not leave them on ice for an extended
period of time. Unused cells can be re-frozen for later use, but they will suffer a
significant loss of efficiency.

2. Pipette 1µl of the pUC19 plasmid DNA and 1µl of ultrapure water into each vial of
E.coli competent cells (50µl) respectively.
Caution: Handle the competent cells by the top of the vial and not at the bottom.
The volume of DNA added should not exceed 5% of the total cell/DNA mixture.

3. Mix the mixture in the vial(s) gently by swirling or tapping it.

Caution: Do not mix by pipetting up and down to prevent cell lysis.

4. Incubate the vial(s) on ice for 15 minutes.

5. After 15 minutes, incubate the vial(s) for exactly 45 seconds in the 42°C dry bath.
Caution: Do not mix or shake.

6. Remove vial(s) from the 42°C dry bath and place them on ice for 2 mins.

7. Immediately add 250µl of S.O.C. medium to each vial and transfer the vial(s) to a
50ml centrifuge tube.
Caution: Sterile technique must be practiced to avoid contamination.

8. Shake centrifuge tube with vial(s) in a 37°C rotary shaking incubator at 250 rpm for
exactly 1 hour to allow expression of the antibiotic resistance.
Caution: Loosen the cap of the centrifuge tube to allow air circulation.

9. Spread 100 µl from each transformation mixture on a separate, labeled, pre-warmed

LB agar and ampicillin plate.

10. Invert the plate(s) and incubate at 37ºC incubator overnight for 16 – 18 hours.

11. On the next day, record the number of colonies that grew on each plate.

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 LOG REPORT (GENE TRANSFORMATION)

Name: Felix Ivander Salim Group: CB04 D Date: 23 AUG 2023

1. Results

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

Figure 1. Positive Control Figure 2. Negative Control

cfu: colony forming units

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

Calculate the transformation efficiency as transformants per 1 µg of plasmid DNA.
For ElectrocompTM cells, use the formula below to calculate transformation efficiency:

# 𝒐𝒇 𝒄𝒐𝒍𝒐𝒏𝒊𝒆𝒔 𝟏𝟎𝟔 𝑻𝒐𝒕𝒂𝒍 𝑽𝒐𝒍

𝒙 𝒙 𝒙 𝑫𝒊𝒍 𝑭𝒂𝒄𝒕𝒐𝒓 = 𝑻𝒓𝒂𝒏𝒔𝒇𝒐𝒓𝒎𝒂𝒕𝒊𝒐𝒏 𝑬𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒄𝒚
𝟏𝟎𝒑𝒈 𝑫𝑵𝑨 𝝁𝒈 𝑽𝒐𝒍 𝒑𝒍𝒂𝒕𝒆𝒅

*Note: The dilution factor in this experiment is 1

The transformation efficiency for the first positive colony can be calculated using the above
equation.

𝟒𝟎 𝟏𝟎𝟔 𝟓𝟎 𝝁𝑳 𝒑𝒍𝒂𝒔𝒎𝒊𝒅 + 𝟐𝟓𝟎𝝁𝑳 𝑺. 𝑶. 𝑪.

𝑬𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒄𝒚 = 𝒙 𝒙 𝒙𝟏
𝟏𝟎𝒑𝒈 𝑫𝑵𝑨 𝝁𝒈 𝟏𝟎𝟎 𝝁𝑳

𝑬𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒄𝒚 = 𝟏. 𝟐 𝒙 𝟏𝟎𝟕 𝒄𝒇𝒖/𝝁𝒈

The transformation efficiency of the positive agar plate is 2 orders of magnitude smaller than
the desired transformation efficiency of chemically competent E. coli (1 x 109 cfu/µg)

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 2. Discussion

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

transformation, host, plasmid, and competent.

Terms Definition
Transformation A method that alters bacteria’s DNA by introducing
foreign genetic material through the membrane cell,
such as DNA fragments, plasmids, etc.
Heat-shock transformation A process of rapidly changing the temperature of the
cell to create membrane pores that will allow the
introduction of foreign genetic material.
Host A cell that is inserted with foreign genetic material.
Plasmid A circular DNA fragment that is essential for
introducing foreign genes into host organisms.
Plasmids can autonomously multiply within the host
cell and are distinct from the chromosomal DNA of
the host organism. For the foreign DNA that
researchers seek to transfer into the host, they act as
carriers.
Competent A cell that has the ability to take in foreign material
from the surrounding environment through the cell
membrane.

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(2) State why E. coli is used in many genetic engineering experiments.
a) Safety Reasons
Even though some strains of E. coli can cause sickness or discomfort, the types of
E. coli utilized in common genetic engineering experiments are non-pathogenic and
represent little to no risk to lab occupants.
b) Simplicity of Genomes Along with Thorough Understanding
Genetic engineering experiments are made easier by the simple genetic makeup of
E. coli. It makes E. coli an appealing choice for genetic engineering experiments
since it lacks many of the intricacies present in other eukaryotic creatures, such as
humans. E. coli is also favoured in genetic experiments due to the depth of
knowledge that has been developed regarding its nucleic acid and protein
manufacturing pathways.
c) Presence of Natural Plasmids
E. coli naturally contains plasmids, which are small, circular DNA fragments that
can be easily manipulated and utilized as vectors to splice in foreign DNA. Scientists
can insert desired genes into plasmids, which E. coli readily accepts, to cause the
plasmids to express the added genes.
d) Versatility
To perform a range of activities, including producing therapeutic proteins and
enzymes and metabolizing certain chemicals, E. coli can be genetically altered. It is
an essential tool for biotechnology and other commercial uses because of its
versatility.
e) Ease of Cultivation
A temperature of 37°C is conducive to E. coli growth and is easy to maintain and
manage for an extended period of time. E. coli can also reproduce in both aerobe and
anaerobe conditions. As a result, very cheap culture medium is needed for E. coli
culture.
f) High Rate of Reproduction
Under ideal conditions, E. coli cells can double every 20 minutes. A parent cell could
produce a million E. coli cells at this rate in just under seven hours. E. coli research
can be completed quickly, conveniently, and economically thanks to fast growth.
g) Genetic Modifications Are Passed Down
Since E. coli primarily replicates asexually, any changes to the genome are preserved
and the consequences of these mutations can be replicated. E. coli is a useful model
organism for molecular genetics because of these aspects.

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(3) Explain why competent cells, ampicillin, and S.O.C. medium were used for the
transformation.

a) Competent cells are bacteria that have undergone a process or preparation that
increases their receptivity to foreign DNA. The cells can take up and incorporate the
desired DNA thanks to their improved permeability. 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.
b) Ampicillin can be used to separate the bacteria that took the pUC19 plasmid
successfully from those that did not. On Agar media, bacteria cannot survive if the
gene transformation was unsuccessful. Bacteria that have been successfully
transformed will also be penicillin resistant, allowing them to live on Agar media.
c) For the growth of E. coli, S.O.C. medium is used as a source of nutrients and other
essential ingredients. It allows the bacteria to develop and recover after a heat-shock
transformation. The S.O.C. medium, also known as Super Optimal Broth with
Catabolite Repression, is frequently used to boost the efficiency of bacterial plasmid
conversions.

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

In this experiment, there are two controls, a positive control and a negative control.
The positive control will undergo gene transformation as a result of the pUC19 plasmid
treatment, while the negative control receives no external genetic material treatment.
Positive controls are organisms that are expected to develop resistance to the antibiotic
ampicillin and subsequently flourish on an agar plate. Meanwhile, a negative control is
being performed to make sure that the colonies seen in the positive control were actually
created by the insertion of the pUC19 plasmid, which is the experiment's independent
variable. Negative controls with no colonies found indicate that the experiment was
successful.

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

According to the colony growth, the E. coli survived on the ampicillin-filled LB agar
plate and carried on with binary fission reproduction. This demonstrates that the
ampicillin-resistant gene on the pUC-19 plasmid was acquired by the E. coli in the
positive sample by homologous recombination during gene transformation. The host
bacterial cell's machinery then assisted in the transcription and translation of the newly
added pUC-19 gene, resulting in the formation of an enzyme that can degrade ampicillin.
Because there is no longer any ampicillin to prevent the development of cross links
between the peptide chains in peptidoglycan, which is essential for the tensile strength
of the cell wall, the new E. coli's cell walls won't rupture under osmotic pressure. As a
result, colonies of bacteria are created as the ampicillin-resistant genes continue to be
passed on to the other freshly synthesized daughter bacterial cells.

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(6) Describe how ionic strength of DNA solution affects electroporation.
Due to the negatively charged phosphate group on the sugar phosphate backbone, the
DNA solution is negatively charged. Similar to human cells, most bacterial cells,
including E. coli, are negatively charged as a result of their abundant phosphate and
amino acid content in the peptidoglycan cell wall. In order to lessen the electrostatic
repulsion between the two potentially charged media, transitory holes are produced
during heat shock, which causes the plasmid DNA to be driven away from the bacterial
cell. Therefore, this might prevent the pUC-19 plasmid from becoming incorporated into
the bacterial cell.

(7) If your transformation efficiency is lower than 1 × 109 cfu/µg, conjecture and explain
potential reasons for the low efficiency.
The transformation efficiency of the positive control in the experiment was far from the
transformation efficiency of optimally competent E. Coli. There are several factors that
could have effected the outcome of the experiment, such as:
a) Prolonged stays in ice-cold environments
Due to long periods of time in cold temperatures (in this case, ice), the competent
cells lose efficiency at a significant rate. Before the start of the experiment, the E.
coli was put in an ice box for an undisclosed period of time. This could have effected
the cell’s ability to take on foreign genetic material.
b) Competence of the E. coli used
The capacity of bacterial cells to absorb DNA from their environment is known as
competence. The level of competence in E. coli varies among strains and is low by
nature. Naturally, some strains are more capable than others.
c) Improper handling during the experiment
The competent cells are extremely fragile, especially after the heat-shock
transformation process. Small vibrations and tremors could cause lysis of the cells.
d) Miscalculation of the number of colonies
The calculation of the colonies was done by manually counting the number of
colonies on the agar plate. Human error is expected. Due to this underestimation, the
calculated efficiency could be greatly effected.
e) Cell age of the E. coli used
The age of the competent cells that are used greatly effect the transformation
efficiency. If the cells that are used are too young or too old, they may not be as
competent in taking up foreign genetic material.

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(8) Discuss current and potential applications of gene transformation techniques in
biotechnology.
a) Production of Recombinant Proteins:
Particular genes that frequently encode proteins of interest are inserted into host
species like bacteria, yeast, or mammalian cells. When these genes are successfully
incorporated and expressed, a lot of the required protein can be produced. For
instance, bacteria like Escherichia coli alter genes to create human insulin, a crucial
therapeutic protein for the treatment of diabetes. Millions of patients around the
world will benefit from this method's stable and high-yield protein source.
b) Treatments for Diseases:
To treat genetic abnormalities and other diseases, for instance, gene therapy includes
inserting therapeutic genes into the cells of the patient. This ground-breaking
strategy shows promise in treating diseases like cystic fibrosis, some forms of
blindness, and cancer. Furthermore, gene modification in stem cell research alters
stem cells for regenerative medicine uses, providing hope for tissue regeneration and
organ transplantation. Gene transformation provides precise control over genetic
material, which is essential for exploring and realizing the possibilities of
personalized treatment.
c) Agriculture:
Genetically modified (GM) crops have been produced thanks to gene-transformation
technology, revolutionizing the agricultural industry. These crops are genetically
modified to have advantageous characteristics, such pest resistance or herbicide
tolerance, boosting agricultural output and sustainability. Additionally, GM crops
can be strengthened with added nutrients to address the issue of malnutrition in many
regions of the world. Gene transformation is essential for ensuring food security and
lowering the demand for dangerous chemical pesticides by increasing crop output
and resilience to environmental stresses.
d) Pharmaceutical Manufacturing
The creation of biopharmaceuticals, which are medicines obtained from living
things, is one significant use. The development of cell lines or microorganisms that
can produce complicated biopharmaceuticals like monoclonal antibodies or
vaccinations is made possible through gene transformation. These GM organisms
work as large-scale bioreactors to produce these essential medications. By providing
more effective and affordable production techniques, this strategy transforms the
pharmaceutical industry.
e) Biotechnology for the environment:
Microorganisms that have been genetically modified are created to carry out certain
functions, such as bioremediation, which entails the detoxification or removal of
toxins from the environment. These bacteria may digest harmful substances, break
down oil spills, and repair contaminated habitats. Additionally, the creation of
biofuels, in which designed bacteria effectively transform organic materials into
renewable energy sources, depends on gene transformation. This helps to create a
future that is greener and more sustainable.

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Conclusion
The method used in this experiment to carry out gene transformation is provided as a
conclusion. Two controls were obtained for this experiment, a positive control using
transformed cells and a negative control using normal cells. After incubation, bacteria
colonies could be detected at the positive control, proving the efficiency of the gene
modification, whereas none could be seen at the negative control. The experiment's
transformation efficiency result is 1.2 x 107, which is significantly less than the
efficiency of fully competent E. coli cells. The lack of efficiency could be attributed to
the factors stated above.

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 REFERENCES

Hanahan, D. (Published in Journal of Molecular Biology, 1983). Efficient

Transformation of Escherichia coli with Plasmids.

R.W. & Primrose S.B. (March 1980). Principles of Gene Manipulation

The National Center for Biotechnology Information (NCBI).

http://www.ncbi.nlm.nih.gov/

Dower, W.J. (Published in Proceedings of the National Academy of Sciences, 1988).

Plasmid Transformation in Escherichia coli: Role of Heat Shock and Calcium Ions.

Stanley Maloy & John Cronan. (January 1994). Microbial Genetics.

Applications of Genetic Engineering (2021). From https://bio.libretexts.org/@go/page/9491

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