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FACULTY OF APPLIED SCIENCES AND

COMPUTING KUALA LUMPUR CAMPUS

BABS 3213 TECHNIQUES IN BIOTECHNOLOGY


BACHELOR OF SCIENCE (HONOURS) IN
BIOSCIENCES WITH CHEMISTRY
Experiment 1
E.COLI COMPETENT CELL PROTOCOL
NAME

STUDENT ID

Chong Khai En

15WAU08702

Goh Yee En

15WAU08280

Khoh Pui Mun

15WAU09813

DATE OF EXPERIMENT: 15TH JUNE 2016


GROUP: RBS 3 (A1)

Introduction:
Bacterial transformation is the creation of pore in the
bacteriums cell membrane to allow foreign plasmid DNA enters the
bacterium. It is the most commonly used way to introduce foreign
DNA into a bacterium that can amplify the gene and clone the
foreign DNA. This bacterium that has the ability to accept or take
up foreign DNA plasmid from its surrounding is called competent
cell. E.coli is the widely used competent cell as it has small genome
size about 4400 genes. The normal bacteria that are undergoing
very rapid growth are made competent more easily than cells in the
other stages of growth. Furthermore, mid-log phase is the efficient
period that the bacteria become competent cells. For example,
competent cells are found at an absorbance of 0.25 using standard
spectrophotometer in an inoculum of E.coli cells. The time required
to reach mid-log phase is proportional to the size of the initial
inoculum.
Bacterial transformation can be naturally occurring in various
types of bacteria. Apart from that, there are many chemical
methods that can make normal bacterium into competent cells.
These chemical methods can artificially induce and enhance a
bacterium cells competency. One of the methods is heat shock.
Heat shock is the widely used method in bacterial transformation by
using calcium chloride and heat. Normal bacterium is made
competent via exposure to a calcium rich environment such as
calcium chloride solution. The positive charges of the calcium ions
will neutralize the negative charge of both foreign plasmid DNAs
phosphate group and bacteriums phospholipid heat. This will
dissipate electrostatic repulsion and weakening the cell wall. When
the bacterium suddenly exposed in high temperature about 47
degree Celsius known as heat shock, a pressure difference between
the outside and the inside of the bacterium is created. Therefore,
the pressure difference will induce the formation of pores and
supercoiled plasmids DNA will be allow entering inside the
bacterium. After bring back the bacterium into normal temperature,
the cell wall will self-anneal.
Besides heat shock, electroporation is another common
technique for bacterial transformation. Electroporation use a
specialized machine known as electroporation. The normal
bacterium mixed with foreign DNA and placed into an
electroporation cuvette, which has electrodes on each side that
make electrical contact with the machine one inserted. The electric

field is applied for a few seconds and causing the voltage passes
through the cell membrane. This leads to a rearrangement of the
phospholipid bilayer that comprises the cell membrane resulting in
pores formation. Therefore, the foreign plasmid DNA will pass
through the cell membrane and enter the bacterium. After that, the
phospholipid bilayers will self-heal.
Objective: To prepare E.coli competent cell by chemical treatment.
Procedure:
1. One colony from LB plate was inoculated into 2 ml LB liquid
medium. It was then shaken at 37oC overnight.
2. 1 ml overnight cell culture was inoculated into 100 ml LB
medium in a 500 ml conical flask. It was shaken vigorously at
37oC to OD600 for about 1.5-2 hours.
3. The culture was chilled on ice for 15 minutes. The 0.1M CaCl2
solution and 0.1M CaCl2 plus 15% glycerol were also ensured on
ice.
4. The cells were centrifuged for 10 minutes at 4000rpm at 4oC.
5. The medium was discarded and the cell pellet was
resuspended in 30-40 ml cold 0.1M CaCl2.
6. The cells were kept on ice for 30 minutes.
7. The cells were centrifuged for 10 minutes at 4000rpm at 4oC.
8. The supernatant was removed, and the cell pellet was
resuspended in 0.5 ml 0.1M CaCl2 solution plus 15% glycerol.
9. 200 L of the cell suspension was pipette into sterile 1.5ml
micro-centrifuge tubes. These tubes were freeze on dry ice and
then transferred to -70oC freezer.

Results and Discussion:


Luria broth (LB) is a nutrient-rich medium that is used to
culture bacteria in the lab to allow fast growth for many species
such as E.coli. Normally, LB medium can support E.coli growth
(OD600 = 0.25-0.30) in a normal shaking incubation conditions. Due
to time constraint, our group did not incubate the cell culture until
OD600 = 0.25-0.30 which take around 2 hours. The optical density
of a sample at 600 nanometres, OD600 reading is referring to the
density of the cell in unit of cells/mL. The OD reading of our cell is
only 0.006 as we only incubate the cell for around 30 minutes. This
indicates that, the cell concentration is very low.
However, we proceed to the next step which was chilling the
culture and centrifuging the cells. Centrifugation aims to separate a
heterogeneous mixture of solid and liquid by spinning it. It works by
using the sedimentation principle, in which the centripetal
acceleration is used to separate the substances of different density.
As a result, the solid precipitate settles to the bottom layer in a
tube, while the solution (supernatant) becomes clear. Anyway, we
did not manage to see the pellet after centrifugation. It may due to
the very low concentration of the E.coli cell or the cells had lysed.
Most of the cell is unable to take up foreign DNA easily. These
cells have to be treated with physical or chemical treatments and
thus their cell walls are altered to take up foreign DNA. This cell is
known as competent cell. The treatment using calcium chloride
(CaCl2) is one of the methods to prepare competent cells. CaCl 2 was
added to a cell suspension to promote the binding of plasmid DNA to
lipopolysaccharide. The positively charged calcium ions attract both
the negatively charged DNA backbone and also the negatively
charged groups in the lipopolysaccharide.
After that, the plasmid DNA can pass into the cell upon heat
shock. This can be done by cooling the cell to a low temperature
(4oC) and a subsequent high temperature (42oC) for a short time.
Therefore, pores are created in the bacterial cell to allow uptake of
plasmid DNA into the cell.

Figure 1: Preparation of competent cell by CaCl2 treatment.


However, we did not manage to carry out this step due to time
constraint. We stored the competent cell in -70 oC freezer for the
next practical.
There are some precautions during this practical. During
centrifugation, we tried to maintain the cells at 4 oC. It was
suggested that not to warm up the bacteria again and work in a cold
room on ice if possible because the quality of the competent cells
will become compensate and lower. All the work should be done
fast, clean and cold to obtain a quality competent cell. When
measuring the OD, the cuvette was ensured to be clean and no gas
bubbles were present because these are the factors which can
affect the OD reading.

Questions:
1. Differentiate between BL21, BL21 (DE3), BL21 (DE3)
pLysS strains.
For the E.coli strains BL21, BL21 (DE3) and BL21 (DE3) pLysS, each
of the following contains different genotype which shows that the
bacterium carries mutant allele.
BL21: E.coli B F dcm ompT hsdS(rB mB ) gal
BL21 (DE3): E.coli B F dcm ompT hsdS(rB mB ) gal (DE3)
BL21 (DE3) pLysS: E.coli B F dcm ompT hsdS(rB mB ) gal (DE3)
[pLysS Camr ]
These B strains of E.coli are general protein expression strains that
lack both the lon protease and the ompT outer membrane protease,
which can degrade proteins during purification.

These three different competent cells provide varying levels of


expression control with T7 promoter-driven vectors, such as the
pCAL vectors and the pET vectors. The BL21(DE3) competent cells
are an all-purpose strain for high-level protein expression and easy
induction. The BL21(DE3)pLysS competent cells provide tighter
control of protein expression for expression of toxic proteins and are
resistant to chloramphenicol. When used with the CE6
bacteriophage, the BL21 cells provide the tightest control of protein
expression. The following table illustrates features of the BL21derived expression strains for protein expression.

Expression strain
BL21 competent
cells

Induction Method
Infection with lambda
bacteriophage CE6

Advantages
Tightest
control of
uninduced
expression

BL21(DE3)
competent cells

Isopropyl-1-thio-Dgalactopyranoside
(IPTG) induction of T7
polymerase from
lacUV5 promoter

High-level
expression

BL21(DE3)pLysS
competent cells

Isopropyl-1-thio-Dgalactopyranoside
(IPTG) induction of T7
polymerase

Ease of
induction

Disadvantages
Induction not as
efficient as DE3
derivatives
Induction
(infection)
process more
cumbersome
Leaky
expression of T7
polymerase can
lead to
uninduced
expression of
potentially toxic
proteins
Slight inhibition
of induced
expression when
compared with
BL21(DE3)

2. Describe the protocol to generate competent cells


through electroporation method.

a) Preparation of Bacterial Cultures, Tools, and Reagents


1. Inoculate 1-5 ml autoclaved LB broth in sterile borosilicate
glass test tubes with a small aliquot of E. coli.
2. Set the inoculated test tubes in a incubator at 37 C
temperature with constant shaking for overnight.
3. Prepare LB-agar without antibiotics and pour into Petri dishes
in preparation of the electrocompetence protocol, and LB-agar
plates containing the appropriate antibiotic (according to the
resistance marker on the vector to be electroporated), and
store at 4 C.
4. Prepare an autoclave ddH2O for E. coli, and store at 4 C.
b) Growth of Electrocompetent Bacteria
1. Deliver 100 l of the overnight bacterial culture onto each LB
agar plate, frozen glycerol stocks may also be utilized and
directly delivered onto the plate.
2. Dip the cell spreader stick in 100% EtOH, briefly drain and
burn off the remaining EtOH to sterilize it prior to use and
spread. Spread the 100 l overnight bacterial culture evenly
with the sterile cell spreader making sure not to disrupt
(break) the agar surface.
3. Incubate the plate at 37 C for 4-6 hours, or until a thin lawn
of bacterial growth becomes distinguishable. Cells are most
competent when actively growing.
c) Preparation of Electrocompetent Bacterial Cells
1. Harvest the bacteria with a sterile inoculating loop without
breaking the surface of the agar.
2. Resuspend the bacterial mass in 1 ml ice-cold sterile ddH 2O
and mix well until no clumps are visible, keep on ice.
3. Centrifuge each bacterial suspension for 5 min at 5,000 x g in
a refrigerated microcentrifuge set to 4 C, or in a
microcentrifuge stored in a cold room set to 4 C.

4. Discard the supernatant, resuspend the bacterial pellet in the


same volume of ice-cold sterile ddH2O, and repeat the
centrifugation step as done before twice more for a total of
three washes.
5. Remove supernatant, resuspend, and then loosen the
bacterial pellet thoroughly in 40 l ice cold 2 mM CaCl 2 or
sterile ddH2O and keep on ice.
3. Why do we need starter culture?
Some of the species grow at lower rate under certain conditions
when the concentration of cells is too low. The log-phase growth of
the bacteria is the most powerful, and so by using starter culture,
we can keep cells in this state for the experiment at hand. Other
than that, another advantage of inoculating with a starter culture is
that it can help in producing reproducible results concerning plasmid
preparations and competent cells preparations. In addition, the
culture may be able to out-compete a contaminant if there is one.
That is more easily accomplished with a starter culture, which is
then used to inoculate a larger culture for scale-up. Inoculating
directing into the large-sized flask may allow the bacteria to enter a
stationary phase, thus giving an opportunity for other species to
out-compete the bacteria.

Conclusion:
The OD reading, OD600 of the cell is 0.006. This indicates a very low
concentration of cell in the culture.

References:
What you need to know about OD600, written by Dr Nick Oswald,
8th December 2008. Retrieved 27th June 2016 from:
http://bitesizebio.com/1005/what-you-need-to-know-about-od600/

Microbial Biotechnology- A laboratory Manual for Bacterial Systems,


S. Das, H. R. Dash, Springer India 2015. Retrieved 27th June 2016
from:
file:///C:/Users/user/Downloads/9788132220947-c1.pdf
Bacterial Transformation: Electroporation|Protocol. 2016. Retrived
27th June 2016 from: http://www.jove.com/scienceeducation/5060/bacterial-transformation-electroporation

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