STUDENT ID
Chong Khai En
15WAU08702
Goh Yee En
15WAU08280
15WAU09813
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.
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.
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)
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/