The document provides an overview of bacterial transformation protocols, highlighting three main steps: preparation, shock, and recovery. Various methods, including chemical transformation and electroporation, are discussed for making bacterial cells competent to uptake DNA, emphasizing the influence of divalent cations and the importance of cell wall structure. It compares the efficiency of electroporation to chemical transformation, noting that electroporation generally offers higher transformation efficiencies albeit at a higher cost and with sensitivity to salt.

1. Overview of bacterial transformation
 Transformation protocols of a bacterial cell
follow three basic steps.
 1- Preparation step, in which the recipient
bacterial cell is made receptive to uptake of
exogenous DNA (made competent), usually by
modifying the cell membrane(s) and weakening
the cell wall to make them more permeable to
macromolecules

2.  2-Shock step, in which the cell, in the presence
of the DNA, is subjected to a non-lethal shock
(usually heat, high voltage, or sonic) that
transiently opens the cell membrane/wall and
allows DNA to permeate into the cell interior.
 3-Recovery step, the cell is incubated in a rich
medium to restore its cell membrane/ wall.
Overview of bacterial transformation

3.  The cells are made competent by being incubated
with DNA in the presence of divalent cations.
 Many cations were tested for their ability to induce
transformation and shown to have a positive effect
in the following order: Mn2+
> Ca2+
> Ba2+
> Sr2+
>
Mg2+
.
 The cations' positive charge may help to condensate
the naked DNA by shielding the negative phosphate
groups, hence making a smaller DNA packet for cell
uptake.
Chemical transformation

4.  The cations can cancel out electrostatic
repulsive forces between DNA and the outer
membrane and thereby facilitate DNA–
membrane contact
 An important part of chemo transformation is
subjecting the cells to a “cold”-shock, e.g., by
incubation on ice.
 This followed by heat-shock at 42°C for 30 s for
E. coli, followed with 60 s on ice.
Chemical transformation

5.  Electrical fields at around 5–10 kV/cm for
5–10 μs can induce pore formation in
different types of cells and hence promote
uptake of different molecules including
DNA.
 This method, termed electroporation.
 The mechanism behind transformation by
electroporation is not entirely clear
Transformation of bacteria with
electroporation

6.  The competence level of the cells varies in
relation to the growth phase.
 This optimum varies depending upon species,
strain, and growth medium.
 E. coli cells become more resistant to the
detrimental effects of electroporation late in the
growth phase.
 At the same time, probably caused by the same
underlying changes in cell membrane structure,
less susceptible to transformation.
 Balancing these two parameters is therefore
important.

7.  In general, the transformation efficiency
increases with higher DNA concentrations until
a saturation level is reached.
 This saturation level seems to be specific to
each species.
 The purity of the DNA is important.
 Removing salt from the exogenous DNA prior to
electroporation can give good results.
 The topology of the transforming DNA may be
important for transformation efficiencies, with
supercoiled DNA being more efficient in
transformation than relaxed DNA.

8.  Membrane components of cells subjected to
short, high-voltage pulses are polarized and that
a voltage potential develops across the
membrane.
 If the potential difference exceeds a threshold
level, local transient pores are formed resulting
in permeability to charged molecules like DNA.
 Formation of the pores happens on timescales
of 10 ns while resealing of the pores happens
on a time scale of seconds

9.  As for other transformation methods, the
structure and density of the cell wall are crucial
to the obtained transformation efficiencies.
 In many cases increasing the fragility of the cell
wall or using protoplasts and spheroplasts
increase the transformation efficiency
significantly.
 Protoplast refers to a bacterial sphere that is
bounded by a single membrane and spheroplast
refers to a sphere that is bounded by two
membranes.

11. Electro-competent cells
 Grow o/n culture in liquid media
 1:100 dilution with fresh media and incubate
for 3 hr (OD 600 is near 0.5).
 Place on ice for 10 min.
 Wash twice with equal volume of ice-cold
Wash solution (10 % glycerol)
 Wash = centrifuge at 5000 x g for 5 min at
4C, then resuspend the pellet in 1 ml of wash
solution and then add wash solution to the
desired volume

12. Electro-competent cells
 Wash once with half volume of ice-cold Wash
solution.
 Resuspend the cell in 1/10 volume of ice-cold
wash solution.
 Divide into small volumes (50-100µl)
 Store on ice for immediate use or at – 70C
for later use

13. Chemically competent cells
 Grow bacterial cells overnight
 Subculture bacterial cells in fresh medium
( dilution 1:100)
 Incubate with vigorous shaking for 3 hours till
OD 600 = 0.4
 Centrifuge @ 5,000 x g 5 min at 4C
 Remove supernatant and resuspend in (0.7
Vol) ice cold ( 80mM MgCl2-20mM CaCl2)
solution.

14. Chemically competent cells
 Centrifuge @ 5,000 x g 5 min at 4C
 Remove supernatant and resuspend in
(0.1 Vol) ice cold (0.1M CaCl2) solution
 Divide into small volumes (50-100µl)
 Store on ice for immediate use or at –
70C for later use

15. Desalt DNA for Electroporation

16. Electroporation
 Thaw your competent cells on ice
 Gently, mix 2-3 µl of your DNA with 50 µl cells.
 Incubate on ice 15 to 30 min.
 Transfer the cells/DNA mix to a pre-chilled
electroporation cuvettes.
 Place the cuvettes in the electroporation
chamber.
 Pulse the cells.
 Add 250 -300 µl recovery media
 Incubate at 37 C for at least 40 min.
 Plate 100 µl on selective media.

17. Chemo-transformation
 Thaw your competent cells on ice
 Gently, mix 5 µl of your DNA with 50 µl cells.
 Incubate on ice 20 to 30 min.
 Transfer to water bath 42C for 30 sec.
 Quickly transfer to ice again for 2 min.
 Add 250 µl SOC medium and keep in incubator
for 45 min at 37C
 Plate 100 µl on selective media.

18. SOC medium (Recovery medium)
 0.5% Yeast Extract
 2% Tryptone
 10 mM NaCl
 2.5 mM KCl
 10 mM MgCl2
 10 mM MgSO4
 20 mM Glucose

19. Electroporation vs. Chemical
Transformation
 Electroporation is less cumbersome than
chemical transformation and generally gives
higher transformation efficiencies (measured in
colonies formed per microgram of DNA).
 However, it is more expensive, requiring
specialized apparatus to deliver the charge and
cuvettes to transfer the charge to the cell
suspension.
 Electroporation is sensitive to salt, so precious
samples can be lost if excess salt is carried over
into the cuvettes.