Bacterial Transformation

Bacterial Transformation

Bacterial Transformation
Bacterial transformation is transfer of free or naked DNA released from donor bacterium into
extracellular environment that results in assimilation and expression of newly acquired trait
in recipient bacterium.
Transformation can happen either naturally or artificially in bacteria.

Competence
The success of transformation depends on competence of the host cell. Competence is the
ability of a cell to incorporate naked DNA in the process of transformation.

Achieving competence in Bacteria

All the cells, whether competent or not, compete for uptake of plasmid but if only competent
cells are used for the transformation, efficiency will be increased up to 50-folds. The practical
approach to acquire competent cells is to make bacterial cells artificially competent using
chemicals or electrical pulses.

 Chilling the cells in the presence of Calcium chloride to make them permeable
 Heat shock treatment at 42°C for 60-120 seconds that causes DNA to enter cells
 Making cells permeable by applying electrical pulses, i.e known as electroporation.

1- sCalcium ion treatment:

E. coli bacteria transformed using two methods;
 CaCl2 treatment followed by heat shock step
 CaCl2 treatment without using heat shock step

DNA being highly hydrophilic molecule, cannot pass through bacterial cell membrane. Bacterial
cells can be made competent to take up DNA by making small holes in bacterial cells by
suspending them in a solution containing high concentration of calcium. The addition of
calcium chloride promotes binding of plasmid DNA to lipopolysaccharides (LPS). Positively
charged calcium ions attract both negatively charged DNA backbone and negatively charged
groups in LPS inner core. The plasmid DNA can then pass into the cell.

DNA entry into E. coli cells is through channels known as zones of adhesion or Bayer's junction.
Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly
(HB) is envisioned to wrap around DNA and is carried in a shield formed by Ca ions.

2-Heat-shock transformation

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Extra-chromosomal DNA will be forced to enter cell by incubating competent cells and DNA
together on ice followed by a brief heat shock that causes bacteria to take up the DNA.
The heat shock step strongly depolarizes cell membrane of CaCl 2-treated cells. Thus, decrease in
membrane potential lowers negativity of cell’s inside potential which ultimately allows
movement of negatively charged DNA into cell’s interior. The subsequent cold shock again
raises the membrane potential to its original value.

Procedure of Ca+ treatment followed by Heat Shock:

 Overnight cultured E. coli Luria Bertani medium is incubated on shaker at 37 ˚C.
 The suspensions are kept on ice for 30 min, then bacteria are pelleted at 1717 × g for 20
min at 4 ˚C.
 The bacterial pellet is suspended in CaCl2 (100mM) and placed on ice for 30 min.
 Next, cell suspensions are centrifuged as above and pellets are dissolved in CaCl 2.
 Thawed bacteria and DNA (isolated from E. coli strain DH5 and qualified with gel
agarose electrophoresis) are added to pre-chilled tube.
 The suspensions are kept on ice for 30 min and after that mixture is heated at 42 ˚C for
45 seconds.
 Following heat shock step tubes are kept on ice for another 5 min.

Advantages of Ca+ ion treatment and heat shock:

 Ca2+ could develop the interaction between DNA molecules and LPS (lipo-
polysaccharide) of outer membrane. Ca2+ divalent cations enhance structural changes in
phosphatidylcholine-cardiolipin bilayers which lead to increased permeability.
 Ca2+ has proven to be most effective both alone and in combinations. A combination of
divalent and monovalent ions, such as calcium and magnesium(increases
transformation efficiency by 15-20 folds) calcium and manganese, calcium and
rubidium, and dimethyl sulfoxide and other alkali metals with a prolonged incubation at
0°C has also been reported to be effective.
 Use of Ca+ ions or heat shock is an economical choice as compared to electrophorises.

Limited range of cells can be transformed:

One of the limitation of chemical methods for transformation is that they are less efficient to
achieve competence in cells having tough cell walls e.g plant cells.

3-Electroporation
Electroporation is a process in which brief electrical pulses create transient pores in plasma
membrane that allow nucleic acids to enter cellular cytoplasm.

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Molecular mechanism of electroporation:

Electroporation causes membrane indentation followed by formation of transient hydrophobic
pores whose diameter fluctuates from minimum of 2 nm to maximum of several nanometers.

Some of larger hydrophobic pores are converted to hydrophilic pores because energy needed
to form an aqueous pore is reduced as trans-membrane voltage is increased and energy
required to maintain circumference of larger hydrophilic pore is significantly lower than that
required to maintain large hydrophobic pore.

Thus, hydrophilic pores have an extended half-life and may be further stabilized by attachment
to underlying cytoskeletal elements. Reclosing of pores can be delayed by keeping cells at 0°C.

Advantages of electroporation
 Low mutation rate: DNA enters directly into cytoplasm, it is not exposed to acid
conditions in endosomes and lysosomes. Thus rate of mutation in DNA introduced to
cells by electroporation is apparently very low compared with DNA transfected as
calcium phosphate coprecipitates.
 Applicable to mammalian cells: It works for very wide variety of mammalian cells,
including those that are difficult to transfect by other means.
 High tranaformation efficiency: For Escherichia coli, 80% of cells in culture can be
transformed to ampicillin resistance by this method, and efficiencies of transfection of
one transfectant per molecule of plasmid DNA have been reported.

Disadvantages of Elactroporation:
1. Electroporation damages or changes the bacterial membrane

When pBR322, carrying genes conferring resistance to ampicillin and tetracycline, is introduced
into E. coli by number of tetracycline-resistant transfectants is ∼100-fold less than number of
ampicillin-resistant transfectants. This is not seen when plasmid is introduced by calcium
chloride method. One possible explanation is electro-poration damages or changes bacterial
membrane so that it can no longer interact efficiently with tetracycline-resistance gene.

2. High electric field strengths cause cell death

Typically, between 50% and 70% of cells exposed to high electric field strengths are killed. Cell
death is dependent on field strength and total time of treatment.

3. It is an expensive procedure as compaired to chemical methods such as diavalent ion

treatment and heat shock.

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References:
Yoshida N, Sato M (2009). “Plasmid uptake by bacteria: a comparison of methods and
efficiencies”. Appl Microbiol Biotechnol 83(5):791–798.

Dagert, M.; Ehrlich, S. (1979). "Prolonged incubation in calcium chloride improves the
competence of Escherichia coli cells". Gene. 6 (1): 23–28.

Hanahan D (June 1983)."Studies on transformation of Escherichia coli with plasmids".

Journal of Molecular Biology. 166 (4): 557–80.

Azka Asif, Hareem Mohsin, Rabia Tanvir, Yasir Rehman (2017). “Revisiting the Mechanisms
Involved in Calcium Chloride Induced Bacterial Transformation’’. Frontiers in Microbiology.
; 8: 2169. doi: 10.3389/fmicb.2017.0269. PMCID: PMC5681917

Maral Rahimzadeh, Majid Sadeghizadeh, Farhood Najafi, Seyed Arab (2016). “Impact of
heat shock step on bacterial transformation efficiency”. Molecular Biology Research
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Naoto Yoshida & Misa Sato (2009). “Plasmid uptake by bacteria: a comparison of methods
and efficiencies”. Applied Microbiology and Biotechnology. Vol. 83, 791–798

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