bacterial transformation

1. MUHAMMED ASHARUDHEEN C P
17LS400111
2ND MSC MICROBIOLOGY

2. INTRODUCTION
• Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist Frederick
Griffith.
• Transformation is one of three forms of horizontal gene transfer that occur in nature among
bacteria ie., TRANSFORMATION, CONJUGATION, TRANSDUCTION.
• It is the incorporation of exogenous genetic material from its surroundings through the
cell membrane(s).
• It is one of the three possible mechanism of HGT (Horizontal Gene Transfer).
• Cells that can be transformed are called competent.

3. • Transformation is a complex process.
• energy-requiring developmental process.
• In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome,
it must become competent.
Competence for transformation is typically induced by:
• high cell density
• nutritional limitation,
• conditions associated with the stationary phase of bacterial growth.

5. NATURAL TRANSFORMATION
• Naturally competent bacteria carry sets of genes that provide the protein machinery to bring
DNA across the cell membrane(s).
• The transport of the exogenous DNA into the cells may require proteins that are involved in the
assembly of type IV pili and type II secretion system, as well as DNA translocase complex at
the cytoplasmic membrane.
• Due to the differences in structure of the cell envelope between Gram-positive and Gram-
negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells

6. GRAM POSITIVE
• The DNA first binds to the surface of the competent cells on a DNA receptor, and passes
through the cytoplasmic membrane via DNA translocase.
• Only single-stranded DNA may pass through, the other strand being degraded by nucleases in
the process.
• The translocated single-stranded DNA may then be integrated into the bacterial chromosomes
by a RecA-dependent process.

7. GRAM NEGATIVE
• Due to the presence of an extra membrane, the DNA requires the presence of a channel formed
by secretins on the outer membrane.
• Pilin may be required for competence, but its role is uncertain.
• The uptake of DNA is generally non-sequence specific, although in some species the presence
of specific DNA uptake sequences may facilitate efficient DNA uptake.

8. GRAM POSITIVE

10. DNA CLONING
• DNA cloning is a molecular biology technique that makes many identical copies of a piece of
DNA, such as a gene.
• In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called
a plasmid.
• The plasmid is introduced into bacteria via process called transformation, and bacteria carrying
the plasmid are selected using antibiotics.
• Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases,
induced to express the gene and make protein.
•

11. STEPS IN DNA CLONING
• Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which
cut DNA) and DNA ligase (which joins DNA).
• Transform the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up
the plasmid.
• Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein.
Harvest the protein from the bacteria and purify it.

12. CUTTING AND PASTING
• A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target
sequence and cuts DNA into two pieces at or near that site.
• Many restriction enzymes produce cut ends with short, single-stranded
overhangs.
• If two molecules have matching overhangs, they can base-pair and stick
together.
• However, they won't combine to form an unbroken DNA molecule until they are
joined by DNA ligase, which seals gaps in the DNA backbone.

15. BACTERIAL TRANSFORMATION AND
SELECTION
• Specially prepared bacterial cells
• a shock (such as high temperature)
take up foreign DNA.
• A plasmid+ antibiotic resistance gene bacteria survive in the presence of a specific
antibiotic.
• Bacteria that took up the plasmid can be selected on nutrient plates +antibiotic.

16. • Thus, Bacteria + no plasmid die.
• Bacteria + plasmid live and reproduce.
• Each surviving bacterium will give rise to a small, dot-like group, or colony, of identical
bacteria that all carry the same plasmid.