Bacterial transformation

bacterial transformation

1. BACTERIAL TRANSFORMATION MUHAMMED ASHARUDHEEN C P 17LS400111 2 nd 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.

4. 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

5. 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.

6. 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.

7. GRAM POSITIVE

8. 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.

9. 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.

10. 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.

11. BACTERIAL TRANSFORMATION AND SELECTION   S pecially 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.

12. 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.