Dna uptake, Entry, andEstablishment in recepient cell Shreya M Modi. MSc Sem-2 Roll no.-11
Introduction• It is a process of genetic transfer that does not require physical contact between the donor and recepient cells.• In the process of transformation the donor cell lysis, releasing its DNA.
DNA UpTake• The process of taking DNA into the recepient cell through the process of transformation from the donor’s cell.• Donor cell – The cell whose gene material is transformed to the donor cell.• Recepient Cell- the cell who receives the genetic material.
Competent cell• When bacteria lyse, they release considerable amounts of DNA into the surrounding environment. These fragments may be relatively large and contain several genes.• If a fragment contacts a competent cell, one able to take up DNA and be transformed, it can be bound to the cell and taken inside.• The transformationfrequency of very competent cells is around 103 for most genera when an excess of DNA is used.• That is, about one cell in every thousand will take up and integrate the gene. Competency is a complex phenomenon and is dependent on several conditions.• Bacteria need to be in a certain stage of growth; for example, S. pneumoniae becomes competent during the exponential phase when the population reaches about 107 to 108 cells per ml. When a population becomes competent, bacteria such as pneumococci secrete a small protein called the competence factor that stimulates the production
Natural transformation in gram negative bacteria• Natural transformation has been observed in some gram negative bacteria. Transformation in Haemophilus influenzae, the first gram negative bacterium in which natural competence was found, is different at least two important respects from that in B. subtilis.• DNA uptake is associated with the formation of small membraneous structures, called transformasomes, which protrude outside the cell. The transforming DNA is taken into these vesicles where it is then internalized into the cell. One of the two strands is degraded while the remaining strand may recombine with the host chromosome.• Unlike gram positive bacteria, DNA uptake in gram negative bacteria appears to require or involve the recognition of specific sequences. The sequences or some bacteria are as follows:
Transformation in Haemophilus influenzae• Transformation in Haemophilus influenzae, a gram- negative bacterium, differs from that in S. pneumoniae in several respects.• Haemophilus does not produce a competence factor to stimulate the development of competence, and it takes up DNA from only closely related species (S. pneumoniae is less particular about the source of its DNA). Double- stranded DNA, complexed with proteins, is taken in by membrane vesicles. The specificity of• Haemophilus transformation is due to a special 11 base pair sequence (5′AAGTGCGGTCA3′) that is repeated over 1,400 times in H. influenzae DNA. DNA must have this sequence to be bound by a competent cell.
• The mechanism of transformation has been intensively studied in S. pneumoniae.• A competent cell binds a double-stranded DNA fragment if the fragment is moderately large; the process is random, and donor fragments compete with each other. The DNA then is cleaved by endonucleases to doublestranded fragments about 5 to 15 kilobases in size.• DNA uptake requires energy expenditure.• One strand is hydrolyzed by an envelope-associated exonuclease during uptake; the other strand associates with small proteins and moves through the plasma membrane.• The single-stranded fragment can then align with a homologous region of the genome and be integrated.
Artificial transformation• Artificial transformation has been demonstrated in a number of bacterial species, most notably in E. coli, where it is used routinely for cloning DNA. However, even in E. coli, the process or mechanism is not well understood.• E. coli cells can be made competent for transformation simply by treating them with calcium chloride. But, even this simple treatment is a bit of an art and variables such as the temperature of growth and the density of the culture seem to be important in determining competence.
Key Steps for Transformation Protein of interest Protein for antibiotic resistance Plasmid DNA enters the bacterial cell and the genes are expressed.• Bacterial cell suspension is placed in CaCl2 solution• Cells must be in log phase of growth.• Cells are kept on ice until heat shock treatment• Heat shock at 42 ˚C for one minute• Recover period in LB broth• Cells are spread on appropriate selection plates
TRANSFORMATION BY ELECTROPORATION
CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE
References• Microbiology, 5th Edition By Lansing M. Prescott