Electroporation ELECTROPORATION It consists of applying short high voltage


It consists of applying short high voltage pulses to a mixture of host cells
in suspension and recombinant vectors in solution. These pulses increase the permeability
of the membranes, allowing the vectors to pass into the cell.
It is a highly efficient method that can be applied to all types of host cells.

Selection and identification of recombinant clones
Once the vector introduction process is finished, a mixture with
three types of cells is obtained:
– Non-transformed cells, which have not captured any DNA molecule.
– Transformed cells that carry the recombinant vector.
– Transformed cells that carry a non-recombinant vector or somemolecule
unwanted DNA.

Therefore, a process of selection and identification of theclones is necessary
recombinant, which will be those that carry the specific recombinant vector.

This selection process is conditioned by genetic markers carried by the
vector: antibiotic resistance genes, enzymes that produce colored reactions,
fluorescent proteins, lethal genes, etc.

It is one of the first selection and identification systems and is based on the use of
vectors with two antibiotic resistance genes, usually ampicillin and tetracycline.
The insertion site of the foreign DNA is inside the sequence of one
of the resistance genes, so that in recombinant vectors that gene is not
functional (its sequence is interrupted by the insert of foreign DNA).

When this vector is introduced into bacteria sensitive to ampicillin and tetracycline,are
three types of cellsobtained:
– Cells not transformed with the vector: sensitive to both antibiotics.
– Cells transformed with the recombinant vector: resistant to ampicillin and
sensitive to tetracycline.
– Cells transformed with the non-recombinant vector: resistant to both

With this system, selection is carried out by cultivating the cells in a culture medium with
ampicillin in which only the transformed bacteria will grow. Identification is done by
replicating the ampicillin plate on another tetracycline plate. Consequently, the recombinant
clones will be the colonies that grow on the ampicillin plate and do not grow on the one
containing tetracycline.

Fluorescent proteins
It is based on the use of vectors with a resistance gene for selection and
a gene encoding a fluorescent protein for identification. The polylinker region is included
in the fluorescent protein gene, so the inserts inactivate the gene. The most widely used
protein is GFP, isolated from a jellyfish.

Antibiotic resistance genes
Selection of transformed bacteria is performed by growth in culture media
with antibiotics, and identification is carried out by illuminating the culture plates with
ultraviolet light.
Colonies of bacteria transformed with non-recombinant plasmids show
green fluorescence while those transformed with recombinant plasmids will not fluoresce.

Lethal genes
It is a relatively recent methodology that usesas a selection marker
antibiotic resistance, and a gene that codes for a lethal protein as anmarker
identification. In recombinant vectors, the lethal protein is not functional because the
gene sequence is interrupted by the foreign DNA insert.

Selection and identification is done simply by growing the bacteria in a medium
with the antibiotic. In this medium, only bacteria that carry the recombinant vector grow,
since non-transformed bacteria do not grow due to sensitivity to the antibiotic, and those
transformed with the non-recombinant vector die due to the effect of the lethal protein.

library is a collection of cloned recombinant vectors
whose foreign DNA sequences have been obtained from a single organism.

Types of DNA library DNA
libraries can be of three types depending on the origin of theDNA

Genomic libraries
These are collections of cloned recombinant vectors that include the entire genome
of an organism.
High capacity vectors are often used to try to containthe entire genome
in as few clones as possible. Specifically, they are usually performed on phages, cosmids,
In theory, a genomic library must contain at least one copy of any
sequence present in the genome of an organism. The minimum number of clones necessary
for this condition to be fulfilled can be calculated based on the size of the genome and the
average size of the cloned inserts, which will depend on the vector used. For reference, a
human genomic λ phage library should contain approximately8105 clones to ensure
representation of the entire genome.

libraries These are DNA libraries constructed from a single chromosome isolated fromcells

Read more here: Source link