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Electroporation
Cell Biology (BIO 108 )
Long Island University
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ELECTROPORATION
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 antibiotics.
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.
DNA LIBRARIES A DNA 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 cloned.
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, BAC or YAC. 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.
Chromosomal libraries These are DNA libraries constructed from a single chromosome isolated fromcells mitotic.
Electroporation
Course: Cell Biology (BIO 108 )
University: Long Island University
