Molecular cloning of desired gene in bacterial and eukaryotic systems and expression of recombinant proteins using different vectors:
Vectors can be described as an autonomously replicating DNA sequence that can be used to carry foreign DNA fragments. Commonly used vectors are bacteriophages and plasmids. Insertion λ vectors are used for constructing cDNA libraries. These vectors are constructed by modifying the λ genome so as to permit insertional cloning into the cl gene.
Characteristics of Vector:
1. Ability to self-replicate.
2. Selectable characteristic so that transformed cells can be recognized from non-transformed cells.
3. Should have origin of replication.
4. Selectable markers.
5. Small size.
6. Plasmid high copy number.
7. Restriction site
E.coli Plasmids E.coli is most commonly used as vector in almost all cases (even with eukaryotes) only because of its following advantages
1. Easy to grow in sample, inexpensive growth medium.
2. Has rapid doubling time of about 20-30 minute during log-phase growth.
3. Its genetics is well understood.
4. Has fully mapped and sequenced genome.
5. Extra-chromosomal copies of DNA can be exploited to carry foreign DNA fragments.
6. It is non-toxic
These genes exist on a circular piece of DNA that replicates independently from the bacterial chromosome, or they can be integrated into chromosome. F stands for fertility.
There are three manifestations of fertility factor:
F+ also called F-episome: Large circular dsDNA molecule that carries only fertility genes.
Hfr The F element has become integrated into E.coli genome. When conjugation occurs, the F-gene start travelling across the pilus, dragging rest of genome behind.
F- Large circular dsDNA molecule that contains the fertility genes and a few other genes. These genes are transferred very efficiently from one bacterium to another.
These are naturally occurring extra-chromosomal DNA fragments that are stably inherited from one generation to another in extra-chromosomal state. Size 1500 bp to over 300 kbp. Shape Closed circular dsDNA molecule that confer particular phenotype onto bacterial cell in which they are replicated.
Plasmids usually carry a gene that codes for resistance to either antibiotics or heavy metals, or that produces DNA restriction and modification enzyme, that bacterium does not often produce.
First vector constructed was pSC101. The selection is done by selectable markers that are called antibiotic resistance genes. When foreign gene is inserted in the plasmids, insertional inactivation occurs. The disadvantage of this vector is that its selection is not easy.
pBR322 p in pBR indicates plasmid; BR represents Bolivar and Rodriguez (Scientist who discovered the plasmid pBR322) and 322 represents the number of plasmid within their stock collection.
Components of the plasmid:
1. Ori Origin of replication, to ensure high plasmid copy number. It can be increased by chloramphenicol amplification.
2. Antibiotic Resistance Genes Two genes; a selectable marker either ampicillin or tetracycline can be used. Each marker includes restriction sites used for cloning.
3. Cloning Sites Plasmid carries number of unique restriction enzyme recognition sites. These are located in Antibiotic resistance genes.
Example AmpR – Pst1, Pur1 and Sca1 TetR – BamH1 and Hind III
Selection: There is a direct selection of recombinants in process called insertional inactivation.
For Example: If we want to insert DNA fragment into BamH1, the insert will disrupt the gene responsible for tetracycline resistance but ampR will not be altered. Transformed cells are first grown on ampicillin plates to kill all cells that do not contain a plasmid. Cells that grow on amp plates are then replica plated onto medium containing amp and tet. Cells that grow in ampicillin presence but dies under tetracycline selection, contains plasmids that have foreign DNA inserts. The insertion of foreign DNA fragment into an antibody resistance gene inactivates gene production and leads to antibody sensitivity.