Fundamentals of Molecular Biotechnology (BIOL4900)

Department of Biology
Memorial University of Newfoundland

Review of DNA Cloning

The cloning of specific DNA fragments usually involve:
    1) Insertion of DNA into a vector (a recombinant vector)
    2) Introduction of recombinant vector into cells (usually E. coli)
    3) Amplification of recombinant vector in the cells
    4) Selection of cells that carry the recombinant vector.
    5) Identification of correct recombinant clone.

Essential Features of Plasmid Vectors


Replication of plasmid DNA is carried by the same enzymes that replicate the E. coli bacterial chromosome.
The number of plasmids per cell range from 1 to a few thousand.
The control of plasmid copy number exists near the plasmid's origin of replication.
Although there is normally only one origin per plasmid, only one is active when there are more.
Derived from the pMB1 plasmid (or the very similar ColE1 replicon), the origin of replication relies upon host genes (for DNA polymerases [DNA polymerizartion], DNA-dependent RNA polymerase [primase function] and dnaB, dnaC, dnaD and dnaZ).
As these gene products are fairly stable, protein synthesis can be stopped (such as through the addition of antibiotics) while plasmid replication continues.
Unidirectional replication is primed by an RNA primer RNA II that is, in turn, negatively regulated by RNA I plus the Rop protein.  Mutations that weaken this interaction result in high copy number plasmids.  These genes also control plasmid incompatibility such that only one version of a set of related plasmids can persist in a cell and its descendants.
In the wild, plasmids are transmitted to new hosts via conjugation.  However, this ability has been deleted in most vectors.

Selectable Markers (Antibiotic resistance)

Ampicillin inhibits synthesis of bacterial cell wall
 amp resistance depends upon production of an enzyme that catalyzes beta-lactam ring degradation in the periplasmic space.

Tetracycline binds to 30S subunit of the ribosome to prevent ribosome translocation
 tet resistance produces a protein that prevents tetracycline from entering the cell.

 Chloramphenicol binds to the 50S subunit of the ribosome to prevent protein synthesis
 cat (chloramphenicol resistance) produces an enzyme system component that converts chloramphenicol to a form that cannot bind the ribosome.

Kanamycin (and the closely related neomycin) are aminoglycosides that bind sub-components of the ribosome and prevent protein synthesis.
kan (and neo) resistance depend upon the synthesis of an aminoglycoside phosphotransferase located in the periplasmic space that inhibits their transport into the cell.

Histochemical identification of recombinant clones

The amino-terminal part of the lacZ gene which produces the "alpha" part of beta-galactosidase has been incorporated into vectors.
To produce functional enzyme, the host must provide the "omega" part of the protein.
Together, the two part functional enyzme can convert the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D- galactoside (X-gal) into a blue compound.
Insertion of cloned DNA into the truncated 5' region of lacZ gene abolishes this alpha complementation to produce a white colony in the presence of X-gal.

Multiple cloning sites (or polylinkers)

The 5' region of the lacZ gene has been extensively modified in sequence to introduce a great number of restriction endonuclease sites that are unique to this region of the plasmid to allow for great flexibility in the cloning of DNA fragments.

Single-stranded DNA production

The presence of the origin of replication of single-stranded phage allows for the possibility of ssDNA production.

Bacteriophage promoters

Promoters from the  T3, T7 and S6 bacteriophages have been included on either side of the multiple cloning sites.
These allow the directed synthesis of RNA using the inserted DNA as template.


        Miniature Preparation of Bacterial Plasmid


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