Seismic Hazard Assessment Software in Python by Prof. Dr. Costas Sachpazis
Plasmids Vectors in recombinant dna technology (pBR322)
1. 1952, Joshua Lederberg coined the term Plasmid : Any
bacterial genetic element that exist in an extra-
chromosomal state for at least part of its replication.
But since this also qualifies the viruses so the definition
was improved and now being accepted as extra-
chromosomal genetic element that replicates
autonomously.
These are now known to be present in most species of
eubacteria and also archea and lower eukarya
Plasmid contributes to bacterial genetic diversity and
plasticity by coding function not specified by bacterial
chromosome e.g. Antibiotic resistance
Plasmid
4. Plasmid Geometry
• Generally plasmids possess circular geometry, now examples of
linear plasmid also exist.
• Circular plasmid have more than one topology ( due to action
ofDNA Gyrase /topoisomerases
• Mostly maintained as covalently closed circular supercoiled
form .
• Topoisomerase convert it to CCC
• Creating a nick in supercoiled convert it to open circular form
• If nicks are introduced to opposite position on both strand a
linearised plasmid form is achieved.
• Homologous recombinase convert monomeric to high ordered
dimeric forms also
5. Copy Number
Size of plasmid
• Vary in size fro 2.3-1354 kb
• less than 10 kb is desirable
for a cloning vector
• Under certain conditions larger
plasmids adapted for cloning
• number of molecules of an individual
plasmid that are normally found in a single
bacterial cell
• one per cell Stringent Plasmids replicated with
chromosomal DNA
• 50 or more copies Relaxed plasmids replicate
independent of Chromosomal DNA
• low copy : 1-10
• high copy :10-50 or more
6. Conjugation and Compatibility
• Plasmids fall into two groups: Conjugative and Non-conjugative.
• Conjugative plasmids are characterized by the ability to promote
sexual conjugation between bacterial cells
• Conjugation and plasmid transfer are controlled by set of transfer
or tra genes, which are present on conjugative plasmids but absent
from the non-conjugative type.
• Several different kinds of plasmid may be found in a single cell,
including more than one different conjugative plasmid at any one
time. In fact, cells of E. coli have been known to contain up to
seven different plasmids at once.
• To be able to coexist in the same cell, different plasmids must be
compatible. If two plasmids are incompatible then one or the other
will be rapidly lost from the cell.
10. The original reference is: Bethesda Research Laboratories. 1986.
BRL pUC host: E. coli DH5α competent cells. Focus 8(2):9.
(see here). DH5α was derived from E. coli DH5, described
in Hanahan, D. 1985. in DNA Cloning: A Practical Approach.
Glover, D. M. (ed.), Vol. 1, p. 109, IRL Press, McLean, Virginia.
"DH" are the initials of Doug Hanahan.
16. Sophisticated Plasmids from pBR322
pBR327, which was produced by removing a 1089 bp segment from pBR322. This
deletion left the ampR and tetR genes intact, but changed the replicative and
conjugative abilities of the resulting plasmid.
• pBR327 has a higher copy number 30–45 molecules per E. coli cell.
• pBR327 became a non-conjugative plasmid that cannot direct its own
transfer to other E. coli cells.
17. A B C D
pUC
X – gal 5-bromo-4-
chloro-3-indolyl-β-D-
galactopyranoside)
