Plasmids Vectors in recombinant dna technology (pBR322)

Plasmids Vectors in recombinant dna technology (pBR322), pUC18, pUC19, and their derivatives.

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

2. Plasmid

3. Integrative and non- Integrative Plasmids

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.

7. Types of Plasmids D- Plasmid

8. The original reference is: Bethesda Research Laboratories. 1986. BRL pUC host:  E. coli  DH5 α competent cells. Focus 8(2):9. (see  here ). DH5 α 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 .

9. pBR322 bla Bla - β Lactam

10. 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.

11. A B C D pUC X – gal 5-bromo-4-chloro-3-indolyl- β- D- galactopyranoside )