Module 1 : INTRODUCTION

Lecture 3 : Types, Biology and Salient Features of Vectors in Recombinant DNA Technology – Plasmid

 

1-3.3.1 RSF1010

•  It is a naturally occurring plasmid isolated from E.coli K-12.

•  This plasmid has broad host range in gram negative bacteria.

•  The size of plasmid is 8694bp.

•  Antibiotic resistance genes for Streptomycin and sulfonamides have present.

•  The replication of RSF1010 starts either bi- or uni-directionally from unique ori-V region (2347-2742).

•  It cannot initiate transformation independently but can be transferred to host bacterium in presence of helper plasmid.

•  Genebank accession no. M28829.

1-3.3.2 ColE1:

• It is a naturally occurring multicopy plasmid obtained from E.coli ( copy number is around 40).

•  The size of this natural plasmid is 6646bp.

•  It forms the basis of many artificial vectors used in molecular cloning.

•  The natural ColE1 plasmid has genes for colicin E1 production. Colicin is an antibacterial toxin produced under stressed condition. Cells harboring the plasmid will have resistance against the toxin.

•  For using in molecular cloning experiment, colicin genes are replaced with selection marker (antibiotic resistant) gene.

•  Genebank accession no. M33100.

1-3.4 Artificial Plasmids:

Naturally occurring plasmids has several limitations; for example, some are stringent and not relaxed (pSC101), some has poor marker genes (ColE1), and some are too large (RSF2124). To overcome the limitations of natural vectors, artificial plasmid are designed by combining different elements from diverse sources.

Artificial plasmids vectors are classified into two broad types based on their use:

  1. Cloning vector
  2. Expression vector

Apart from the following, there is another class of vectors known as shuttle vector. Shuttle vectors can be propagated in two or more different host species (both in prokaryotes and eukaryotes). Hence, inserted DNA can be manipulated and replicated in two different cellular systems.

Cloning vectors are designed for efficient transfer of foreign DNA into the host. Expression vectors have efficient machinery for cloning and expression of foreign gene in the host system. Selection of a vector depends upon various criteria decided by the experimental goal.