Features of different plasmids: There are minimum molecular components to assemble in a bacterial plasmid to perform the function of vector are as follows-
1. Origin of replication-Like any other replicating DNA, plasmid DNA needs its own independent origin of replication to provide replication start site to make more copies. It decides the range of bacterial host strain can be use with the particular plasmid vector. The plasmids containing ori region from Col E1 can be able to grow in limited bacterial species such as E.Coli etc. In contrast, plasmid containing ori from RP4 or RSF1010 can be able to grow in gram (-) bacteria and gram (+) bacteria.
2. Selection marker- Selection marker in the form of either antibiotic resistance gene or enzymatic gene is essential to give phenotypic changes in host after entry of the plasmid.
3. Promoter- Plasmid replication in host is performed by the host provided proteins such as DNA gyrase, helicase, polymerase and DNA ligase. But proteins required for conferring antibiotic resistance or enzyme use for selecting transformed host cells is present on plasmid and a promoter adjacent to it is required to express genes present on plasmid DNA. In addition, promoter is also needed to express gene present on foreign DNA.
pBR322: In early days, naturally occurring plasmids such as col E1 and RSF1010 were used for cloning. But these plasmids had several limitation such as number of selection marker and recognition sites for restriction sites. To facilitate the cloning, a much improved cloning vector was produced and named as pBR322. It is produced after taking structural elements from RSF2124, pSC101 and pMB1. Plasmid pBR322 received ampicillin (ApR) and teracyclin (TcR) resistance gene from RSF2124 and pSC101. Origin of replication is derived from pMB1 and a detail of construction of pBR322 with multiple steps of recombination is given in the following article. [Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977 ;2(2):95-113. PUBMED ID: 344137] . A vector map of pBR322 with regions derived from different plasmid is given in Figure 17.2. pBR322 is a 4359 bp long plasmid and has 40 unique restriction sites (Figure 17.3). Eleven restriction sites are present within Tetracycline resistance gene and six sites are within ampicillin resistant gene. In addition, two sites are present within promoter of the Tetracycline resistance gene. Cloning any DNA fragment into these sites will disrupt the resistance gene and as a result it can be used as a criteria for selecting recombinant plasmid. The details of different selection methods are discussed in future lecture.