Promoter cloning vectors

Shimada, T. et al (2004) Classification and strength measurement of stationary-phase promoters by use of a newly developed promoter cloning vector. J. Bacteriol, 186 (21), 7112-7122. 

Fig. 7.11 Schematic diagram of foreign gene expression in plant. P promoter, T terminator, pGA482 plant cloning vector, Km kanamycin resistance gene, Tc tetracycline resistance gene.

In some circumstances the introduction of a cloning vector into a host cell is a trivial process. For example, phage vectors are designed so they introduce recombinant DNA in an infective process called transfection, and some bacteria take up plasmids unaided. However, most host cells must be induced to take up foreign DNA. Several methods are used. In some prokaryotic and eukaryotic cells, the addition of Ca2+ to the medium promotes uptake. In others, a process called electroporation, in which cells are treated with an electric current, is used. One of the most effective methods for transforming animal and plant cells is the direct microinjection of genetic material. Transgenic animals, for example, are created by the microinjection of recombinant DNA into fertilized ova. 

A cloning vector is also cut with the same pair of restriction endonucleases downstream of the promoter element (p) (see Figure 3.5). In cloning vector, pDNA sense strand is dark grey complementary strand is in light grey. In heterologous DNA, sense strand is in red complementary strand is in yellow. 

An expression vector, such as pET 5 plasmid, has the components of any normal cloning vector (e.g., origin of replication, selectable marker, multiple cloning site), but it also has the ability to have the inserted DNA be transcribed. It has a promoter for RNA polymerase, such as T7 polymerase, and a termination sequence. These border the multiple cloning site. These vectors are used with a cell line that makes T7 RNA polymerase when induced. 

Once the affinity of the nucleic acid pool has plateaued, individual sequences can be cloned and sequenced from the pool for further characterization. The final pool is PCR amplified for eight cycles to ensure that all sequences are double stranded. Primers should not be 5 labeled with biotin or a fluorescent tag since this will prevent ligation of the sequences into the cloning vector. dsDNA is ligated into a pGEM vector and transfected into DH5 Escherichia coli and colonies are raised. Plasmids from approximately 30 (or more) colonies are chosen at random and the sequences of individual clones are determined using the T7 promoter sequence. 

FIGURE 3. The scheme of cyanobacterial promoter-selection vector construction and the chloroplast promoter cloning in Synechocystis... 

It may be desirable to further process purified proteins by removal of the respective affinity tags. The cloning vectors associated with each system allow for this possibility, by incorporating recognition sites for site-specific proteases into the respective constructs. In our experience, this is not necessary for most of the characterizations that have been described. Indeed, for at least one plant polyadenylation factor subunit, proteolytic removal of the (MBP) tag results in precipitation of the polyadenylation factor subunit. More generally, affinity tags are known to promote solubilization of fusion proteins... 

Cloning vector a plasmid with a convenient reporter (chloramfenicol acetyl transferase, luciferase, alkaline phosphatase) that is useful in cotransfection experiments, e.g., pUTKAT (10), or the pGL2-Promoter and pCAT-Promoter vectors (Promega, Madison,WI)... 

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