For conventional DNA sequencing templates of signal stranded DNAs are used. PCR-based methods can be employed to manufacture them. Also vectors based on certain bacteriophages whose genomes assume a single stranded DNA at some stages in their life cycle can be used.

M₁₃ Bacteriophage

M₁₃ is a filamentous bacteriophage. Its genomes bear single-stranded circular DNA (∼6.4 kb long), associated to each other in DNA sequence. A protein coat covers the genomes forming a long filamentous form. The single-stranded form of genome of the phage converts into a double stranded or replicative form in the host cell after getting adsorbed on the host E. coli.

Later a phage genome product triggers DNA synthesis to obtain single-stranded DNA, which is surrounded by a protein coat and migrates towards the cell membrane. Likewise, many mature phage particles are extruded from the host cell without lysis.

M₁₃ Vectors

M₁₃ vectors are synthesised by double-stranded replicative form with multiple cloning sites that can produce double-stranded recombinant DNA circles. These vectors transfect the host, E. coli. Later the phage particles are harvested, and their protein coat is removed to liberate the single-stranded recombinant DNA. These DNA serves as templates for DNA sequencing.

Expression Vectors

There are many conditions where apart from amplification and propagation of the cloned DNA, expression of the gene is also needed. For this, the cloning system should provide appropriate expression signals. Various expression cloning vectors have been designed for use in different host cell systems, bacterial and mammalian cells.

An ideal expression vector to be used for producing bioactive molecules should have the following features:

1. It should have a high plasmid stability.
2. The plasmids should not exert any harmful effects on the host.
3. Vector re-arrangements and deletions should occur at low frequency.
4. The plasmids should transform cells at high frequency.
5. The expression vector should be capable of incorporating and maintaining large quantities of foreign DNA.
6. Recovery of the intact plasmid from the transformed cell should be easy.
7. The copy number for high level expression of genes should be high.
8. Large varieties of host should be available.
9. The transcriptional and translational elements should show compatibility with the host organisms.

The expression vectors comprise of a strong promoter (which is subjected to external conditions of temperature), the level of specific inducer or repressor, and a ribosomal binding site sequence. In the vector a restriction site is incorporated where the gene to be expressed is inserted.

The origin of replication and a marker gene resistant to antibiotic are also incorporated. Expression vectors are made using a few promoters, namely, lambda PL promoter, lac Z promoter, trp promoter, and lac promoter. Each of these promoters has been incorporated in different vector systems, regulated by their corresponding repressors.

By placing the inserted gene in the expression vector in correct phase with respect to initiation codon, the correct polypeptide product can be obtained. However, the eukaryotic proteins within the prokaryotic cells are not always stable, and thus the proteins should be protected using vectors which contain a prokaryotic structural gene so that it will be expressed. This results in a hybrid polypeptide, in which the part of the prokaryotic product is attached to the N-terminus of the foreign polypeptide. This extra sequence at the N-terminus stabilises the polypeptide.

General Design Features of an Expression Vector

To simplify manipulation, plasmids are mainly preferred to construct an expression vector. Plasmids have been modified to incorporate control elements [such as strong promoters, efficient ribosome binding site (rbs), multi-restriction insertion sequences, and terminator sequence] intended for high level expression of inserted target gene. The genes for origin of replication (ori) and for selectable markers (antibiotic resistance) were also retained.

This expression vector includes multi-cloning short segment (McS) that encodes several restriction sites where foreign gene coding segments are to be inserted. Initiation codon (ATG) is positioned downstream from rbs. This excludes the inclusion of rbs and initiation codon on insert segment. Prokaryotes do not have these sequences. Downstream McS is a strong transcription terminator signals sequences.