Botany · Ch 4 — Principles and Processes of Biotechnology
Vectors
Vectors
A vector, or cloning vehicle, is a small DNA molecule with one defining ability: it can replicate itself autonomously once inside a host cell, which is exactly why it can be used to carry a foreign DNA insert along for the ride and keep multiplying it. Not any small DNA molecule will do the job well, though - a genuinely useful vector needs to be small and of low molecular weight (under about 10 Kb, so it enters host cells easily), to carry an origin of replication (ori, the specific sequence a cell's replication machinery recognises as 'start copying here'), to carry a selectable marker - almost always an antibiotic-resistance gene - that lets transformed cells be told apart from untransformed ones, and to have a Multiple Cloning Site (MCS, or polylinker): a short stretch containing several different, usually unique, restriction sites clustered together, so a researcher has a choice of which restriction enzyme to use for a given insert. Vectors also split functionally into two types: cloning vectors, whose whole job is simply propagating a DNA insert inside a suitable host, and expression vectors (e.g. pUC19), which additionally carry the transcription/translation signals needed to actually produce the protein the insert encodes, in bulk, inside the host. The chapter then works through a whole family of named vector types built around this basic template. Plasmids - extrachromosomal, self-replicating circular DNA molecules a bacterium carries in addition to its main chromosome - are the classic example, and pBR322 (4361 bp, built by Bolivar and Rodriguez, hence the 'BR', carrying both ampicillin- and tetracycline-resistance genes and multiple unique restriction sites) is the best-known, most widely used cloning plasmid of all. A very different plasmid, the Ti (tumour-inducing) plasmid of Agrobacterium tumefaciens, is naturally capable of transferring a defined segment of itself, the T-DNA, into a plant cell's own genome, which the chapter later exploits directly for plant genetic engineering. Transposons ('walking' or 'jumping' genes) are DNA sequences able to insert themselves at a new genomic location with no sequence relationship to that target site, and are used as tools for studying gene and protein function, well characterised in Arabidopsis thaliana and E. coli. Beyond plasmids and transposons, the chapter also introduces cosmids (hybrid vectors combining a plasmid with a lambda-phage cos site, letting DNA be packaged into a phage particle for very efficient delivery), bacteriophage vectors (viruses that infect bacteria, such as lambda phage and M13, capable of carrying inserts up to about 25 Kb, larger than a typical plasmid can), phagemid vectors (reconstructed plasmids that carry both …
What this figure shows. A three-panel labelled diagram of a generic cloning vector, illustrating its three required properties in turn: (1) an origin of replication (ori) recognised by the host cell, so the vector replicates along with any DNA it carries; (2) a selectable marker gene, whose trait lets cells that took up the vector be identified/selected; (3) a set of unique restriction-enzyme cleavage sites (labelled with example enzymes Sall, EcoRI, HindIII, BamHI, PstI) providing a single cut site for each comm …
What this figure shows. A schematic of a bacterial cell interior showing the large circular bacterial chromosome (nucleoid) coiled in the centre of the cell, with several much smaller, independent circular plasmid molecules scattered separately in the cytoplasm around it - illustrating that plasmids are extra-chromosomal, self-replicating DNA distinct from t …
What this figure shows. A circular restriction map of the pBR322 cloning vector (4361 bp), marking around its circumference: the ori (origin of replication), the rop gene (encoding a protein involved in plasmid replication), the ampR gene (ampicillin-resistance) and tetR gene (tetracycline-resistance) - the two selectable antibiotic-resistance markers - and unique restriction sites cut by HindIII, BamHI, PvuII, EcoRI, SalI, PstI and ClaI positioned at their respective points around the map, several of them sitting inside the tetR or ampR …
What this figure shows. A circular map of the Ti (Tumour-inducing) plasmid of Agrobacterium tumefaciens, marking its origin of replication (ORI), its Virulence Region (vir genes, needed for T-DNA transfer), and its T-DNA Region containing the genes actually transferred into the plant genome - shown further subdivided into genes for auxin and cytokinin synthesis (which drive uncontrolled plant cell growth/tumour formation) and opine synthesis/opine catabolism genes (which let th …
What this figure shows. A simplified linear diagram of a chromosomal DNA sequence with a transposon (transposable/mobile genetic element) shown inserted into and interrupting it - illustrating how a transposon can insert itself at a new genomic location without any sequence relationship to that target locus, which is why transposons are …
What this figure shows. A circular map of a bacterial expression vector, labelling around it: the ori, a selectable genetic marker (e.g. antibiotic resistance), a restriction site (cloning site) for inserting the foreign gene, a ribosome-binding site, bacterial promoter (P) sequences, an operator (O) sequence, a gene encoding a repressor protein that binds the operator and regulates the promoter, and a transcription-initiation sequence and transcription-termination sequence flanking the insert site - the operon-style regulatory elements that let the host transcr …