2. What is vector ? a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types: Plasmids, Bacteriophages and other viruses, Cosmids, and Artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. expression vectors specifically are for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene. Insertion of a vector into the target cell is generally called transfection, although insertion of a viral vector is often called transduction.
3. Expression vectors An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA.
4. Expression vectors require not only transcription but translation of the vector's insert, thus requiring more components than simpler transcription-only vectors. Expression vectors require sequences that encode for: Polyadenylation tail: Creates a polyadenylation tail at the end of the transcribed pre-mRNA that protects the mRNA from exonucleases and ensures transcriptional and translational termination: stabilizes mRNA production. Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors. Kozak sequence: Vectors should encode for a Kozak sequence in the mRNA, which assembles the ribosome for translation of the mRNA.
5. After expression of the gene product, the purification of the protein is required; but since the vector is introduced to a host cell, the protein of interest should be purified from the proteins of the host cell. Therefore, to make the purification process easy, the cloned gene should have a tag. This tag could be histidine (His) tag or any other marker peptide.
6. DNA vectors that are used in many molecular-biology gene-cloning experiments need not result in the expression of a protein. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin that are important for medical treatments of specific diseases like diabetes. Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).
8. Plasmid vectors A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomycescerevisiae).
9. Plasmids used in genetic engineering are called plasmid vectors The plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive , since the plasmid makes them resistant. Now these bacteria can be grown in large amounts, harvested and lysed to isolate the plasmid of interest. However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.
10. There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate Integrate into the host chromosome.
11. pBR322 Derived from E. coli plasmid ColE1), which is 4,362 bp DNA and was derived by several alterations in earlier cloning vectors. pBR322 is named after Bolivar and Rodriguez, who prepared this vector. It has genes for resistance against two antibiotics (tetracycline and ampicillin), an origin of replication and a variety of restriction sites for cloning of restriction fragments obtained through cleavage with a specific restriction enzyme. It has unique restriction sites for 20 restriction endonucleases. Certain restriction sites for eg., BAM HI in the tetrgenes of the plasmid are present within thegene in such a way that the insertion of foreign segment of DNA will inactivate the tetrgene. The recombinant plasmid will allow the cells to grow only in the presence of ampicillin but will not protect them against tetracyclin. Thus recombinant plasmids selection will be easily carried out.
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13. pUC vectors Another series of plasmids that are used as cloning vectors belong to pUC series (after the place of their initial preparation I.e. University of California). These plasmids are 2,700 bp long and possess Ampicillin resistance gene The origin of replication derived from pBR322 and The lacz gene derived from E.coli. Within the lac region is also found a polylinker sequence having unique restriction sites. When DNA fragments are cloned in this region of pUC, the lac gene is inactivated. These plasmids when transformed into an appropriate E. coli strain, which is lac(JM103, JM109), and grown in the presence of IPTG (isopropyl thiogalactosidase, which behaves like lactose, and induced the synthesis of b-galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise to white or clear colonies.
14. On the other hand, pUC having no inserts are transformed into bacteria, it will have active lac Z gene and therefore will produce blue colonies, thus permitting identification of colonies having pUC vector with cloned DNA segments. The cloning vectors belonging to pUC family are available in pairs with reverse orders of restriction sites relative to lac Z promoter. pUC8 and pUC9 are one such pairs. Other similar pairs include pUC12 and pUC13 and pUC18 and pUC19.
15. Sal I BamHI BamHI PstI Sma I Sal I Sma I PstI EcoRI Hind II Hind II EcoRI pUC8 (2768 bp pUC9 (2768 bp Operator LacZ Promoter HaeII B-galactosidase gene pUC Hae II Ampicillinresistance
16. Ti Plasmid The most commonly used vector for gene transfer in higher plants are based on tumour inducing mechanism of the soil bacterium A. tumefaciens, which is the causal organism for crown gall disease. The disease is caused due to transfer of DNA segment from the bacterium to the plant nuclear genome. The DNA segment which is transferred is called T-DNA and is part of a large Ti plasmid found in virulent strains of A. tumefaciens.
17. Most Ti plasmids have four regions in common Region A: comprising T-DNA which is responsible for tumour induction leading to production of tumors with altered morphology (shooty or rooty mutant galls). Sequences homologous to this region are always transferred to plant nuclear genome, so that the region is described as T-DNA (transferred DNA) Region B: This region is responsible for replication Region C Responsible for conjugation Region D: Responsible for virulence, so that mutation in this region abolishes virulence. This region is therefore called virulence (vir) region and plays a crucial role in the transfer of T-DNA into the plant nuclear genome.
18. The T-DNA consist of following regions: An ONC region: Consisting of 3genes (2 genes tms1 and tms2 rep shooty locus and one gene tmr rep rooty locus) responsible for 2 phytohormones, namely IAA and isopentyladenosine-5’-monophosphate (a cytokinin). These genes encode the enzymes responsible for the synthesis of these phytohormones so that the incorporation of these genes in plant nuclear genome leads to the synthesis of phytohormones in the host plant. These phytohormones in turn alter the development programme, leading to the formation of crown gall.
19. An OS region: responsible for synthesis of opines. These are derivatives of various compounds (eg. Arginine + pyruvate) that are found in plant cells. Two most common opines are octopine and nopaline. The enzymes for sytnhesis of these 2 opines, are encoded by (octopinesynthase and nopalinesynthase) T-DNA. Depending upon the opine it codes, they are described as octopine type Ti plasmid or nopaline type Ti plasmid.
20. The T-DNA region in the plasmid is flanked on both the sides with 25 bp direct repeat sequences, which are essential for T-DNA transfer, acting only in its cis orientation. Any DNA sequences flanked by these 25 bp repeated sequence in the correct orientation, can be transferred to plant cells. This is successfully utilized for Agrobacterium mediated gene transfer in higher plants.
