ti plasmid derived vector.pptx

Ti plasmid derived vector

1. Submitted by G. Ponsutha II MSc Microbiology REG :20201232516113 Submitted to, G. Ramanathan Assistant professor Department of microbiology Semester – iv bioinformatics Seminar topic : ti plasmid derived vector

2. 1907 smith &Townsend postulated that a bacterium was the causative agent of Crown gall tumor A tumifaciens infects wounded or damaged plant tissues, it induces the formation of a plant tumor called crown gall. Ti plasmid is a tumor-inducing or tumor induction plasmid It is plasmid of agrobacterium tumifaciens bacteria, the cells of virulent strains of A. tumifaciens contain, besides the chrismal DNA, a small circular non-chromosomal DNA called Ti-plasmid, which carries ancillary g enes such as those affecting pathogenicity and metabolism of opines. I ntroduction

3. Using Ti plasmid as a vector it is possible to insert a desires DNA sequence (gene) into the T DNA region of Ti plasmid. There are several limitation to use Ti plasmid directly as cloning vectors Ti plasmid are large in size(200-800kb) smaller vectors are preferred for recombinant experiment. Absence of unique restriction enzyme site on Ti plasmid. Presence of oncogene or tumor causing genes (auxins & cytokinin) . Ti plasmid derived vector system Two type of Ti plasmid derived vectors are used for genetic transformation of plats :- Co integrate vectors Binary vectors

4. Need 3 vector: Disarmed Ti plasmid capable for infection Intermediate vector with T-region and gene of interest (transferred by conjugation ) Needs 2 vectors: Disarmed Ti plasmid with gene of interest (no vir genes ) Helper vector for infection (with vir genes) Genetically engineered Ti-plasmid vectors Form co-integrated plasmid after homologous recombination Helper vector for transfer of intermediate plasmid into A.tumifaciens Binary vector Co-integrated vectors

5. In the cointegrate vector system, the disarmed and modified Ti plasmid combines with an intermediate cloning vectors to produce a recombinant T i plasmid. Production of disarmed T i plasmid:- The T-DNA genes for hormone biosynthesis are removed. In place of the deleted DNA, a bacterial plasmid(pBR322) DNA sequence is incorporated. This disarmed plasmid, also referred to as receptor plasmid , has basic structure of T-DNA (right &left borders, virulence genes etc.) COINTEGATRE VECTOR

6. Construction of intermediate vector:- The intermediate vector is constructed with the following components. An intermediate vector is made using E.coli plasmid, vir region, T-DNA, origin of replication, pBR322sequence A plant transformation marker(PTM) A gene coding for neomycin phosphotransferase 2 (npt2), These gene confer resistance to kanamycin in the plant cells and this permits their isolation.

7. A bacterial resistance marker:- A gene coding for spectinomycin resistance. This gene confers spectinomycin resistance to recipient bacterial cells and thus permits their selective isolation. A multiple cloning site(MCS) Where foreign genes can be inserted . A co-E1 origin of replication which allows the replication of plasmid in E.coli but not in agrobacterium. An oriT sequence with basis of ,mobilization (bom)site for the transfer of intermediate vector form E.coli to Agrobacterium

8. Target genes can be easily cloned. The plasmid is relatively small with a number of restriction sites. Intermediate plasmid is conveniently cloned in E.coli and transferred to Agrobacterium. Advantages

9. A binary vector system consists of an agrobacterium strain along with disarmed T i plasmid called vir helper plasmid (the entire T-DNA region including borders deleted while vir gene is retained.) It may be noted that b oth of them are not physically linked (or integrated.) A binary vector with T-DNA can replicate in E.coli and agrobacterium. binary vector is consist of a pair vector. The binary vector has following component:- The disarmed ti plasmid: this plasmid has T-DNA with gene of interest and ori for both E.coli and agrobacterium. Also called as mini-Ti plasmid or micro plasmid eg.Bin 19 Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to the plant . BINERY VECTORS

10. A bacterial resistant marker e.g. tetracycline resistant gene for selecting binary vector colonies in E.coli and agrobacterium. oriT sequence for conjugal mobilization of the binary vector from E.coli agrobacterium. A broad host-range origin of replication such as RK2 that allows the replication of binary vector in agrobacterium.

11. The binary vector system involve only the transfer of a binary plasmid to agrobacterium without any integration. This is in contrast to cointegrate vector system. where in the intermediate vector is transferred and integrated with disarmed Ti plasmid. Due to convenience, binary vectors are more frequently used than cointegrate vectors Advantages of binary vectors

12. The target(foreign) gene of interest is inserted into the multiple cloning site of the binary vector. In this way, the target gene is placed between the right and left border repeats and left border repeats and cloned in E.coli. By a mating process, the binary vector is mobilized from E.coli to Agrobacterium. Now, the virulence gene proteins of T-DNA of the vectors into plant cells . Uses of binary vectors

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