Co integrated vector

In study of Ti plasmid based vector we study about co integrated vector and binary vector. Here i have tried to share basic about Co-integrated vector

1. Co-Integrated Vector Name – Nirbhayaditya Vaghela Roll number - 233

2. Introduction to Co-integrated Vector This vectors were among the first types of modified and engineered Ti plasmids devised for Agrobacterium - mediated transformation. These vectors is made by the homologous recombination of bacterial plasmid with  T-DNA region of  Ti plasmid in Agrobacterium. Agrobacterium is also known as natural genetic engineer. The bacterium transfers part of its DNA to the plant, and this DNA integrates into the plant’s genome, causing the production of tumor and associated changes in plant metabolism. But this Co-integrated vector method is not widely used today. Three  type of vectors are necessary in this system. Disarmed Agrobacterium Ti plasmids Intermediate vectors Helper vectors

3. 1) Disarmed Agrobacterium Ti plasmids In this we remove the oncogenes present in T-DNA of Agrobacterium responsible for tumor formation. We replace the oncogenes with  exogenous DNA. The example of this vector is  pGV series. In pGV series we replace the  phytohormone genes with the part of pBR322 vector sequence. The left and right border sequences, the nopaline synthase gene and vir regulon of the Ti plasmid are conserved.

4. 2) Intermediate vectors T-DNA isolated from a parent Ti plasmid was subcloned in conventional  E.Coli plasmid vector.  It's used to overcome the problems derived from the large size of disarmed Ti plasmids and their lack of unique restriction sites. Intermediate vectors are replicated in E. coli and are transferred into Agrobacterium by conjugation.  They cannot replicate in A. tumefaciens.  It carries the pBR322 sequence which is also present in disarmed  Ti plasmid. It also have our gene of interest .  This pBR322 sequence is responsible for homologous recombination between intermediate and disarmed Ti plasmid 

5. 3) Helper Vector These are the plasmid present in E.Coli  which contain transfer and mobilization gene. This gene allow the transfer of the conjugation-deficient intermediate vectors into Agrobacterium

6. Procedure Conjugation between the two E.Coli will take place in which the helper vectors will transfer to E.Coli having intermediate vector.   Which was in turn mobilized and transferred to the recipient Agrobacterium. Homologous recombination between the T-DNA sequence of the disarmed  Ti plasmid and intermediate vector takes place and forms large co-integrate plasmid. Now with the help of sequences present in our co-integrated plasmid (example-  vir gene) the recombinant T-DNA (which have our gene of interest)  will transfer to the plant genome.

7. Co-integrated Plasmid The normal co-integrated plasmid which is assembled by in vitro manipulation contains  vir genes  The left and right T-DNA borders  Exogenous DNA sequence between the two T-DNA borders Plant and bacterial selectable markers.

8. Drawbacks Long homologies required between the Ti plasmid and the E. coli plasmids making them difficult to engineer and use. The transfer of gene is  relatively inefficient compared to binary vectors.

9. References The basic principles of gene cloning and DNA Analysis Google images