Agrobacterium intro and plan

1. Agrobacteriumintro and plan

2. intro • This slide deck is the result of my conversations with Chris and our discussions at iGEM meetings. One of the best ways to see if you understand a proposal is to try to present it. • Please take this slide deck as a starting point and try it yourself!

3. Problem statement • The binary vector system using E. coli and Agrobacteriumtumefasciensis slow, cumbersome, riddled with patent constraints, and not suited for all target genes or organisms • The mechanism is reasonably well understood, though, so let’s try to engineer a better solution

4. Approach • E. coli is our preferred chassis. The most iGEMbiobricks are available on that platform and it is a convenient organism to work with. • The type 4 secretory system that allows Agrobacterium to be such an effective vector for plant transformation doesn’t exist in E. coli so let’s put it there.

5. Approach details • Ti plasmid in Agrobacterium contains most of the virulence factors needed to move the T-DNA from the host bacterium to the target cells • This plasmid can be inserted into E. coli but a working gene delivery system has not yet been engineered • Attachment of the host bacterium to the target cells is crucial

6. Approach details • virA and virG – involved in signalling and enabling the remainder of the vir genes – we will exclude these and use a simpler system • virB and virD4 – make up the type 4 secretory system that transfers the T-DNA • Other vir genes – virD2, virE, virC – enhance transformation efficiencies • PC – Agrobacterium uses PC in it’s host interactions and E. coli does not produce PC. Some evidence implies it’s necessary.

7. Get Ti Plasmid Attachment factors virB D4 vir C virD Other vir PC PCR virus qPCR B2 Ab 2D gel Attachment mutants Test in E. coli Agro transformation control Plant Yeast HeLa Gene of interest combine Transform w/ E. coli