1. Agrobacterium intro and plan 1

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! 2

3. Problem statement The binary vector system using E. coli and Agrobacterium tumefasciens is 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 3

4. Approach E. coli is our preferred chassis. The most iGEM biobricks 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. 4

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 5

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. 6

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