Agrobacterium intro and plan 1 intro This slide
Agrobacterium intro and plan 1
intro • This slide deck is the result of my conversations with Chris and our discussions at i. GEM 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
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
Approach • E. coli is our preferred chassis. The most i. GEM 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
Approach details • Ti plasmid in Agrobacterium contains most of the virulence factors needed to move the TDNA 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
Approach details • vir. A and vir. G – involved in signalling and enabling the remainder of the vir genes – we will exclude these and use a simpler system • vir. B and vir. D 4 – make up the type 4 secretory system that transfers the T-DNA • Other vir genes – vir. D 2, vir. E, vir. C – 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
Get Ti Plasmid vir. B D 4 B 2 Ab vir C vir. D Other vir Attachment factors PCR PC virus q. PCR 2 D gel Test in E. coli Attachment mutants Agro transformation control Plant Yeast He. La Gene of interest combine Transform w/ E. coli 7
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