Transcript Agrobacterium plan

Agrobacterium intro and plan
1
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
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 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
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
• 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
Get Ti Plasmid
virB
D4
B2
Ab
vir C
virD
Other
vir
Attachment factors
PCR
PC
virus
qPCR
2D
gel
Test in E. coli
Attachment
mutants
Agro transformation control
Plant
Yeast
HeLa
Gene of interest
combine
Transform w/ E. coli
7