California State University Northridge
Biology 470 - Biotechnology
Plant genetic engineering
Resource: Glick and Pasternack slides from Chapter 17
The crown gall, and the wounded plant - acetosyringone and hydroxyacetosyringone
The distribution of the vector (requires shockwave)
|Agrobacterium tumefaciens, chromosome and plasmid
Rough draft of Ti plasmid (requires shockwave)
The military analogy: Is Agrobacterium like a "mercenary commando"?
Resource pictures: The Pathogenesis of Crown Gall, by Sforza et al.
Octopine-type Ti plasmid
T-region - the segment transferred to plants, between Right and Left border
Outside the T-region - the segment not transferred
What is the difference between an element required in cis, and one required in trans?
...a trans element can be moved to a different location, for example from the Ti plasmid to the genome, and it will still function effectively. A gene that makes a diffusible protein product can be moved generally, and that is an example of a "trans" element. An example of a "cis" element is an origin of replication. That cannot be moved, and still serve its purpose. It must be "on" the DNA that needs replicating.
A T-border sequence is a "cis" active element, whereas the vir genes are "trans" active. Vir genes do not need to be on the plasmid carrying the T-DNA region.
From CAMBIA Intellectual Property Resource, a 262 page "white paper" with detailed information on Agrobacterium transformation, intellectual property, and patents: (pdf format)
Binary vector systems - widely used
1. A wide host-range plasmid with
2. A "helper" Ti that lacks the T-DNA, but contains the vir genes
Co-integrated or hybrid Ti vector system - oldies but goodies
1. A disarmed Ti plasmid
2. An ori-less plasmid that can integrate into the Ti plasmid, containing (typically) cis elements necessary for T-DNA mobilization. (Fig 17.6)
Depends on a single crossover event
Modified Co-integrated vectors - using cre/lox to prepare for directed recombination
1. In vitro methodology
2. Transformation of E. coli
3. Transformation or conjugation (with cis/trans function) into A. tumifaciens
4. Transformation of plants
General Plant Applications:
Microprojectile bombardment - biolistics
Biorad's Helios Gene Gun
The gene is in the plant, so now what?
Detection of foreign DNA expression - Table 17.4
Selectable markers vs. Reporters
GUS - beta-D-glucuronidase (from E. coli) plus 5-bromo-4-chloro-3-inolyl B-D-glucuronic acid
Reading assignment: Chapter 18