MadSci Network: Molecular Biology

Re: Will a plasmid like pGL2 be compatible with Agrobacterium tumefaciens?

Date: Sat Oct 21 16:44:48 2006
Posted By: Shireef Darwish, Grad student, Department of Plant Science, McGill University
Area of science: Molecular Biology
ID: 1160604772.Mb

Hi Stephanie!
The pGL2 plasmid will be compatible with both E. coli and Agrobacterium
competent cells. However, there are several reasons why plasmids designed
for E. coil will be unsuitable for your project goals. First, E. coli and
Agrobacterium will display different sensitivities to the various
antibiotics used to screen successfully transformed cells... remember, most
plasmids used for cloning contain an “antibiotic-resistance” gene that will
allow the selection of transformed cells by culturing the bacteria on Petri
plates containing the antibiotic. Therefore, antibiotics appropriate for E.
coli may not be appropriate for Agrobacterium. Second, Agrobacterium
contains a unique plasmid, called the Ti (tumor-inducing) plasmid, which
has the ability to transfer its DNA into the plant genome... most other
plasmids lack this ability. It is the Ti plasmid, containing the T-DNA
(transfer DNA) that makes Agrobacterium such a great tool for plant
transformation. Therefore, even if you are able to transform Agrobacterium
with the pGL2 plasmid and screen successfully transformed cells, there is
no mechanism for the luciferase gene to be transferred to the plant. Also,
because you want the luciferase gene transcribed in the plant, you must
include a plant-specific promoter upstream of the luceiferase insert; the
cauliflower-mosaic virus 35S RNA (CaMV 35S) promoter sequence is often used
(see Schneider et al, 1990. Plant Molecular Biology, 14: 935-947). (see for a brief overview).
Together, these factors will prevent you from using a ready-made plasmid
designed for an E. coli cloning system in an Agrobacterium model without
significant modification. However, there are several papers that describe
transforming plants with the luciferase gene using an Agrobacerium system,
and I suggest you read their materials and methods sections to see how
others have succeeded in making glowing plants! Note the species typically
used for transformation (i.e., Arabidopsis, tobacco).

Koncz et al (1987). Expression and assembly of functional bacterial
luciferase in plants. PNAS USA, 84: 131-135

Van Leeuwen et al (2000). The use of the luciferase reporter system for in
planta gene expression studies. Plant Molecular Biology Reporter, 18:

Kovalchuk et al (2003). Pathogen-induced systemic plant signal triggers DNA
rearrangements. Nature,  423: 760-762 

Forreiter et al (1997). Stable transformation of an Arabidopsis cell
suspension culture with firefly luciferase providing a cellular system for
analysis of chaperone activity in vivo. The Plant Cell, 9: 2171-2181. 

Think about contacting the researchers directly with specific questions
that may arise concerning the design of a suitable plasmid, if you continue
along this route.

NOTE: the amount of light produced by the enzymatic cleaving of luciferin
is very low, and the pictures that you may have seen online were taken with
very long exposure times. Successfully transformed plants will therefore
not glow in front of you eyes! If you are interested in generating plants
with an immediate glowing effect, there is an alternative, still using
Argrobacterium, that provides a pretty great effect under UV light. It is
called “agroinfiltration”, and it’s pretty cool. 

First, Agrobacterium is transformed with the gene encoding the green
fluorescent protein (GFP), a protein that fluoresces green when exposed to
blue light. Transformed bacteria are cultured (liquid), washed, and
suspended in a buffer solution that is placed in a syringe (without the
needle!). Then, you just press the syringe against the bottom of the leaf
(tobacco, potato, Arabidopsis) and squeeze the liquid into the leaf through
the stomata. You can actually see the liquid enter the leaf like a sponge!
The Agrobacterium will then transfer the GFP to the plant genome (2-3 days)
and the plant will begin to make the protein. When you view the plant under
UV light, you can immediately see the transformed leaves/tissues light up!
However, the transformation is transient, lasting only a few days. This is
a simplified overview of the process, but it’s pretty straight-forward.
Check out the following:

McIntosh et al (2004). A rapid Agrobacterium-mediated Arabidopsis thaliana
transient assay system. Plant Molecular Biology Reporter, 22: 53-61.

I hope this helps answer your questions!
Good luck, Stephanie!


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