| MadSci Network: Molecular Biology |
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 http://opbs.okstate.edu/~Melcher/MG/MGW4/MG4373.html 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: 143a-143t 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: http://www.apsnet.org/mpmi/pdfs/2000/0210-02R.pdf 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!
Try the links in the MadSci Library for more information on Molecular Biology.