1. Designing your own experiment with a GFP reporter gene
Stokes S. Baker
Associate Professor of Biology
University of Detroit Mercy
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2. Introduction
Acclimation is the process where changes in physiology allow organisms to prepare for a stress.
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3. Arabidopsis thaliana “Fruit fly” of the plant world Small
Grows well indoors
Prolific
Short life cycle
Well established genetics
The Arabidopsis Information Resource Center (Tair) aboutarabidopsis.jsp.
Figure 1: A photograph of Arabidopsis thaliana. Photo by S.S.B.
Creative Commons Copyright: Stokes S. Baker

4. Dehydration inducible genes
Dehydration induced changes in gene expression are associated with increased drought tolerance (Harb et al., 2010).
Some genes induced by dehydration are involved with other acclimation processes such as low temperature induced freezing tolerance (Harb et al., 2010).
Acclimation processes can be interconnected.
Hanin M, Brini F, Ebel C, Toda Y, Takeda S, Masmoudi K Plant dehydrins and stress tolerance: Versatile proteins for complex mechanisms. Plant Signaling & Behavior 6(10):
Harb A, Krishnan A, Ambavaram MMR, Pereira A Molecular and Physiological Analysis of Drought Stress in Arabidopsis Reveals Early Responses Leading to Acclimation in Plant Growth. Plant Physiology 154(3):
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5. Eukaryotic structural genes
Promoter
Transcribed region
Terminator
Figure 2: A simplified diagram of the structure of a eukaryotic gene that encodes a protein.
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6. GFP (green fluorescent protein) coding region
Chimeric genes
Genetic engineering involves using recombinant DNA technology to create new genes. One way to do this is to create a chimeric gene.
Promoter
Transcribed region
Terminator
Figure 3: A simplified diagram of the structure of a eukaryotic gene that encodes a protein.
drt2-promoter
GFP (green fluorescent protein) coding region
NOS-ter
Figure 4: Structure of a cold inducible chimeric gene. The promoter region of the Arabidopsis thaliana drought inducible gene drought2 (drt2) was inserted in front of the coding region of red shifted jellyfish green fluorescent protein (GFP) using recombinant DNA technology. The transcription termination signal is encoded from the Agrobacterium NOS gene.
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7. GFP Reporter Gene
Figure 5: GFP and chlorophyll fluorescence in Arabidopsis leaves. Panel A, wild type leaf viewed under white light (chlorophyll reflection). Panel B, wild type illuminated with visible blue light and viewed through a orange filter (chlorophyll fluorescence). Panel C, wild type leaf illuminated with visible blue epifluorescence lighting as in the next slide. Panel D, transgenic plant expressing GFP illuminated as in Panel C. Photographs by Haittam Greib (Panels A & B) and Cleo B. Vidican (Panels C and D).
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8. Epifluorescence lighting system
Figure 6: Digital single lens reflex (SLR) camera with epifluorescence attachment. Light from a blue light emitting diode (LED) (A) passes through condensing lenses and an excitation filter (B). A 45O dichroic beam splitting filter (C) reflects blue light (blue down arrow) onto the specimen and allows contaminating green light (green dashed arrow) to pass through. The blue light reflecting off the specimen (blue dashed arrow) is blocked by a combination of the beam splitting filter (C) and a yellow barrier filter (E). Red light from chlorophyll fluorescence (red dashed arrow) can be blocked if a cyan filter (D) is included with the barrier filter set. Green light produced by green fluorescent protein fluorescence (solid green arrow) passes through the beam splitting filter (C) and both barrier filters (D and E) and enters the camera.
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9. Detecting GFP in Arabidopsis
Place plant samples under a camera with epifluorescence attachment.
Focus lens while under white light illumination.
Turn off white light and turn on the blue light. Remove extraneous background lighting (i.e., close door to light tight box.)
Photograph image for 1 to 5 seconds.
Transfer digital image to a computer.
View image with ImageJ software.
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10. Dehydration Assay Excise leaves with scissors.
Immediately photograph using the epifluorescence attachment.
One to two hours later, photograph with the epifluorescence attachment.
Leaf
Time (hours)
Wild type 2
35S/GFP
drt/GFP
Figure 7: Typical results from leaf dehydration experiment. Panel A: Treatments and times that photographs were taken. Panel B: Photographs of leaves using white light and epifluorescence.
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11. Graphing GFP Expression
Average pixel intensity
(counts/s)
Stress: Before After Before After Before After
Plant: drt2/gfp wild type S/gfp
Figure 8: Quantification of surface fluorescence during leaf dehydration experiment. The chimeric genes used are drt2/gfp and 35S/gfp. Wild type plants do not express the gfp gene. Range bars are 95% confidence intervals.
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12. Your Assignment
During the next few weeks, you and your table partners will be jointly performing experiments with transgenic Arabidopsis that express the chimeric drt2/gfp gene. You will be investigating the following issue:
Is expression of drt2 specific to dehydration or is it induced by multiple stresses?
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13. Your group will need to turn in a written research proposal next week
1. Make sure to schedule a time for your group to plan the proposal before leaving class.
, discussion boards, and cloud services are great mechanisms to work on a joint document.
2. You need to do some background research.
3. Hand in written proposal for critique by the professor at the next laboratory session.
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14. Scientific Method
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15. Your Groups proposal
1. What issue is being addressed? What is your hypothesis?
2. Describe the experiment.
- What variable will be altered?
- What will be your controls?
3. Describe how you will quantify your results.
- Describe the statistical analysis.
4. Describe the alternative outcomes and what they mean.
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16. Include both positive controls and negative controls
Average pixel intensity
(counts/s)
Stress: Before After Before After Before After
Plant: drt2/gfp wild type S/gfp
Figure 8: Quantification of surface fluorescence during leaf dehydration experiment. The chimeric genes used are drt2/gfp and 35S/gfp. Wild type plants do not express the gfp gene. Range bars are 95% confidence intervals.
Creative Commons Copyright: Stokes S. Baker

17. Acknowledgements
The instructional materials were developed and tested at the University of Detroit Mercy
This material is based upon work supported by the National Science Foundation under Grant number and number