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This lab is deigned to introduce students to the most common method of inserting genes into plants. In addition, this lab will demonstrate how transgenic plants can be used to study biology and how they can be used in agricultural biotechnology. Students will also gain further practice with PCR and running gels. By the end of this lab and the next lab (Labs 7 and 8), students should:
Know how to extract DNA from plant material
Know how to avoid contamination of DNA samples using clean lab techniques
Be able to determine whether transgenes are present in plant material
Understand how transgenic plants are used in agriculture and in basic plant research.
II. Safety Considerations
As in all labs, tie back long hair and be careful in handling the bleach spray solutions. The mist will irritate eyes and mucous membranes and damage clothes.
A. AGROBACTERIUM: A NATURAL GENE TRANSFER SYSTEM
As is the case with many of the major discoveries in science, the techniques that are used today to transfer genes into plants were developed as a result of basic research for which this outcome was not even imagined by the researchers who began the work.
Over many years, farmers have noticed that trees often form galls (tumor-like growths) near the soil line. Since they form near the top of the root system, they are referred to as crown galls. Careful observation indicated that these crown galls typically grew after the plant was wounded. (For example, it is common for trees to be nicked by a plow at the soil line). Scientists who set out to understand what was occurring at these wound sites eventually discovered that the organism Agrobacterium tumefasciens was responsible for the formation of these galls. As they investigated further, it was discovered that the galls resulted from a hijacking of the plant's normal growth and development. The bacterium accomplishes this by transferring genes into the plant. The researchers were amazed to discover that the genes were transferred from the bacterium into the nucleus of the plant cell where they were inserted into the genome of the plant! Once inserted, they behave exactly like a normal piece of plant DNA--they are said to be stably integrated into the genome--and are inherited normally. Normal (wild-type) Agrobacterium causes gall formation by inserting genes that direct the plant to produce hormones and other signals that cause uncontrolled growth at the site of infection, promoting and aiding the bacteria's ability to infect the plant. Plant biologists quickly realized that if those genes were removed it might be possible to replace them with other genes and use Agrobacterium to insert them into the plant genome. Indeed, this has proven to be very straightforward to do. The ability to insert genes into plants has created a revolution in plant biology and has opened the door for transgenic plants to be used in agriculture. Other techniques have been developed to transfer genes into plants, but Agrobacterium-based transformation remains the most commonly used method.
The genes that "Agro" transfers to plants were found to be carried on a plasmid (a circular piece of extrachromosomal DNA). That Agro utilizes a plasmid for its mobile genes makes the use of Agro even more convenient for generating transgenic plants since plasmids are much simpler to work with than genomic DNA (due to their relatively small size). Study of the plasmid (which was named the "Ti plasmid" for Tumor inducing) demonstrated that only a portion of the plasmid was transferred into the plant genome and that this region (the T-DNA) was defined by two flanking sequences (defined as the left and right T-DNA borders--TL and TR). Removal of the tumor inducing genes from the Ti plasmid and their replacement with other genes allowed experiments to be developed that proved that any genes inserted between the left and right border could be inserted into plant genomes by Agro.
B. ASSEMBLING GENES FOR TRANSFER INTO PLANTS
The basic techniques of molecular biology allow the easy assembly of DNA constructs for insertion into plants by Agro. As we have seen in earlier labs, restriction enzymes can be used to cut DNA at specific sites. The enzyme DNA ligase can be used to connect fragments that have been cut by restriction digestion, which allows assembly of larger DNA molecules, including the specific pieces of DNA that the researcher has chosen. A wide array of clever techniques using restriction enzymes, PCR, and other enzymes have expanded our ability to copy, cut and paste pieces of DNA together to generate exactly the DNA constructs that we would like to study. You will have the opportunity to explore many of these techniques in upper division courses such as Molecular Biology Laboratory (BIO 181). Most techniques utilize E. coli plasmids for the assembly of DNA. E. coli plasmids were the first developed for recombinant DNA work and continue to be the easiest to work with since they are relatively small and have been modified to facilitate DNA assembly and high yields of DNA upon purification. Since this is the case, most assembly of DNA for use in Agro is first carried out in E. coli and (usually following verification by DNA sequencing) the assembled DNA is then transferred into Agro plasmids for subsequent transfer into plants.
While we will not assemble DNA in this lab, we hope to help you understand what is necessary for genes to be expressed in plants and how these techniques can be used in research and in agricultural biotechnology.
As described above, Agro will transfer any DNA construct into plants, as long as it is placed in the T-DNA region of the appropriate Agro plasmid. In addition, if DNA we place into Agro is to be functional in the plant genome, it must have the minimal components of a normal plant gene that are necessary for function.
What do you think are the minimal necessary components for a gene to be expressed (in addition to a coding region)?
Add the components that you think are necessary to the diagram below:
The ability to generate transgenic plants opened the door for researchers to test what the minimum components necessary for expression are in addition to testing much about the basic biology of plants. In developing plasmids to use in the transfer of genes, it was demonstrated that, for expression, a coding sequence just required a promoter (to direct the initiation of transcription) and a terminator to direct the termination of transcription and to direct the addition of a poly (A) tail to the mRNA.
In addition to transferring a functional gene or genes of interest into plants using Agrobacterium, it is also normally necessary to include a selectable marker--i.e. some gene that allows the researcher to select plants that have been successfully transformed (i.e. that have received the transgene). Selectable markers are also typically included on plasmids to allow selection of bacteria that have successfully received plasmids that are being manipulated. Most commonly, antibiotic resistance genes are used to select for the presence of plasmids and for the successful transformation of plants. When used in bacteria, bacteria are spread on plates or grown in liquid cultures that include antibiotics. Only those bacteria that have plasmids that provide genes that allow them to be resistant to the antibiotic are able to grow. Likewise, following plant transformation, if an antibiotic resistance gene has been used as a selectable marker, plants are grown on media containing antibiotics, and only those that have picked up the transgene (and are expressing it!) are able to grow on antibiotics. Most commonly, the bacterial NPT II (neomycin phosphotransferase) gene has been used as a selectable marker. This gene confers resistance to the antibiotic kanamycin. To direct expression of this gene, an Agrobacterium promoter is most commonly used (the NOS promoter from the Agro nopaline synthase gene), along with the NOS terminator.
Based on what you have learned so far, sketch and label the elements that you think are likely to be present on this Agrobacterium plasmid between the TL and TR borders of the Ti plasmid shown schematically below:
NPT II My gene
C. Agricutural Biotechnology
The ability to transfer genes of interest into plants also made it possible for plant biologists to modify plants in ways that could not be accomplished with conventional plant breeding. Some of the first changes that were made have now gained widespread acceptance by farmers around the world. Two of the most common changes have been to: 1) insert genes that code for a protein from Bacillus thuringensis (BT) that is toxic to many lepidopteran larva (including corn borers, cotton boll worms and many other larva that have a huge impact on agricultural yields) and 2) insert genes that confer resistance to herbicides such as Round-upÂ® (sold by Monsanto â€¦ as are the resistant plant varieties!). Table I (on the next page) shows the extent to which transgenic plants have been adopted in recent years.
BT toxin has been used by organic farmers for many years and is thought to have no negative health affects as a result of its consumption. As a result, it was an early target for Ag biotech
companies. While some early studies suggested that plants expressing Bt toxin might have a
Table I. Major GM (genetically modified) Crops in the U.S. Percentage of total planting that is genetically engineered. (Source: U.S. Dept. of Agriculture, Economic Research Service (ERS) http://www.ers.usda.gov/Data/BiotechCrops/)
negative impact on beneficial insects, to date, there is no strong evidence that this is the case. The net affect of the use of Bt is that farmers are able to greatly reduce the amount of pesticides used.
Round-upÂ® was chosen as a target by Monsanto for the obvious reason that they produce and sell the herbicide. In addition, Monsanto focused on this herbicide because it is thought be very safe for humans and to have very little environmental impact. Being able to plant a Round-upÂ® -resistant crop and then spray to the kill normal, Round-upÂ®-sensitive weeds that otherwise might compete for the crop's resources reduces the need for cultivation. This, in turn, has the benefit of reducing the loss of soil due to erosion--a major problem in agriculture. In addition, farmers have been able to substitute Round-upÂ® for other, more toxic, herbicides.
While acceptance has been high and there appear to be clear benefits to these uses of transgenic plants, the use of biotechnology in agriculture has also encountered lots of opposition. Early predictions were that weeds would develop resistance to Round-upÂ®--and incidences of this have now occurred (evolution never sleeps!). While many of these changes seem to have helped the small farmer, there certainly is not universal agreement that small farmers are benefiting from this technology. As new changes are proposed--producing pharmaceuticals in plants, using crops that are exported to countries that oppose the technology (rice to Japan), etc.--issues continue to arise (see pages 406-408 of your text). Your Activity instructor may raise some of these concerns for your contemplation and discussion.
In the next two lab periods, we will isolate DNA from plant material and use PCR to test for the presence of transgenes. We will also see how transgenic plants can be used to study basic questions in biology.
D. Using Transgenic Plants to Study Biology
The ability to transfer genes into plants made possible a very wide array of experiments to test hypotheses in plant biology. In parallel with this revolution in plant biology, most other areas of biology were taking advantage of similar techniques to address fundamental questions in biology.
A casual examination of journals in plant biology, such as the Plant Cell, Plant Physiology and the Plant Journal (all available in our library) demonstrates that a large percentage of studies utilize transgenic plants in one way or another. As mentioned above, gene constructs were used to test the minimum components required for expression. Using that same basic approach, gene constructs have been used to understand many aspects of the control of transcription, translation and protein localization. Gene constructs have also been used extensively to study the function of specific gene products. For instance, to test the function of a protein, transgenic plants can be generated that over-express the protein of interest. Often, over-expression provides evidence of the function of the protein. Conversely, DNA constructs can be prepared that will cause a decrease in the level of a specific protein, which can also be very informative in helping to unravel the functional role of a protein.
E. Using Reporter Genes to Study Gene Expression
In attempting to understand basic biology, we often seek to understand how a specific gene is regulated. A major piece of this picture is to understand how the promoter of the gene regulates its expression--i.e what level of expression does the promoter confer, in what cells does the promoter direct synthesis, is the promoter responsive to specific signals, etc.
One powerful way to address these questions is through the use of reporter genes. Reporter genes were developed as markers that are readily visualized and quantified since it can be very difficult or even impossible to measure the level of expression of the products of some genes directly. One way to utilize reporter genes is to fuse the promoter of the gene of interest to a reporter gene (along with a transcriptional terminator) and transfer this construct back into the organism of interest. The promoter will then direct expression, as it would for the gene with which it is normally associated, and the reporter gene allows this pattern of expression to be observed and quantified. In plant biology, two commonly used reporter genes are GFP (green fluorescent protein) and GUS (ß-glucuronidase--an enzyme encoded by a bacterial gene). If GFP is used as a reporter gene, its pattern of expression can be observed directly using fluorescence microscopy. If GUS is used, plants expressing the gene can be stained to detect the specific sites of GUS expression. Crude extracts of proteins can also be measured in enzyme assays to quantify the level of expression.
In our lab exercises, we will utilize GUS reporter gene constructs to illustrate how this approach can be used to study gene expression. Specifically, we will utilize transgenic plants that are expressing GUS under the control of the promoter of a tomato gene. (The reporter gene constructs and transgenic plants were generated by graduate and undergraduate students in Dr. Ewing's lab. The goal of this research is to understand how the promoter of this gene--the tomato plasma membrane H+-ATPase, LHA2--regulates its expression to control plant growth and development.)
Here is a schematic diagram of the construct expressed in these plants:
(a) NPT II (b) LHA2 promoter GUS (c)
To what do you think a, b and c refer?
When this construct is expressed in plants, GUS will be expressed wherever LHA2 is normally expressed--providing a powerful means for understanding the regulation of the LHA2 gene.
How is GUS detected? GUS encodes the enzyme ß-glucuronidase, a bacterial enzyme that is able to cleave a number of different substrates. In our assay, we will use GUS enzymatic activity to cleave the colorless compound X-gluc (an abbreviation for 5-Bromo-4-chloro-3-indolyl-ß-D-glucuronic acid) to yield a dark blue product (Figure 1). By incubating plants in a solution containing X-gluc we will be able to observe blue staining wherever GUS is expressed. This will enable us to see where LHA2 would normally be expressed in the plant as well as to see the relative levels of expression of the gene in different tissue/cell types.
Figure 1. GUS cleavage of X-Gluc. X-Gluc (5-bromo-4-chloro-3-indolyl-Î²-D-glucuronide), a substrate of Î²-glucuronidase, is cleaved to produce glucuronic acid and chloro-bromoindigo. When oxidized, chloro-bromoindigo dimerizes to produce the insoluble blue precipitate dichloro-dibromoindigo. (From: Karcher, S. Blue Plants: Transgenic Plants with the Gus Reporter Gene. www.ableweb.org/volumes/vol-23/3-karcher.pdf)
F. OVERVIEW OF THE TRANSGENIC PLANTS LABS
Today's lab will consist of two separate experiments. Both of these experiments will be started today and finished in our next lab. In Part A of today's lab, we will extract DNA from a known non-GMO food (organically-grown soybeans) and from "test foods" purchased at the grocery store. We will then set up PCR reactions using these extracts to test for the presence of the promoters discussed above. As a positive control for the PCR reactions, you will be provided with a DNA extract from a known transgenic plant (but you will not extract this DNA yourself).
To understand and interpret your PCR results, you will need some information about the "Instagene Matrix" DNA extraction technique and the PCR primers you will be using. "Instagene Matrix" is essentially the same method as Chelex extraction (described in the Introduction to Lab 5). The cells are lysed by heat and the Matrix chelates out magnesium to protect the DNA from attack by nucleases present in the cells' cytoplasm. The result is a very crude extract that is too impure for many downstream applications but is sufficiently pure for PCR.
We will use two different primer sets for PCR. The first primer set (Plant PSII primers) targets a region of the Photosystem II chloroplast gene that is common to most higher plants. Thus, if a food contains any plant material, it will probably produce a product during a PCR reaction that contains this primer set. The expected product size is 455 bp.
The second primer set (GM primers) actually contains two different primer sets. There is a primer set that amplifies a 203 bp fragment of the 35S promoter of the Cauliflower mosaic virus (CMV 35S promoter) and a primer set that amplifies a 225 bp fragment of the terminator of the nopalene synthase gene of Agrobacterium tumefaciens. (NOS terminator). Some GMO plants will only have one, some may have both, and some may not have either one. However, 85% of all GM foods are detectable with these primers. The CaMV 35S primer set alone can only detect ~70% of GM foods, so there's a very good chance that if you have a food that contains genetically modified plant material, you will get a PCR product with the GM primer set.
Note that since the two product sizes from the GM primer sets are very close in size, they are going to be indistinguishable on an agarose gel.
In Part B of today's lab, we will treat reporter gene expressing plants so that we can observe the distribution of the reporter gene in our next lab.
I. Things to Do
PART A. Prepare DNA EXTRACTS AND SET UP PCR REACTIONS
Wear gloves throughout (to protect you and to keep your DNA out of the samples).
Retrieve a mortar and pestle and rinse it thoroughly with tap water to remove any residual plant material from previous groups.
Obtain sterile distilled water and serological pipettes from the front bench.
Weigh out 1 gram of the non-GM food (obtain from front bench), and place this non-GM control food into your mortar.
Add 5 mL sterile distilled water to the mortar and food, using one of your 5 mL serological pipettes. Take care to NOT let the tip of the pipette touch the food sample! Grind the food and water together thoroughly with the pestle for at least 2 minutes to form a concentrated "slurry".
Add 5 more mL of sterile, distilled water (using the same, hopefully still clean, 5 mL pipette) and mix/grind the food further until smooth enough to pipette. Another 2 minutes of grinding should suffice.
Use a micropipettor to transfer 50 ÂµL of this final, diluted slurry to the "non-GM" tube, which should already contain 500 ÂµL of "InstaGene Matrix."
Choose a source of food you would like to test from the front bench or bring some from home.
Indicate your test food choice in your lab notebook. Also write a clear hypothesis for your experiment in your lab notebook.
Clean your mortar and pestle again. Dispose of the 5 mL serological pipette you used to transfer the water for the last sample.
Weigh out exactly 1 gram of the "test" food, and place it in your mortar.
Add 5 mL sterile distilled water to the mortar and food, using your second 5 mL serological pipette (the clean, unused one). Grind the food and water together thoroughly with the pestle for at least 2 minutes to form a concentrated "slurry".
Add 5 more mL sterile, distilled water and mix/grind the food further until smooth enough to pipet. Another 2 minutes of grinding should suffice.
Use a micropipettor to transfer 50 Âµl of this final, diluted slurry to the "test" tube which should already contain 500 ÂµL "InstaGene Matrix."
Vortex each tube (non-GM & test) and place in 95°C water bath for 5 minutes.
Place your tubes in the microcentrifuge in a balanced fashion. Spin the tubes at maximum speed for 5 minutes. When removing tubes from the centrifuge, be careful not to disturb the pellet. (Keep tubes upright, don't drop them, don't shake them, etc.) The DNA is in the supernatant and we want it to remain separate from the material that has been bound by the InstaGene Matrix.
17. Obtain a tube of "Reporter DNA" (DNA from a known transgenic plant) from the front bench. This will serve as your positive control DNA for the PCR reactions.
18. Obtain 6 autoclaved 0.2 ml PCR tubes and number them on the top 1 - 6. Then label them on the side with this number and your lab group's number and/or a symbol you can identify. Place these labeled tubes in your tube rack. The tube numbers should correspond to the following table:
Master Mix / Primer Combo
20 Âµl Plant MM (green)
20 Âµl non-GM food DNA
20 Âµl GM MM (red)
20 Âµl non-GM food DNA
20 Âµl Plant MM (green)
20 Âµl Test food DNA
20 Âµl GM MM (red)
20 Âµl Test food DNA
20 Âµl Plant MM (green)
20 Âµl REPORTER DNA
20 Âµl GM MM (red)
20 Âµl REPORTER DNA
19. Using a fresh tip for each addition (read that again!), add 20 Âµl of the indicated Master Mix/primer combo to the appropriate PCR tubes and cap them.
20. Using a fresh tip for each tube, add 20 Âµl of the indicated DNA extract to each PCR tube. Be careful to NOT disturb the InstaGene pellet at the bottom of the tubes when you are withdrawing this sample! (The "Instagene Matrix" inhibits PCR reactions.) DNA from the reporter gene expressing plants is in the tube that was provided for you.
21. Once the indicated extract/DNA is placed in a given PCR tube, gently mix the DNA with the Master Mix/primer combo by flicking (do not be too vigorous since Taq can be damaged by by contact air). If necessary collect the contents in the bottom of the tube with a quick spin in the mini-centrifuge.
22. When all of your tubes are ready, you may load them into the thermocycler. Load them in order: 1-6 and note the row in case numbers get rubbed off.
23. The PCR program we will be running is as follows:
Step 1. 94° C 5 min
Step 2. 94° C 1 min
Step 3. 59° C 1 min
Step 4. 72° C 2 min
Step 5. CYCLE steps 2-4, 25 X
Step 5. 72° C 10 min
Step 6. 4° C HOLD
PART B: Detection of Reporter Gene Expression
As described in the introduction, transgenic plants have many uses in studying biology. The plants that you will examine are expressing the reporter gene GUS under the control of the promoter of a gene being studied in Dr. Ewing's lab. The goal of this part of the lab is to determine the pattern in which this promoter directs expression of the transgene GUS.
The Arabidopsis plants that you will use have been grown under sterile conditions so that there will not be any bacteria present. Why might bacteria cause a problem with this experiment?
1. Obtain a plate of seedlings from the front bench.
2. Record the identifying number marked on the plate (this identifies the transgenic line that you are testing).
3. Obtain two 1.5 mL microfuge tubes containing 0.5 mL of GUS staining solution and label each with the identifying number on your plate along with one or more of your initials (to distinguish them from the others in the class.)
4. Carefully transfer 2 or 3 plants into each tube.
5. Place tubes in the 37o C water bath that is labeled for this use. These will be removed after 24 hrs and stored at 4o C for you until the next lab period.
V. Lab Clean-Up
Liquid waste can be disposed down the sink.
Dry waste can be thrown in the trash.
Chilton, M.-D., M. H. Drummond, D. J. Merlo, D. Sciaky, A. L. Montoya, M. P. Gordon, and E. W. Nester. 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263-271.
Gelvin, S.B. 2003. Agrobacterium-Mediated Plant Transformation: the Biology behind the "Gene-Jockeying" Tool Microbiol Mol Biol Rev. 67(1): 16-37.