An Investigation of Genetically Modified Food Stuffs

Transcription

1 Please print Full name clearly: BIOL 305L Laboratory Five An Investigation of Genetically Modified Food Stuffs Introduction: Genetically modified foods are foods derived from genetically modified organisms (GMOs), such as genetically modified crops. GMOs have had specific changes introduced into their DNA by genetic engineering techniques. These techniques are much more precise than mutagenesis (mutation breeding) where an organism is exposed to radiation or chemicals to create a non-specific but stable change. Other techniques by which humans modify food organisms include selective breeding; plant breeding, and animal breeding, and somaclonal variation. A genetically engineered plant is generated in a laboratory by altering its genetic makeup. This is usually done by adding one or more genes to a plant's genome using genetic engineering techniques. Most genetically modified plants are generated by the biolistic method (particle gun) or by Agrobacterium tumefaciens mediated transformation. Plant scientists, backed by results of modern comprehensive profiling of crop composition, point out that crops modified using GM techniques are less likely to have unintended changes than are conventionally bred crops. Corn, (Zea mays), known throughout most of the world as maize (and NO, I do NOT know why it has two names!), represents the most remarkable plant breeding achievement in the history of agriculture. The modern manifestation of this ancient plant bears very little resemblance to its original ancestor, a wild grass from southern Mexico called teosinte. This transformation from an inconspicuous grass to a diverse, highly evolved and productive food plant is a story of co-evolution and interdependence between humans and corn that spans thousands of years and involves millions of farmers (and YES, I do get this enthusiastic about this topic I am a plant biologist, after all!). Teosinte is a tall, drought-tolerant grass that produces, instead of a cob, spikes close to the ground, filled with two rows of small, triangular-shaped seeds within an enclosed husk. A hard shell around each seed protects them once they fall to the ground. Perhaps within 100 years after discovering that teosinte was

2 edible, people began selecting spikes to plant near their homes, which were close to irrigation sources. These selected plants continued to be developed in isolation from wild teosinte that was growing in the surrounding forests, and thus the process of developing corn had begun. The oldest known corncobs, distinctly different from teosinte, were found in the highlands of Oaxaca in southwestern Mexico and are estimated to be 5,400 years old. They had two to four rows of kernels firmly attached to the cob and were only one-inch long. Yet, even by then, there had already been at least two thousand years of human manipulation. This attachment of kernels to a central cob and the lack of a hard, inedible coat around the seed are the key factors that have inextricably bound corn with humans ever since those early times. Firm attachment meant that ears must be harvested and kernels manually removed from the cob for corn to successfully reproduce and disperse. Lack of a hard seed coat required protection, namely cool and dry storage, until the following spring planting. Figure 1: How natural breeding lead to modern corn The difference between Teosinte and corn is about 5 genes. One of the first scientists to fully appreciate the close relationship between teosinte and corn was George Beadle. In the 1930s, Beadle studied teosinte-corn hybrids and showed

3 that their chromosomes are highly compatible. Later, he produced large numbers of teosinte-corn hybrids and observed the characteristics of their offspring. By applying basic laws of genetic inheritance, Beadle calculated that only about 5 genes were responsible for the most-notable differences between teosinte and a primitive strain of corn. Using more-modern techniques, another group of scientists analyzed the DNA from teosinte-corn offspring. They too noticed that about 5 regions of the genome (which could be single genes or groups of genes) seemed to be controlling the most-significant differences between teosinte and corn. In recent years, geneticists have used advanced molecular-biology tools to pinpoint the roles of some of the genes with large effects, as well as many other regions across the genome that have had subtle effects on maize domestication. Figure 2: Maize cobs uncovered by archaeologists show the evolution of modern maize over thousands of years of selective breeding. Even the oldest archaeological samples bear an unmistakable resemblance to modern maize. Despite all of this plant breeding, corn still needed to be improved for a growing world population - and that is where genetic modification comes into the story.

4 The corn line Bt11 was developed through a specific genetic modification to be resistant to attack by the european corn borer (Ostrinia nubilalis), a major insect pest of corn in agriculture. The novel variety produced the insecticidal protein, Cry1Ab, derived from Bacillus thuringiensis subsp. kurstaki (B.t.k.) HD-1 strain. Deltaendotoxins, such as the Cry1Ab protein expressed in Bt11, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ab is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. In other words, this is the "Roundup Ready", or glyphosate-resistant genetic modification we talked about in class!. The vector details are shown below in figure three. Bacillus thuringiensis Delta endotoxin crystal Bt gene Ti plasmid Ori Ti genes Figure three: The make up of the plasmid to transfer the Bt gene

5 Lab Activities: So, for this lab we will be looking for the Bt gene to indicate a genetic modification of corn food stuffs. Remember, the Bt gene was developed through a specific genetic modification to be resistant to attack by the european corn borer (Ostrinia nubilalis), a major insect pest of corn in agriculture. Also, remember that there are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to the gene product of the activated gene in these modified corn lines. Objectives for week one: During this lab period you will extract DNA from a non-modified corn and from various corn chips which may contain this particular gene insert. Extraction of DNA from samples: a. Find your screwcap tubes and label one non-gmo and one test. b. Weigh out g of certified non-gmo food and put it into the mortar. c. Add 5 ml of distilled water for every gram of food. To calculate the volumes of water you need, multiply the mass in grams of the food weighed out by 5 and add that many milliliters. Mass of food = g x 5 = ml d. Grind with pestle for at least 2 min to form a slurry. e. Add 5 volumes of water again and mix or grind further with pestle until smooth enough to pipet. f. Pipette 50 µl of ground slurry to the screwcap tube containing 500 µl of InstaGene labeled non-gmo using the 50 µl mark on a graduated pipette. Recap tube. g. Repeat steps 2 5 to prepare the test food sample. h. Pipette 50 µl of ground test food slurry to the screwcap tube labeled test. Recap tube.

6 i. Shake or flick the non-gmo food and test food InstaGene tubes and place tubes in 95 C water bath for 5 min. j. Place tubes in a centrifuge in a balanced conformation and centrifuge for 5 min at max speed. Set Up PCR Reactions: a. Number PCR tubes 1 6 and initial them. The numbers should correspond to the following tube contents: Tube 1: 20 µl Plant MM (green) 20 µl Non-GMO food control DNA Tube 2: 20 µl GMO MM (red) 20 µl Non-GMO food control DNA Tube 3: 20 µl Plant MM (green) 20 µl Test food DNA Tube 4: 20 µl GMO MM (red) 20 µl Test food DNA Tube 5: 20 µl Plant MM (green) 20 µl GMO positive control DNA Tube 6: 20 µl GMO MM (red) 20 µl GMO positive control DN b. Place each tube in a capless microtube adaptor and place in the foam float on ice. c. Referring to the table and using a fresh tip for each addition, add 20 µl of the indicated master mix to each PCR tube, cap tubes. d. Referring to the table and using a fresh tip for each tube, add 20 µl of the indicated DNA to each PCR tube, being sure to avoid the InstaGene pellet at the bottom of the tubes. Mix by pipetting gently up and down; recap tubes. e. Get PCR tubes ready for input into the thermal cycler. The PCR reaction will be run for you and be ready for the next lab section.

7 Objectives for week two: Time to run and stain your gels to separate the DNA and potentially identify any food stuff which contains the Bt gene insertion. a. Set up your gel electrophoresis apparatus as instructed. b. Obtain your PCR tube from the thermal cycler and place in the capless microtube adaptor. Pulse-spin the tube for ~3 seconds. c. Using a fresh tip each time, add 10 µl of Orange G loading dye (LD) to each sample and mix well. d. Load 20 µl of the molecular weight ruler and 20 µl each sample into your gel in the order indicated: e. The run time and voltage will depend on the type of gel you are running. Run an agarose gel for about30 min at 100 V. f. Stain gel, get picture and analyze banding pattern. LASTLY: For the following week: Individual 9-10 page write up of the lab section (1 inch margin, 12 pt font, 1 ½ spacing). You know what to include! Just in case: Intro with background info and hypothesis: at least three references Methods in detail Written description of data, including picture of gel(pasted into your work document, legends and titles! Detailed conclusion, explain results, include info from introduction. Did the experiment work? Possible errors and/or improvements. All references listed. No required format for this just be consistent!