Genetic Transformation Part 1

Transcription

1 Genetic Transformation Part 1 The beginning of an exploration of genetic transformation and the influence of environment on gene expression. * CONTENTS 1 Objectives Experimental Goal Prerequisite skills and Knowledge Research Skills Learning Objectives Pre-Experiment Genetic Transformation and Gene Expression Regulation of Gene Expression The pglo Plasmid Genetic Transformation Gene Expression Experiments to Come Prepare for the Experiment Laboratory Manual Materials Check Off List Safety and Waste Disposal Protocols, including waste labels to be prepared Experimental Procedure Transformation Protocol Post-Lab Assignment EXPERIMENTAL GOAL 1 OBJECTIVES In this experiment students will begin an exploration of the effect of the environment on gene expression. They will transform the DNA of E. Coli, using the pglo plasmid and examine the effect of the sugar arabinose on the expression of the pglo gene. * Some instructions, explanations, and images adapted from Bio-Rad Biotechnology Explorer TM pglo TM Bacterial Transformation Kit.

2 2 G e n e t i c T r a n s f o r m a t i o n PREREQUISITE SKILLS AND KNOWLEDGE Students should have some experience with basic molecular structures, a sense of the physical interactions between charged species, and a familiarity with interactions between molecules. Prior experience with genes and gene expression is helpful, but not required. 1.3 RESEARCH SKILLS After this lab, students will have had practice in: Bacterial culture Bacterial transformation Sterile technique 1.4 LEARNING OBJECTIVES After this lab, students will be able to: Distinguish between genotype and phenotype Explain how to make bacteria competent Suggest theories for the physical mechanism of transformation Distinguish between positive and negative controls 2 PRE-EXPERIMENT 2.1 GENETIC TRANSFORMATION AND GENE EXPRESSION Regulation of Gene Expression Our bodies contain thousands of different proteins which perform many different jobs. Digestive enzymes are proteins; some of the hormone signals that run through our bodies and the antibodies protecting us from disease are proteins. The information for assembling a protein is carried in our DNA. The section of DNA which contains the code for making a protein is called a gene. There are over 30, ,000 genes in the human genome. Each gene codes for a unique protein: one gene, one protein. The gene that codes for a digestive enzyme in your mouth is different from one that codes for an antibody or the pigment that colors your eyes. If each cell in your body contains all of the same genes, does that mean that the cells in your mouth are currently making eye color pigments or the cells in your eyes are making digestive enzymes? Organisms regulate expression of their genes and ultimately the amounts and kinds of proteins present within their cells for many reasons, including developmental changes, cellular specialization, and adaptation to the environment. Gene regulation not only allows for adaptation to differing conditions, but also prevents wasteful overproduction of unneeded proteins which would put the organism at a competitive disadvantage. How would that be disadvantageous? The genes involved in the transport and breakdown (catabolism) of food are good examples of highly regulated genes. For example, the sugar arabinose is both a source of energy and a source of carbon. Escherichia coli bacteria produce three enzymes (proteins) needed to digest arabinose as a food source. A good part of this section as well as the transformation experiment, and techniques is adapted from Bio-Rad pglo manual

3 G e n e t i c T r a n s f o r m a t i o n 1 3 The genes which code for these enzymes are not expressed when arabinose is absent, but they are expressed when arabinose is present in their environment. Regulation of the expression of proteins often occurs at the level of transcription from DNA into RNA. This regulation takes place at a very specific location on the DNA template, called a promoter, where RNA polymerase sits down on the DNA and begins transcription of the gene. In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. These clusters of genes controlled by a single promoter are called operons. The three genes (arab, araa and arad) that code for three digestive enzymes involved in the breakdown of arabinose are clustered together in what is known as the arabinose operon. These three proteins are dependent on initiation of transcription from a single promoter, PBAD. Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, the DNA binding protein called arac, and the sugar arabinose. arac binds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon). When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with arac which is bound to the DNA. The interaction causes arac to change its shape which in turn promotes (helps) the binding of RNA polymerase and the three genes arab, araa, and arad are transcribed. Three enzymes are produced; they break down arabinose, and eventually the arabinose runs out. In the absence of arabinose the arac returns to its original shape and transcription is shut off.

4 4 G e n e t i c T r a n s f o r m a t i o n The pglo Plasmid In the genetically engineered pglo plasmid DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene that codes for green fluorescent protein GFP. When bacteria that have been transformed with pglo plasmid DNA are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow brilliant green when exposed to UV light. This is an excellent example of the central molecular framework of biology in action; that is, DNARNAPROTEINTRAIT. When arabinose is absent from the growth media, the GFP gene remains turned off and the colonies appear white under the UV light. In addition to the GFP gene, the pglo plasmid also carries a gene (-lactamase, bla) for a protein that gives the bacteria resistance to the antibiotic ampicillin. This gene has its own promoter which is always on. It is not part of the arabinose operon, and thus is not regulated by the presence or absence of arabinose.

5 G e n e t i c T r a n s f o r m a t i o n Genetic Transformation This transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. To move the pglo plasmid DNA through the cell membrane you will: 1. Use a transformation solution of CaCl2 (calcium chloride) 2. Carry out a procedure referred to as heat shock For transformed cells to grow in the presence of ampicillin you must: 3. Provide them with nutrients and a short incubation period to begin expressing their newly acquired genes Gene Expression Experiments to Come In the pglo system you are using, arabinose is used to induce expression of the GFP gene. In future experiments, you will examine bacteria grown in liquid LB media under different concentrations of arabinose, or other sugars of your choice, and observe the effect on gene expression. In the weeks that follow you will use a variety of biotechnology techniques to examine the regulation of gene expression in transformed E. coli. When you have completed the experiments, you should have a good understanding of how gene expression is regulated, as well as knowledge of the techniques used to study it. Each molecular biology experiment builds upon the previous experiment, making it essential to keep up with concepts from week to week as we go through this section. The experimental tools you will use in the next few weeks include spectrophotometric measurement of fluorescence, epi-fluorescence microscopy, and reverse transcription polymerase chain reaction (RT-PCR) analyzed by agarose gel electrophoresis. The PNAS paper by Deborah A. Siegele and James C Hu describes the results of experiments similar to what you will be doing. This paper is available in PDF format in the Transformation folder on e-learning. Take advantage of the weekend to get started on working your way through it. In this first experiment of the cycle, you will use the pglo plasmid to transform ordinary E. coli cells into ampicillin resistant, GFP-expressing cells. Overview: Part 1 Transform E. coli cells with the pglo plasmidyou are here Part 2 Observe transformation results and plan gene expression experiment Part 3 Observe expression through spectrophotometry and microscopy

6 6 G e n e t i c T r a n s f o r m a t i o n 1 Part 4 Extract RNA from transformed E. coli cells and Part 5 Make cdna and run PCR reaction Part 6 Run agarose gel electrophoresis on the products of the PCR reaction 2.2 PREPARE FOR THE EXPERIMENT Read through the entire laboratory procedure so that you can prepare your lab notebook. Plan your time schedule and record a preliminary schedule in your lab notebook. Read the questions in blue and think about how you will answer them. Start to work your way through the paper by Siegele and Hu. Check your preparation with the Pre-Experiment Quiz.

7 G e n e t i c T r a n s f o r m a t i o n MATERIALS CHECK OFF LIST 3 LABORATORY MANUAL For the transformation experiment, each small group of (2-3) students will have: Laptop computer Gas burner w/gas and striker E. coli LB starter plate Poured agar plates containing: o LB nutrient agar only (1 plate, labeled LB) o LB nutrient agar and ampicillin (2 plates, labeled LB/amp) o LB nutrient agar, ampicillin and arabinose (1 plate, labeled LB/amp/ara) Tube with pglo plasmid DNA stock solution Tube with 500 L LB nutrient broth Tube with 500 L transformation solution Each large group of 1-2 small groups will have Cooler for ice Disposables (place in pan of 10% bleach by sink): Bag of sterile pipettes 2 microcentrifuge tubes 10 sterile innoculation loops Each class will have: Hot water baths Ice 3.2 SAFETY AND WASTE DISPOSAL PROTOCOLS, INCLUDING WASTE LABELS TO BE PREPARED Any disposable material that has come in contact with E. coli in this lab is a biohazard and needs to be placed into the pan by the sink containing 10% bleach. Wear UV protective goggles when handling the UV pen light. Your chemical resistant lab goggles will block UV light EXPERIMENTAL PROCEDURE Recall that the goal of genetic transformation is to change an organism s traits (phenotype). Before any change in the phenotype of an organism can be detected, a thorough examination of its usual (pretransformation) phenotype must be made. Look at the colonies of E. coli on your starter plates. The following pre-transformation observations of E. coli might provide baseline data to make reference to when attempting to determine if any genetic transformation has occurred. a) Number of colonies b) Size of : 1) the largest colony 2) the smallest colony

8 8 G e n e t i c T r a n s f o r m a t i o n 1 3) the majority of colonies c) Color of the colonies d) Distribution of the colonies on the plate e) Visible appearance when viewed with ultraviolet (UV) light f) The ability of the cells to live and reproduce in the presence of an antibiotic such as ampicillin Q1. Here, and in your lab notebook, list all observable traits or characteristics that can be described: Discuss these questions with your extended group before answering them. Q2. Based on your reading of the experimental procedure, predict what you will see on each of your plates. a. +pglo LB/amp b. +pglo LB/amp/ara c. pglo LB/amp d. pglo LB Q3. In step 7 of the Transformation Protocol, why are you not adding DNA to the pglo tube? Q4. According to the DNA Transformation video, what is thought to be the role of the calcium chloride solution in DNA bacterial transformation? Q5. What is the purpose of the temperature changes described in the Heat Shock section? Describe what happens to the E. coli cells. Q6. Why is LB nutrient broth added after the heat shock sequence? Transformation Protocol 1. Prepare your sterile field. 1. Label one closed microcentrifuge tube +pglo and another -pglo. Label both tubes with your group s name. Place them in the foam tube rack. 2. Open the tubes and, using a sterile pipette tip, transfer 250 μl of transformation solution (CaCl 2) into each tube. Go to: to view the animation.

9 G e n e t i c T r a n s f o r m a t i o n Place the tubes on ice. 4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pglo tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the pglo tube. Close the pglo tube. 5. While wearing goggles, examine the pglo DNA solution with the UV lamp in the dark. Q7. Note your observations here and in your notebook. 6. Immerse a new sterile loop into the pglo plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. If there is no ring, dip your loop into the stock tube again. 7. Mix the loopful into the cell suspension of the +pglo tube. Close the tube and return it to the rack on ice. Do not add plasmid DNA to the pglo tube. 8. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes down in the rack so the bottom of the tubes stick out and make contact with the ice.

10 10 G e n e t i c T r a n s f o r m a t i o n 1 9. While the tubes are sitting on ice, label your five LB nutrient agar plates on the bottom (not the lid) as shown below as well as with your group name and date Heat shock 1. Using the foam rack as a holder, transfer both the +pglo and pglo tubes into the water bath, set at 42 C, for exactly 50 seconds. The timing of this step is critical! 2. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. 3. When the 50 seconds are done, immediately place both tubes back on ice. For the best transformation results, the transfer from the ice (0 C) to 42 C and then back to the ice must be rapid. Incubate tubes on ice for 2 minutes. 4. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipette, add 250 μl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipette for the other tube. Incubate the tubes for 10 minutes at room temperature. 5. Tap the closed tubes with your finger to mix. Using a new sterile pipette for each tube, pipette 100 μl of the transformation and control suspensions onto the appropriate nutrient agar plates.

11 G e n e t i c T r a n s f o r m a t i o n Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the LB nutrient agar by lightly sliding the flat surface of a new sterile loop back and forth across the plate surface. It is important not to dig the loop into the agar. 7. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37 C incubator until the next lab period. Make sure the plates are upside down. Lid up would allow condensation to drop onto the plate, placing water on the surface. Single colonies cannot grow in this situation. Q8. What do the labels (LB, LB/amp, LB/amp/ara) on the plates stand for? Describe the purpose of each of the four plates. Q9. Why are the plates placed upside down in the incubator?

12 12 G e n e t i c T r a n s f o r m a t i o n 1 Q10. On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed? Explain your prediction. Q11. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your prediction. Q12. Which organism would be the best choice for a genetic transformation: a bacterium, earthworm, fish or mouse? Describe your reasoning. Q13. Describe how you could use two LB nutrient agar plates, some E. coli, and some ampicillin to determine how E. coli cells are affected by ampicillin. What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells? 3.4 POST-LAB ASSIGNMENT Discuss the following questions with your extended group and be ready to share your answers with the class. Q14. What would happen if you cultured the transformed cells under a different enantiomer of the arabinose; if instead of L-arabinose, you used D-arabinose? Would you expect the chirality of the sugar to affect the expression of GFP? Explain your answer. L-(+)-arabinose D-(-)-arabinose Q15. What if you cultured the transformed bacteria under other sugars? For example, D-fructose, or D- glucose? Q16. What evolutionary advantage is there for an organism to turn on and off particular genes in response to certain conditions (environmental or otherwise)?