Experiment 8: Bacterial Transformation

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1 Experiment 8: Bacterial Transformation Objectives: At the end of this exercise, you will be able to 1. Understand the concepts of plasmids and genetic transformation. 2. Perform a bacterial transformation with appropriate laboratory techniques. 3. Describe the function of a selectable marker in the isolation of transformants. 4. Analyze the results and calculate the transformation efficiency. This topic was adapted from pglo TM Bacterial Transformation kit, Bio-Rad Laboratories, Inc. Duplication of any part of the document is permitted for classroom use only. One of the most important techniques of modern molecular biology is the ability to introduce a specific gene into an organism and have that genetic information expressed by the organism. This process, called genetic transformation, played a critical role in the history of molecular biology and the discovery that DNA was the genetic material. Four scientists, Griffith in the 1920's and Avery, McCarty, and MacLeod in the 1940's, showed that the apparent "transformation" of one bacterial type into another required DNA and no other biological molecule. Today, transformation is not limited to bacteria, and in fact, is used routinely by molecular biologists to manipulate and study the behavior of genes. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person s cells with healthy copies of the defective gene that causes the disease. You will use a procedure to transform bacteria (Escherichia coli K-12:HB101) with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. In this activity, you will learn about the process of moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids. Bio-Rad s unique pglo plasmid (p=plasmid) encodes the gene for GFP and a gene (bla) for betalactamase that provides resistance to the antibiotic ampicillin. pglo also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells nutrient medium (see Appendix ). Selection for cells that have been transformed with pglo DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type 1

2 phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium. Arabinose (C 5 H 10 O 5 ) is found in nature and can be metabolized by microorganisms as a carbon source. In the lab, E. coli cells are more likely to incorporate foreign DNA if their cell walls and cell membrane are altered so that DNA can pass through more easily. Such cells are said to be "competent". E. coli cells are made competent by a process that uses calcium chloride and heat shock. In addition, cells from exponential phase of growth are typically used in preparation of competent cells since they give better transformation efficiency. The transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the host cells and provide an environment for the cells to express their newly acquired genes. Incubation: the cells mixed with a transformation solution of CaCl 2 (calcium chloride) and the plasmid. Heatshock: the cells and DNA mixture are then subjected to a treatment at 42ºC for 50 seconds; the temperature changes destabilize cell membranes and create thermal imbalance. Recovery: a short incubation period to begin expressing their newly acquired genes Appendix MAP of pglo 2

3 The Arabinose Operon: 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, P BAD. Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called arac and 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 (actually helps) the binding of RNA polymerase and the three genes arab, A and D, 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. The Expression of Green Fluorescent Protein: The DNA code of the pglo plasmid has been engineered to incorporate aspects of the arabinose operon. Both the promoter (P BAD ) and the arac gene are present. However, the genes which code for arabinose catabolism, arab, A and D, have been replaced by the single gene which codes for GFP. Therefore, in the presence of arabinose, arac protein promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliant green as they produce more and more GFP. In the absence of arabinose, arac no longer facilitates the binding of RNA polymerase and the GFP gene is not transcribed. When GFP is not made, bacteria colonies will appear to have a wild-type (natural) phenotype of white colonies with no fluorescence. This is an excellent example of the central molecular framework of biology in action: DNA RNA PROTEIN TRAIT 3

4 Procedure Day 1 1. (TA) Streak E. coli HB101 on LB plate and incubate overnight at 37 C. LB plates should be streaked for single colonies hours before the transformation activity is planned. Under favorable conditions, one cell multiplies to become millions of genetically identical cells in just 24 hours. There are millions of individual bacteria in a single 1 mm bacterial colony. Procedure Day 2 Your workstation: (two students per group) E. coli starter plate (shared by four groups) LB nutrient broth Inoculation loops Glass spreader Ethanol beaker with lid Bunsen burner and striker Sterilized tip box Micropipets: P200 Foam container full of crushed ice Marking pen Two sterilized microtubes Microfuge-tube rack Masking tape Timer 1 LB plate (one black line) 2 LB/amp, (One black line, one red line) 1 LB/amp/ara (one black line, two red line) Common workstation. Transformation solution (CaCl 2, 50 mm ) pglo plasmid (0.05 μg/μl) 42 C water bath with floating tube rack, Timer 37 C incubator 1. Label one closed micro test tube +pglo and another -pglo. Label both tubes with your group s name. Place them on ice. 2. Use P1000 micropipet to transfer 400 μl of transformation solution (CaCl 2 ) into the tube labeled with -pglo. Place the tubes on ice. 3. Use a sterile loop (flamed by using Bunsen burner) to pick up a single colony of bacteria from your starter plate. 4. Immerse the loop into the transformation solution in the -pglo 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. Transfer 200 μl of cell suspension from the -pglo tube to the +pglo tube. Discard the used yellow tips in a small biohazard trashcan. 5. In dark room, examine the pglo DNA solution with the UV lamp. Note your observations. Would you see fluorescence in DNA tube? Why or why not? (On TA bench) Transfer 5 μl pglo DNA into the cell suspension of the +pglo tube. Close the tube and return it on ice. Do not add plasmid DNA to the -pglo tube. 6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down to make contact with the ice. 4

5 7. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom (not the lid) as follows: Label one LB/amp plate: + pglo Label the LB/amp/ara plate: + pglo Label the other LB/amp plate: - pglo Label the LB plate: - pglo Label all plates with sign-in numbers, section and the host strain. 8. Heat shock. Transfer both the (+) pglo and (-) pglo tubes into the water bath, set at 42 o C, for exactly 50 seconds. 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. When the 50 seconds are done, 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. 9. Remove the tubes from the ice and place on the bench top. Open a tube, add 200 μl of LB nutrient broth to one tube and reclose it. Repeat with a new tip for the other tube. Close the tube of LB and return it to TA. Incubate the tubes for 10 minutes at room temperature. 10. Tap the closed tubes with your finger to mix. Using a new tip for each tube, pipet 100 μl of the cell suspensions from (+) pglo and (-) pglo tubes onto the appropriate nutrient plates. 11. Spread the suspensions evenly around the surface of the LB nutrient agar using glass-rod spreader. Sterilize the spreader by dipping in the alcohol and passing through flame before and after each spreading. Dispose excess cell suspension, used yellow tips in the biohazard container. 12. 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 day. TA will store your plates at 4 o C till next week. You will analyze the results during your lab next week. 5

6 Procedure Day 3 A. Data Collection 1. Carefully observe and draw what you see on each of the four plates under normal room lighting and the ultraviolet light, respectively. 2. What color are the bacteria? 3. How many bacterial colonies are on each plate (use marking pen to count the colonies on the bottom of plates). 4. Plasmid added Plasmid added No plasmid No plasmid Group 1 LB/Amp LB/AmpP/Ara LB/Amp LB Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Group 11 Group (no fluorescene) 0 TNTC (too numerous to count) B. Analysis of Results The goal of data analysis for this investigation is to determine if genetic transformation has occurred. 1. Which of the traits that you originally observed for E. coli did not seem to become altered? Which seem now to be significantly different after performing the transformation procedure? 2. From the results that you obtained, how could you prove that the presence of colonies on LB/Amp/Ara is due to the effect of pglo plasmid? 3. What are the two possible sources of fluorescence within the colonies when exposed to UV light? Note: Recall what you observed when you shined the UV light onto a sample of original pglo plasmid DNA and describe your observations. Which of the two possible sources of the fluorescence can now be eliminated? 6

7 4. Calculate Transformation Efficiency The formula used below is one of the ways to calculate transformation efficiency. It tells us the total number of bacterial cells transformed by one microgram of DNA. The transformation efficiency is calculated using the following formula: Transformation efficiency = Total number of cells growing on the agar plate Amount of DNA spread on the agar plate (in μg) 1) Determine the Total Number of transformed Cells Count the colonies on your LB/amp plate. 2) Determine the Amount of pglo DNA in the Bacterial Cells Spread on the LB/amp Plate a. Determining the Total Amount of pglo plasmid DNA (DNA in μg) = (concentration of DNA in μg/μl) x (volume of DNA in μl) In this experiment you used 5 μl of pglo at concentration of 0.05 μg/μl. Calculate the total amount of DNA used in this experiment. Total amount of pglo DNA (μg) used in this experiment = b. Determining the fraction of pglo plasmid DNA (in the bacteria) that actually got spread onto the LB/amp/ara plate: Fraction of DNA used = Volume spread on LB/amp plate (μl) Total sample volume in microfuge tube (μl) You spread 100 μl of cells containing DNA from a test tube containing a total volume of 405μl of solution. Fraction of DNA = pglo DNA spread = Total amount of DNA used in μg x fraction of DNA used pglo DNA spread on the LB/amp plate (μg) = Now use the data above to calculate the efficiency of the pglo transformation. Report your calculated transformation efficiency in scientific notation. Transformation efficiency = Total number of cells growing on the agar plate Amount of DNA spread on the agar plate Transformation efficiency = transformants/μg (Optional) If a particular experiment were known to have a transformation efficiency of 3 x 10 4 cells/μg of DNA, how many transformants would be expected to grow on the LB/amp plate? You can assume that the concentration of DNA and fraction of cells spread on the LB/amp plate are the same as that of these procedures. Number of colonies on LB/amp plate = 7

8 Laboratory Report: (20 pts total) Turn in 2-a) Data analysis (printed on page 10) to your TA for grading at least 30 minutes before the end of the lab session. You don t have to repeat this section (part a) in your typed-up report. Part (b) should be included in your report. 1. Include a title page and present data in a table format (see below) and drawings (or photographs) with appropriate labeling. (2 pts) Plates Characteristics Plasmid added Colony number Colony fluorescence 2. Data analysis: (7 pts total) a). During the lab, calculate the transformation efficiency of the following experiment using the information and the hypothetical results listed below. Show your work and units. DNA plasmid concentration: 6 x 10-3 μg/μl Cells incubated with 560 μl CaCl 2 transformation solution and split into two tubes 20 μl pglo plasmid solution to the tube of +pglo 200 μl LB broth 50 μl cells spread on agar 213 colonies of transformants on the LB/amp plate Total amount of pglo DNA used (1pts) = Fraction of DNA used (1pts) = Micrograms of DNA spread on the plates (1pt) = Transformation efficiency (1pts) = b) Calculate the transformation efficiency of your OWN experiment after you obtain the results the following week. Number of colonies on LB/amp plate = Total amount of pglo DNA used = Fraction of DNA used (1pts) = Micrograms of DNA spread on the plates (1pt) = Transformation efficiency (1pts) = 8

9 3. Discussion and conclusions: (Include the questions in your report) 1) Based on the experimental design, on which plate(s) should the genetically transformed bacterial cells be located? Which plates should be compared to determine if successful genetic transformation has occurred? What should growth on each of these plates look like? (2 pts) 2) What allows the transformed cells to grow on LB/Amp? How? (1 pt) 3) Why do transformed cells display fluorescence in the presence of arabinose? Explain the mechanism. (2 pts) 4) Based on the analysis of your results, describe the evidence that indicates your transformation experiment was successful or not successful? Offer a possible explanation if your result was unsuccessful? Explain the possible errors resulting in the data of group 12 on page 6 (2 pts). 5) Which plate in your experiment is the negative control plate? What might cause growth on the negative control plate? (1 pt) 6) What purpose does spreading pglo on the LB plate serve? What might cause your cells to fail to grow on the LB plate? (1 pt) 7) If you spread 100 μl of the +pglo transformation solution onto an LB plate, what should you see after incubating the plate at 37-degrees for a few days and why? If you used the velvet stamping method demonstrated in lab 3 to replica-plate the growth from this LB plate onto an LB/Amp plate, what would the LB/Amp plate look like after a few days of incubation at 37 degrees. (2 pts) 9

10 Section Name Points Turn in Part-a) Data analysis below to your TA for grading 30 minutes before the end of the lab session. You don t have to repeat this section in your typed-up report, but you may want to copy your work and answers on page 8 for future reference. a). During the lab, calculate the transformation efficiency of the following experiment using the information and the hypothetical results listed below. Show your work and units. DNA plasmid concentration: 6 x 10-3 μg/μl Cells incubated with 560 μl CaCl 2 transformation solution and split into two tubes 20 μl pglo plasmid solution to the tube of +pglo 200 μl LB broth 50 μl cells spread on agar 213 colonies of transformants on the LB/amp plate Total amount of pglo DNA used (1pts) = Fraction of DNA used (1pts) = Micrograms of DNA spread on the plates (1pt) = Transformation efficiency (1pts) = 10

11 Biol 2281, Expt. 8 The Central Dogma of Molecular Biology Bacterial Transformation BIOL 2281 DNA Transcription RNA Translation Protein Genetic transformation A process that introduces a specific gene into an organism and have that genetic information expressed by the organism. Transformation Griffith s experiment Griffith Avery, McCarty, and MacLeod "transformation" of one bacterial type into another required DNA and no other biological molecule. Genetic transformation (cont.) Transformation (cont.) Most naturally transformable bacteria develop competence (being able to take up DNA) only under specific conditions (nutritional stress). Cells are passively made permeable to plasmid DNA using laboratory procedures and are called competent cells. calcium chloride and heat shock. electroporation, protoplast formation, and microprojectiles Drs. Wenju Lin & Elizabeth Pickett, Spring

12 Biol 2281, Expt. 8 Plasmids Small DNA molecules that are separate from the chromosomal DNA Often contains 1. Gene of interest 2. Genes that confer a selective advantage to the bacterium harboring them Antibiotic resistance 3. Origin of replication Shuttle vector 4. Polylinker area (multiple endonuclease cleavage sites) Recombinant DNA technology 1. Obtain Plasmid DNA and DNA containing the gene of interest. 2. Insert the gene of interest into plasmid. 3. Put recombinant plasmid into bacterial cells. Efficiency? Selection mechanism? 4. Obtain more of recombinant plasmid by growing bacterial cells. 5. The resulting recombinant plasmid can be used to transform another organism cells (bacteria or yeast cells) to change their phenotype. Bacterial Transformation A successful transformation requires: Host cells: Escherichia coli K-12:HB101 A plasmid containing a selectable marker and a gene of interest. the gene (bla) for beta-lactamase the gene for GFP Procedures of placing the plasmid DNA into the host Incubation heat shock recovery A selection mechanism: growth on media containing antibiotics (ampicillin, a member of the β-lactam family) The Host Cells Escherichia coli K-12:HB101 Is a single-celled organism Is an organism which reproduces quickly. Is not able to infect plants or animals. Is not naturally antibiotic resistant Can t grow on antibiotic plates (LB/Amp) LB, HB101, -pglo LB/Amp, HB101 -pglo The Plasmid We Will Use pglo Selectable marker: the ampicillin resistant gene (bla or Amp r ) which codes for beta-lactamase protein Origin of replication (ori) A polylinker area The gene for GFP (Green Fluorescent Protein) A special gene regulation system for GFP Promoter (P BAD ) and arac gene Drs. Wenju Lin & Elizabeth Pickett, Spring

13 Biol 2281, Expt. 8 pglo plasmid Green Fluorescent Protein (GFP) Originally isolated from the bioluminescent jellyfish, Aequorea victoria. Excitation occurs when protein is exposed to ultraviolet light and emission is in the green portion of the visible spectrum. Mutation of Chromophore Alters Color Genetics Terms Refresher Gene expression Constitutive Inducible RNA Polymerase Promoter A region of DNA that initiates transcription of a particular gene Expression of GFP in pglo Inducible system Arabinose present in growth media The gene for GFP will be switched on Transformed cells will appear fluorescent green under UV light No arabinose present in growth media The gene for GFP will remain switched off Transformed cells will appear white under UV light How Does Our Inducible System Work? Uses components of the arabinose operon Drs. Wenju Lin & Elizabeth Pickett, Spring

14 Biol 2281, Expt. 8 In E.coli The Arabinose Operon Three enzymes are needed to digest arabinose as a food source. The genes coding for these enzymes are not expressed in the absence of arabinose. The genes are expressed when arabinose is present. This cluster of genes controlled by a single promoter, P BAD, and is called The Arabinose Operon. Transcription of these three genes requires the DNA template, RNA polymerase, a DNA binding protein called arac and arabinose. Inducing GFP Expression from pglo The pglo plasmid Contains the promoter (P BAD ) and the arac gene from the arabinose operon. The three genes which code for arabinose catabolism have been replaced by the gene which codes for GFP. Inducing GFP Expression From pglo The arac protein binds the pglo DNA near the P BAD and blocks binding of RNA Pol. Arabinose interacts with arac, changing its shape to a formation that helps recruit RNA Pol to the P BAD Cells transformed with pglo In the presence of the ampicillin Cells can grow on media containing ampicillin In the presence of arabinose Cells fluoresce brilliant green as they produce more and more GFP. In the absence of arabinose Bacteria colonies will appear to be white colonies with no fluorescence. Once RNA Pol binds, transcription starts. Translation follows and GFP is produced. Procedures of transformation Steps to enhance the transformation efficiency 1. Incubation: cells mixed with CaCl 2 2. Heat shock: quick temperature changes 3. Recovery: short incubation In the lab Two groups of plates in this controlled experiment Transformation plates - the factor being tested is applied. Control plates - the factor being tested is not applied. Cells growing at log (exponential) phase are often used to increase the transformation efficiency Drs. Wenju Lin & Elizabeth Pickett, Spring

15 Biol 2281, Expt. 8 Results of Transformationnormal room lighting Results of Transformationultraviolet light LB, -pglo LB/Amp, -pglo LB, -pglo LB/Amp, -pglo LB/Amp, +pglo LB/Amp/Ara, +pglo LB/Amp, +pglo LB/Amp/Ara, +pglo Data analysis Transformation efficiency = Total number of Transformants on LB/Amp plate Amount of plasmid DNA (in μg) used in transformation Read procedures Study the steps of calculation on page 7 Turn in Part-(a) in Data analysis (p 10) for grading 30 minutes before the end of your session in the lab. Quiz samples What are the three main steps in procedures of transformation? What methods can be used to make cells permeable to plasmid DNA? Describe the important characteristics of the pglo plasmid and host cells in this experiment. Which is the selectable media for transformants and which media will produce glowing colonies? Why? Drs. Wenju Lin & Elizabeth Pickett, Spring