Biology 2180 Laboratory #7 Name Bacterial Growth and Transformation Introduction: Most aspects of molecular biology require the use of basic microbiological methods. This section is included for those individuals who are unfamiliar with techniques or were introduced to them in the distant past. Techniques of Microbiology The microbiological procedures required for molecular cloning are quite simple and should present no problems to anyone with training in basic sterile techniques. The two difficulties most commonly encountered are crosscontamination of strains and loss or gain of genetic markers. Both of these problems can be minimized by colony or plaque purification, followed by verification of the genotype of the strain. Serial passaging of strains should be avoided by preparing working stocks from verified master cultures kept in longterm storage. Even in the best-run laboratories, there is always a possibility of contamination. It is important to check for bacterial colonies with an altered morphology, color, or odor, or for bacteriophage plaques with an unusual appearance or size. Such plates, as well as any that become contaminated with mold or other fungi, should be sealed, autoclaved, and discarded. Sterile (Aseptic) Technique With any type of microbiology technique (i.e. working with and culturing bacteria), it is important not to introduce contaminating bacteria into the experiment. Because contaminating bacteria are ubiquitous and are found on fingertips, bench tops, etc., it is important to avoid these contaminating surfaces. When working with the inoculation loops, pipettes, and agar plates, remember that the round circle at the end of the loop, the tip of the pipette, and the surface of the agar plate should not be touched or placed onto contaminating surfaces. While some contamination will not likely ruin the experiment, students would benefit from an introduction to the idea of sterile technique. Using sterile technique is also an issue of human cleanliness and safety. 1
Estimating Cell Density The cell mass can be measured by determining the dry weight of a culture, or by recording the turbidity. In cases where most of the cells are viable, both the dry weight and the turbidity are directly related to cell number. Bacterial cultures exhibit light scattering, which is approximately proportional to cell mass. At wavelengths where the ratio of absorbance to light scattering is low (e.g. 550-600 nm), the optical density of a growing culture can be followed in a spectrophotometer, and is proportional to the number of cells. The exact relationship of optical density at a particular wavelength to cell number varies with the particular strain used and the growth conditions, because the mass/cell number ratio varies with the medium. It also varies with the brand of spectrophotometer being used since one is really measuring scattering not absorbance (which all spectrophotometers measure essentially the same). The degree to which light is scattered in any given instrument depends on the distance from the cuvette to the phototube which differs among different instruments. Thus, one should not assume that if a protocol calls for growing the cells to A550 0.6, that 0.6 will be the optimal absorbance on a different model spectrophotometer. If the exact concentration is critical for the success of a method it is always better to report the concentration as 4x108 cells/ml rather than A 550 0.6. As a rough estimation, Effects of Nutrients -- Aeration 1 OD 600 = 8 x 10 8 cells/ml E. coli can grow on a simple chemical medium in which glucose is the carbon and energy source, provided the medium is buffered at a ph near 7.0 and contains magnesium, phosphate, and a nitrogen source (usually ammonium chloride or ammonium sulfate). Strains of E. coli grow more rapidly in rich broth media such as LB, 2XYT or TB (Terrific Broth) than in this minimal medium because these media supply many of the compounds which the cell would otherwise have to synthesize. The rich media vary from one another largely in the amount of yeast extract and tryptone (an enzymatic digest of casein). Addition of several percent agar to the medium still allows growth, only now this is confined to the surface of a solid medium. This is convenient for use in Petri dishes. E. coli is capable of growing either in the presence of air (aerobic) or in its absence (anaerobic). However, the growth rate in the absence of aeration is significantly poorer than that achieved with good aeration. After a density of 10 7 cells/ml is reached, air must be supplied either by shaking the liquid or by bubbling air through the cultures to allow rapid growth. As a rule of thumb, a titer of 10 7 cells/ml is just visible in a standard test tube. Logarithmic Growth Growing bacteria, dividing by binary fission, exhibit exponential or logarithmic growth kinetics until a point of saturation of the culture is reached. During this time the increase in the number of bacteria per unit time is proportional to the number of bacteria present in the culture. 2
An equation that describes the increase in cell number is: F = S(2) t/x Where F is the final number of cells, S is the starting number of cells, t is the time that has passed between the F and S measurements and x is the generation (doubling) time. Often we are interested in the generation time of the culture, the time required for the cells to double in number. The above equation can be solved for x : x = 2tS F Using this equation, we can determine the doubling time for a particular strain of bacteria, in a certain medium and using available equipment (shaker, flasks etc...). If we can determine the number of cells at two distinct points in the log phase of the growth curve, and determine how much time has passed between them, we can calculate the doubling time. Growth Curves When exponentially growing cells are transferred to fresh medium, they continue growing with exponential kinetics. If instead the inoculum is taken from a saturated culture consisting of half-starved, slowly multiplying cells, there is a lag time (usually consisting of 30-60 minutes) before the cells resume rapid growth. Bacterial Growth Curve Most experiments begin by growing an "overnight" culture. An overnight is made by inoculating several milliliters of broth with a single colony and then growing to saturation overnight. On the day of an experiment, the overnight is subcultured by transferring several drops into a fresh broth tube. The dilution factor must be at least 1:20 if the cells are to subsequently attain exponential growth. Continued subculturing should be 3
avoided, and it is always preferable to prepare a new culture from a single colony. Otherwise, mutants and faster growing contaminants have a chance to accumulate and overgrow the original strain. Strains with unstable properties, for instance, certain highly fertile male strains (Hfr's) of E. coli are readily lost by continued subculturing. Determination of Bacterial Doubling Time 1) With the spectrophotometer wavelength set at 600nm, measure the optical density of the growing log-phase bacterial culture. Zero the tube with 3 ml growth medium then replace the medium with 3 ml of the growing culture. What is the optical density? 2) Measure the optical density again after 45 minutes. 3) Use the above equations to calculate the doubling time for this bacterial culture. 4) What would the optical density be if you measured it after another 45 minutes? 5) Imagine that you pick a single bacterial colony into a flask containing LB medium and start shaking at 37 degrees. Every 30 minutes you remove some of the culture and measure the optical density with the spectrophotometer. The results are tabulated as follows: Time after inoculation Percent Absorbance (A 600 ) 30 min 0.00 60 min 0.00 90 min 0.02 120 min 0.05 150 min 0.12 180 min 0.30 210 min 0.74 240 min 1.00 270 min 1.00 4
Graph these results below and determine the generation time for these bacteria. Indicate on the graph, the lag phase, log phase and saturation. Time (minutes) -------> 6) You place the saturated culture in the refrigerator for a couple of weeks then decide to measure the Percent Absorbance again. What do you expect to see? Why? Introduction to Bacterial Transformation In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA that provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation literally means change caused by genes, and it involves the insertion of a gene into an organism in order to change an organism's trait. Genetic transformation is used in many areas of biotechnology. 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 their disease. You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. The 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 under ultraviolet light. In this activity, you will learn about the process of 5
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 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 encodes the gene for the Green Fluorescent Protein (GFP), and a gene for 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 the Green Fluorescent Protein can be switched on in transformed cells by adding the sugar arabinose to the cells' nutrient medium. Selection for cells that have been transformed with pglo DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar. 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 plasmid DNA pglo through the cell membrane you will: 1.Use a transformation solution of CaCl 2 (calcium chloride) 2.Carry out a procedure referred to as heat shock For transformed cells to grow in the presence of ampicillin you must: 1.Provide them with nutrients and a short incubation period to begin expressing their newly acquired genes Transformation Procedure 1.Label one closed sterile micro test tube +DNA and another -DNA. Label both tubes with your group's name. Place them in the test-tube rack. 6
2.Open the tubes and, using a sterile pipette tip, transfer 250 µl of transformation solution (CaCl 2 ) into each tube. 3.Place the tubes on ices. 4.Use a sterile loop to pick up a single colony of bacteria from the fresh starter plate. Pick up the +DNA 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 (no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the -DNA tube. 5.Using a sterile pipette tip, add 10µl of the p-glo plasmid DNA solution to the cell suspension of the +DNA tube. Close the tube and return it to the rack on ice. Also close the -DNA tube. Do not add plasmid DNA to the - DNA tube. 6.Incubate the tubes on ice for 10 minutes. 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 ice. 7.While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid) as follows: Label one LB/amp plate "+ DNA" Label the LB/amp/ara plate "+ DNA" Label the other LB/amp plate "- DNA" Label the LB plate "- DNA" 8.Heat shock. Using the foam rack as a holder, transfer both the (+) and (-) tubes into the water bath, set at 42 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 up, place both tubes back on ice. For the best transformation results, the change 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 rack containing the tubes from the ice and place on the benchtop. Using a new sterile pipette tip, add 250 µl of LB 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. 10.Tap the closed tubes with your finger to mix. Using a new sterile pipette tip for each tube, pipette 100 µl of the transformation and control suspensions onto the appropriate plates. 11.Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. 12.Stack up your plates and tape them together. Write your group name and class period 7
on the bottom of the stack and place it upside down in the 37 C incubator until the next lab session. Data Collection (Next Lab Session) Observe the results you obtained from the transformation lab under normal room lighting. Then turn out the lights and hold the ultraviolet light over the plates. 1. Carefully observe and draw (also record in a table) what you see on each of the four plates. Write down the following observations for each plate. How much bacterial growth do you see on each, relatively speaking? What color are the bacterial colonies? How many bacterial colonies are on each plate (count the spots you see). Analysis of the Results Questions 7) Why does the p-glo plasmid need to have: Ori The bla gene The ara promoter The GFP gene The plates were as follows: Plate number 1 -DNA LB only Plate number 2 -DNA LB+amp Plate number 3 +DNA LB+amp Plate number 4 +DNA LB+amp+ara 8) On which of your LB plates did you expect to find no bacterial colonies? What is this plate a control for? 8
9) If you only obtained colonies on plate number 1 what do you think might have gone wrong? 10) If you only obtained colonies on plates 1 and 3 what do you think might have gone wrong? 11) On plate number 3 you see some large colonies with some tiny colonies around them. If you picked one of the large colonies and streaked it onto a new plate that is LB+amp, would you expect new colonies to form? Why? What if you tried this with one of the tiny colonies? REFERENCE: Sambrook et al., Molecular Cloning, 1989, Ausabel et al., 1991 BioRad Explorer Bacterial Transformation 9