Green Fluorescent Protein - Bacterial Transformation INTRODUCTION Green Fluorescent Protein, also written as GFP, is a protein that’s made by a jellyfish— Aequoria Victoria. This specific protein has a green fluorescence when it’s exposed to UV light. The jellyfish itself contains the protein aequorin that gives off blue light. The green fluorescent protein then changes the light to green light, which is what we see when the jellyfish lights up. Purified GFP looks yellow under normal room lights, but they glow with a bright green color when taken outside and exposed to sunlight. The protein takes in UV light from the sunlight, and then gives it off as a low-energy green light. GFP is a ready-made protein, so it’s fairly simple to use. GFP is extremely useful in scientific research, because it allows to look directly into the “core” workings of cells. It’s also very easy to find where GFP is present in any given sample at any given time—if UV light is shone on the sample, any GFP that’s present will radiate bright green. GFP is generally non-toxic and can be expressed to high levels in different organisms with minor effects on their physiology (Chalfie et al., 1994). Furthermore, when the gene for GFP is fused to the gene of a protein to be studied in an organism of interest, the expressed protein of interest retains its normal activity and, likewise, GFP retains its fluorescence, so that the location, movement and other activities of the studied protein can be followed by microscopic monitoring of the GFP fluorescence (Wang and Hazelrigg, 1944). The DNA of most bacteria is in a single molecule, called the bacterial chromosome. In addition to the chromosome, bacteria often contain plasmids—small circular DNA molecules (SLH, 2014). Bacteria can pick up new plasmids from either the environment or from the other bacterial cells. However, they can also easily lose them—like when a bacterium divides in two, resulting in one of the daughter cells losing a plasmid. Every plasmid has DNA that makes sure it gets replicated by the host bacteria. Because of this, plasmids can replicate themselves independently, which results in numerous copies of a plasmid within a bacterial cell. Replicating the DNA of a plasmid uses up energy, which results in the plasmid becoming hard to keep by the bacterial cell. Under certain conditions, bacteria that contain plasmid will live longer, and will also have more opportunity to pass on the plasmid. Bacteria that lack plasmid find it more difficult to survive and reproduce. All of the plasmids that are used to deliver DNA have genes that are used for antibiotic resistance. Once bacteria has been treated with a plasmid, they can be grown alongside an antibiotic. Only the cells that actually contain the plasmid will be able to survive and reproduce while the others will be killed off by the antibiotic. The purpose of this lab was to treat pGLO DNA to make them “competent” (ready to take DNA from the environment), and to “transform” a part of the sample of the pGLO DNA so it becomes resistant to ampicillin and produces GFP. We hypothesized that the plate containing +pGLO with the Luria Bertani broth, ampicillin and arabinose would grow to have colonies and have all the colonies have a fluorescent green color. MATERIALS The materials that we used for this lab included 1 P1000 Pipetman, 1 P200 Pipetman 1 P20 Pipetman, a Pipetman stand, 1 2 mL micro test tube containing +pGLO solution, 1 2 mL micro test tube containing -pGLO solution, 1 sharpie of any color, a sterile transfer pipet, 500 µl CaCl2 (transformation solution), Styrofoam tray filled with crushed ice, 12 sterile loops, starter plate containing bacteria, UV lamp, pGLO plasmid, incubator, stopwatch, 4 agar plates, 42°C water bath, 500 µl of LB nutrient broth, and tape. METHOD We started off by setting the P1000 pipetman to draw 250 µl, the P200 to draw 100 µl, and the P20 to draw 10 µl and placed them back on the stand until we were ready to use them. We then labeled on close, 2 mL micro test tube “+pGLO” and another “-pGLO” with our sharpie and labeled both with our group name. We then placed them back in the foam tube rack. We then opened the tubes and using a sterile transfer pipet, transferred 250 µl of CaCl2 (transformation solution) into each tube. We then placed the tubes on crushed ice. Next, we used a sterile loop to pick up a single colony of bacteria from our starter plate. We picked up the +pGLO tube and immersed the loop into the transformation solution at the bottom of the tube. Next, we spun the loop between our index finger and thumb until the entire colony is dispersed in the transformation solution with no floating chunks. We then placed the tube back in the tube rack in the ice. Using a new sterile loop, we repeated it for the -pGLO tube. We then examined the pGLO plasmid DNA solution with the UV lamp and recorded our observations. Then, using the P20 pipette, we then transferred 10 µl of pGLO plasmid into the +pGLO tube and mixed. We then closed the +pGLO tube and returned it to the rack on ice. We didn’t add the plasmid DNA to the -pGLO tube, closed the lid and returned it to the rack on ice. Next, we incubated the tubes on ice for 10 minutes. We made sure to push the tubes all the way down in the rack so the bottom of the tubes stuck out and made contact with the ice. Then, we labeled two agar plates LB/amp, one LB/amp/ara, and one LB. We then labeled the one agar plate +pGLO and -pGLO (on the bottom) that had LB/amp written on it. On the one labeled LB/amp/ara, we labeled that one +pGLO, also on the bottom. For the one labeled just LB, we labeled that one -pGLO, also along the bottom. Then, using the foam rack as a holder, we transferred both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds making sure to push the tubes all the way down in the rack so the bottom of the tubes stuck out and made contact with the warm water. When the 50 seconds had passed, we then placed both tubes back on the ice. We then incubated the tubes on ice for 2 minutes. We then removed the rack containing the tubes from the ice and placed it on our table. We open a tube and, using a new sterile pipet, added 250 µl of LB nutrient broth to the tube and closed it. We then repeated this with a new sterile pipet for the other tube. Next, we incubated the tubes for 30 minutes at room temperature. We then gently flicked the closed tubes with our finger to mix. Using a new sterile pipet for each tube, we pipetted 100 µl from each of the tubes to the corresponding plated. We made sure to put the cells on the jello-like agar medium, not the agar lid. Using a new sterile loop for each plate, we then spread the suspensions evenly around the surface of the agar by quickly stroking the flat surface of a new sterile loop back and forth across the plate surface, and closed the lid. We then stacked up our plates and taped them together. Next, we placed the stack upside down— the lids were on the bottom—in the 37°C incubator until the next day. We then took our plates and observed the results. DATA AND RESULTS Shown below is a table that depicts what our group expected the results to be before we took out our plates and observed. EXPECTED RESULTS Growth? Plate: None/Colonies/”Lawn” +pGLO LB/amp +pGLO LB/amp/ara -pGLO LB/amp -pGLO LB Colonies Fluorescence Plate: None/some colonies/all colonies/”Lawn” None Colonies None None will grow None will glow Many colonies None Shown below is a table that depicts what the actual results were when we took out the plates and observed. ACTUAL RESULTS Growth? Plate: None/Colonies/”Lawn” +pGLO LB/amp +pGLO LB/amp/ara -pGLO LB/amp -pGLO LB Colonies Fluorescence Plate: None/some colonies/all colonies/”Lawn” None A lot of colonies Glowing None None “Lawn” None CONCLUSION When we observed the pGLO plasmid DNA solution under UV light it didn’t fluoresce green because it had no presence of sugar to show the fluorescence within it even with the UV light. All of the plates didn’t have bacteria growing on them because of the ampicillin. The bacteria couldn’t grow in the presence ampicillin unless it contained the plasmid, so in turn, there was no growth on the LB/amp agar plate because it had none of the plasmid on it. On the first plate, the bacteria was able to grow because of the presence of the ampicillin, but it didn’t show any fluorescence because there was no sugar present. On the second plate, the bacteria grew to show numerous colonies because ampicillin was present, and it practically glowed under the UV light because arabinose was also added—which was the sugar needed to help it fluoresce green. On the third plate, because it was -pGLO, the ampicillin prevented the bacteria from growing, so it ended up having no colonies and consequently didn’t have a fluorescence. On the final plate, there was no ampicillin present so it allowed the bacteria to grow normally and formed a “lawn” on the entire plate, but there was no fluorescence because there was no sugar present. The bacterial colonies that were transformed fluoresced green when UV light was shown on them. There was no fluorescence when the UV light was shone only on the plasmid alone in the tube because it’s not the necessarily the plasmid that reacts to the light. It’s the protein that’s made when the plasmid is translated—which only occurs when the plasmid and bacteria get incorporated. The purpose of using the LB only plate was because it contained no DNA—it allowed for all cells, even ones that hadn’t been transformed yet, to be able to grow on it. The purpose of using the plate that contained LB and ampicillin without the pGLO was to provide a “control” for ampicillin—because the presence of ampicillin would kill the bacteria that hadn’t been transformed yet. The purpose of using the plate that contained the LB and ampicillin with the pGLO was to use it as a “reverse-control” mechanism for the ampicillin. Since the pGLO was added alongside the LB and ampicillin, it virtually rendered the ampicillin useless, which allowed the bacteria to grow but there was no fluorescence present. The purpose of using the plate that contained the LB and ampicillin with arabinose alongside pGLO was to see how much growth and fluorescence there would be if arabinose was added. Since the arabinose was present, it enabled the plate to practically glow because there was sugar present, and bacteria was also able to grow because the ampicillin was also rendered useless in the sample.