Connor Lauffenburger 3/17/13 pGlo Transformation Lab Report I Introduction The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids.
It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies will be located on the LB/amp/ara and LB/amp agar plates. Cells that have been transformed with +pGlo plasmids have the ability to grow in ampicillin plates, and the arabinose sugar allows the colonies to be visibly fluorescent under ultraviolet light. The GFP is able to resist ampicillin because of the Beta- Lactamase protein that is produced and secreted by the bacteria that have been transformed to include it in their plasmids. Arabinose is a carbohydrate, normally used as a source of food by bacteria.
Bacterial colonies are not able to grow on –pGlo plates because they are sensitive to ampicillin. In this lab, I will move the GFP gene from one organism to another with the use of pGlo plasmids. II Materials and Procedures The first procedure of the lab was to obtain two microtubes and label one +pGlo and the other –pGlo with a marker. Next, the tubes were placed in the microtube rack and 250 ? l of CaCl2 was transferred into each tube using a sterile pipette. After placing the microtubes in ice, a sterile inoculation loop was used to pick up a single colony of bacteria from the E. coli starter plate.
Next, the +pGlo microtube was removed from the ice and, to ensure that the colony was efficiently dispersed into the transformation solution, the loop was gently swirled at the bottom of the tube. Once the bacteria was completely dissolved in the solution, the microtube was placed back in the ice and this procedure was repeated for the –pGlo using a new, sterile inoculation loop. From here, the pGlo DNA solution was viewed under the ultraviolet lamp and observations regarding color were recorded. Using another sterile loop, a loopful of plasmid solution was extracted from the pGlo plasmid DNA stock tube and mixed into the +pGlo tube.
However, the plasmid DNA was not added to the –pGlo solution because it is serving as the control group in this experiment. After the solution was mixed completely, both tubes were closed and put on ice for 10 minutes. During the 10 minutes of ice time, the four LB nutrient agar plates were labeled in the following manner: the LB/amp plate was labeled +pGlo, the other was labeled –pGlo, LB/amp/ara was labeled +pGlo, and LB was labeled –pGlo. Ampicillin and arabinose provide for experimental groups to see if the pGlo gene can be shown in the bacteria.
After placing the tubes into the microtube rack, both the +pGlo and –pGlo tubes were put into the 42? water bath for 50 seconds then quickly put back on ice for 2 minutes. Next, the rack was removed from the ice and 250 ? l of LB nutrient broth was added to both the +pGlo tube and –pGlo tube using a sterile pipette; both tubes were incubated for 10 minutes at room temperature. Once the tubes were removed from the incubator, they were both flicked to mix the solution inside and 100 ? l of the transformation and control suspensions was transferred from the tubes to the appropriate nutrient agar plates using a sterile pipette.
Using a new sterile loop for each plate, the suspensions were spread gently and evenly around the surfaces of the LB nutrient agar. After stacking the plates and taping them together, they were placed upside down inside of the 37? incubator until the next day. The following day, the agar plates were removed from the incubator and observed. The color and number of colonies contained by each plate was recorded and each one was examined under the ultraviolet light to see if any of the colonies had received the pGlo gene. Next, the transformation efficiency was calculated by dividing the total amount of DNA on the agar plate in ?
l by the number of cells growing on the +pGLO LB/amp/ara plate. During the purification section of this lab, the LB/amp/ara agar plate was examined for well-isolated green colonies and the LB/amp plate was observed for white colonies with space between each other. These colonies were circled on the outside of the plates using a marker. Next, two 15 milliliter culture tubes containing 2 milliliters of nutrient growth media were obtained and labeled “+” and “-“. Using a new inoculation tube, the circled colonies from each plate were scooped out and immersed in their respective culture tubes.
Once the bacteria was mixed into the solution, the tubes were sealed and placed horizontally into the 32? incubator for 24 hours. After labeling a new microtube with a marker, the culture tubes were removed from the incubator and observed under the ultraviolet lamp. Using a sterile pipette, the contents of the (+) culture tube were transferred to the 2 milliliter mictrotube and the (-) culture tube was disposed of. The (+) tube was then spun in the centrifuge at maximum speed for 5 minutes. Once it was removed from the centrifuge, a noticeable bacterial pellet had formed at the bottom of the tube and the remaining liquid was disposed of.
After the pellet was observed under the UV lamp and color was noted, 250 microliters of TE solution was added to the tube using a sterile pipette. The pellet was re-suspended by rapidly pipetting the solution up and down. Once again using a new pipette, a single drop of lysozyme was added to the solution and the tube was capped and mixed. Before placing the tube in the freezer until the next lab period, the pellet was viewed under the UV light. After thawing the tube out by hand, the tube was placed in the centrifuge for 10 minutes at maximum speed.
While waiting on the centrifuge, the chromatography column was shaken to re-suspend the beads. Next, the cap and bottom of the chromatography column was removed and the liquid buffer was drained. After the buffer completely drained, 2 milliliters of Equilibration Buffer was added to the top of the column, and then drained until there was only 1 milliliter using a pipette. The top and bottom of the column were sealed and the column was placed at room temperature until the next lab period. After the tube was done with centrifugation, it was immediately placed under the UV light and examined for a pellet at the bottom.
Notes were taken on the color of the pellet and liquid and, using a sterile pipette, 250 microliters of the supernatant was transferred into a new microtube. Next, 250 ? l of Binding Buffer was added to the microtube containing the supernatant and the tube was refrigerated until the following lab period. After labeling 4 collection tubes 1, 2, 3, and waste, the top and bottom of the chromatography column were removed and its contents were drained into the liquid waste container. Once the buffer reached the surface of the HIC column bed, the column was placed at the top of collection tube 1.
Next, 250 ? l of the supernatant from the microtube was added to the top of column by placing the pipette on the side of the column wall and dripping the liquid down the side. While waiting for the supernatant to flow into the collection tube, the column was viewed under the UV light. After transferring the column to collection tube 2, 250 ? l of Wash Buffer was added to the top of the column using the previous technique and observed under the UV lamp. Finally, the column was moved to collection tube 3 and 750 ? l of TE Buffer was added to the top of the column.
After examining collection tube 3 under the ultraviolet light and taking notes, the collection tubes were all sealed with Seran Wrap and placed in the refrigerator until the next lab period. III Results The results of this experiment were exactly as hypothesized. +pGlo LB/amp contained 44 small, non-glowing colonies that were tan in color, +pGlo LB/amp/ara contained 83 small colonies that glowed a bright green color, -pGlo LB/amp lacked any bacterial growth, and –pGlo LB contained a single mass of non-glowing bacteria that was tan in color and covered the circumference of the agar.
The transformation efficiency of the LB/amp plate was 293 transformants/microliters and 553 transformants/microliters for the LB/amp/ara agar plate. Once the (+) capped tube was transferred to a microtube and spun in the centrifuge for 5 minutes, a bacterial pellet formed that glowed a bright green color when observed under the ultraviolet lamp. The addition of the lysozyme to the pellet solution had no effect on the glow, but once the tube was spun in the centrifuge again, the pellet lost its glow and became white, while its supernatant glowed brightly.
When the supernatant was drained into the collection tubes, you could still see a glow around the column beds of tubes 1 and 2. After the solution finished draining into collection tube 3, it glowed a bright green while the previous collection tubes had completely lost their glow. IVConclusion The hypothesis that +pGlo LB/amp/ara and +pGlo LB/amp would be the only agar plates to grow bacterial colonies was partially supported. While –pGlo LB contained a single, massive colony, the previous two plates were able to grow multiple colonies as they should have.
Also, since the +pGlo LB/amp/ara plate was the only one that glowed, it can be concluded that the sugar is essential for the GPF protein to activate. The fact that the bacteria with the pGlo gene was able to grow on the plates with ampicillin shows that the bacteria is able to resist the antibiotic once it receives the gene. During purification, the final glow in collection tube 3 with no other tubes glowing showed that the GFP gene was isolated.
This has been a useful process for applying insulin to diabetic patients and to obtain other medications that can’t be produced solely by the human body. Transformation, which clones and targets specific genes and proteins in bacteria, is an important role in biotechnology because it provides new organisms to be manipulated and experimented on. Overall, the experiment was a success. However, it is crucial to be extremely sterile during the phases of the lab in order to avoid cross-contamination.