Monday, March 17, 2014

pGLO Lab

pGLO Lab


Genetic transformation is an alteration within genes that is a direct result of exogenous DNA. One can insert a specific gene into an organism in order to change its trait. In this lab, we transformed bacteria into a gene that codes for GFP, or Green Fluorescent Protein. This specific protein allows for a glowing of bright green under a ultraviolet light. In this experiment, the pGLO is resistant to the antibiotic ampicillin in the plasmid DNA. Transformed cells will only grow on the dishes with LB/amp and will not show on those without it 


The purpose of this pGLO lab was to insert a plasmid into the DNA of a bacteria. Dependent on the presence or absence of the sugar, arabinose, determined whether the bacteria glowed or not. We transformed this bacteria into a gene that codes for Green Fluorescent Protein. This protein is what makes the certain ecoli glow under the ultraviolet light.  We then compared the absence/presence of the pGlo and sugars.


First we had 2 test tubes, we labeled one +PGLO and the other one -PGLO. We also labeled the 4 LB agar plates. The first plate was LB/AMP/+PGLO. The second one was LB/AMP/ARA/+PGLO. The third plate was LB/AMP/-PGLO. The last plate was LB/-PGLO. AMP was the antibiotic and the ARA was sugar. Then we used a sterilized pipet and put 250 micro liters of transformation solution in both test tubes labeled PGLO + and -. Both tubes were then placed on ice. A single colony of bacteria put in the +PGLO. We do this by taking a sterile loop and putting it in the bacterial colony and the placing the sterile loop in the test tube. We swirl the loop in the tube to make sure the bacteria colony is completely immersed in the PGLO. This was repeated for -PGLO. Then we added a DNA plasmid in the +PGLO and not the -PGLO. We added the plasmid by using another sterile loop and taking the plasmid for a DNA stock tube, after we did that we swirled the loop in the +PGLO. Next we put both test tubes in a foam rake on ice for 10 minutes. After the 10 minutes we needed to heat shock the test tube. We did this by transfer the test tubes in the foam rake into a water bath that was 42 degrees Celsius for 50 seconds, and then putting the test tubes back on ice for 2 minutes. After the heat shock we used a sterile pipet to but 250 micro-liters of LB broth into both test tubes (+PGLO and -PGLO). The next thing we did was incubate the test tubes at room temperature for 10 minutes. We tapped the test tube with our fingers to make sure the LB was spreading out. Our next step was to pipet 100 micro liters of +PGLO  suspension to plate the nutrient plate 1 and 2. Then we pipeted 100 micro liters of -PGLO suspension  was added to plate 3 and 4. For each plate a new sterilized pipet was used. Next we used a sterile loop at spread the suspension around the plate. We did this by rubbing the loop all over the plate but we couldn't press too hard on the agar. We repeated this step for all 4 plates using new loops each time. We then flipped the plates upside down and stacked them to let the plates incubate in a 37 degree Celsius for the night. The next day we unflipped the plates to check the growth. Then we used a UV to check the plate LB/AMP/ARA/+PGLO to see if any of the bacteria were glowing. We took pictures of all the plates, to record the growth.


After letting the E.coli incubate over night, we were able to determine our results the next day. The Petrie dish with LB and -pGlo grew a big amount compared to all the other dishes. This happened because the dish only had LB (a type of broth) and -pGlo. This -pGlo did not have an effect on the cells. The bacteria grew like normal bacteria would. We made this dish even though we knew it would not glow in UV light to show that our bacteria did grow normally; it showed that the bacteria worked essentially. The next tray had LB/ AMP and -pGlo. This tray had the broth (LB) and an antibiotic (AMP). This tray did not experience any growth at all because it had the antibiotic and was missing the plasmid,+pGlo, which prevented it from growing. Another tray contained +pGlo, LB, and AMP. This had the broth and antibiotic as well as the plasmid, +pGlo. The dish grew approximately six colonies of DNA. It was able to grow because it had the antibiotic and plasmid. This tray did not glow in the UV light because it did not have the sugar, ARA, present that makes it glow. This dish acted as our control for the experiment. The last dish had the plasmid, +pGlo, the LB broth, the antibiotic, AMP, and lastly the sugar, ARA. The ARA sugar in this dish separated it from all the other ones. This tray glowed and grew about five colonies of bacteria. This tray glowed in the UV light because of the ARA sugar, plasmid, and antibiotic AMP. 
After seeing the growth in the different dishes, our group calculated the transformation efficiency of our experiment. The transformation efficiency is the extent to which we genetically transformed the bacteria (in this case, E.coli). When doing an experiment, of course, the more cells you genetically transform the better, so the higher the transformation efficiency the better as well. In this experiment's case, the transformation efficiency represents the total number of bacterial cells that express the green protein, divided by the amount of DNA used in the experiment. This then tells us the total number of bacterial cells transformed by one microgram of DNA. The formula used is: 

Transformation efficiency = total number of cells growing on the agar plate
                                             Amount of DNA spread on the agar plate 

Our transformation efficiency was 14.16 which is very off of the standard amount. Standard amounts are usually way higher, around about 8.0 x 10^2. Clearly, we did not genetically transform many of the DNA cells.  


From these results, we can come to two conclusions. E Coli can only grow in the presence of an anitbiotic when it has been treated with pGLO which causes the bacteria to become antibiotic resistant. The glowing characteristic of the pGLO, however, is only activated in the presence of a sugar. We know this is true because the bacteria only treated with pGLO and anitbiotic was able to grow but not glow but the bacteria treated with both grew and glowed.