Introduction & Purpose:
For this lab, we were trying to genetically transform colonies of E.coli so that they would display a trait that allowed them to become bioluminescent (glow) as well as become antibiotic resistant. To do this we took a gene called Green Fluorescent Protein (GFP) that is originally found in jellyfish and inserted it into the dish of E.coli colonies. We also inserted a gene that allows bacteria to overpower antibiotics. Although the E.coli bacteria were never designed to become bioluminescent, they have the ability to transfer plasmids, causing them to share the new genes we inserted into them. The purpose of this experiment was to help us understand the procedure of genetic transformation. We were able to observe how different factors allowed the bacteria to transform. We learned how to insert new genes into the bacteria, and why the bacteria colonies were able to display the new traits.
Analysis:
Through the lab it was determined that E. coli can be genetically modified to glow in the dark under UV light. Through the method used, it was determined that this could be done by inserting E.coli into an agar plate that contained quantities of the plasmid known as pGLO, along with ampicillin and arabinose. The plasmid pGLO contains the GFP gene which is what allowed the E. coli to glow in the dark by coding for a green fluorescent protein. It is also important to note the total amount of growth for each plate, especially the plates that had the least and most growth.
As predicted, the two control plates, those labeled -pGLO and -pGLO LB/amp exhibited the least amount of growth, with the -pGLO exhibiting no growth. The plates that exhibited the most amount of growth were labeled +pGLO LB/amp and +pGLO LB/amp/ara, with the latter plate having the most growth out of the four plates. This may be due to the bacterias increased resistance to ampicillin.
This would also explain why the control plates has less growth than those with the pGLO plasmid. The plate with the -pGLO had no growth because it had no LB broth or arabinose to stimulate growth. The plate labeled -pGLO LB/amp did not have the plasmid necessary for antibiotic resistance to ampicillin, and therefore the E. coli could not grow. However, the plate labeled +pGLO/amp/ara had both the plasmid necessary to encode for antibiotic resistance, as well as ambroise which will induce the bacterial cell to express the fluorescent trait and grow.
To make the environment within the the plate most suitable for cell growth it is then necessary to add the pGLO plasmid, the ampicillin, and the arabinose. It is plausible to believe that the E. coli cells will grow with the added presence of arabinose because this chemical is what begins to stimulate the E. coli growth with both the expression of the fluorescent trait, along with antibiotic resistance. For the transformation efficiency of the experiment it was determined that there were transformants/μg. Because it is common for the transformation efficiency calculations to result in higher numbers it is possible that there might have been an error while conducting the experiment. It is plausible to believe that too little of LB broth or arabinose was added to the agar plate. It is also possible that too much ampicillin was added.
While we obtained the results predicted, to obtain more accurate results, it would be suggested to take more accurate measurements of the time the plates were in ice or into the warm water, along with perhaps better, more detailed written observations of the transformation and control plates. It would also be beneficial to make sure that each plate contains very precise amount of LB broth and ampicillin to get the most accurate calculations of the efficiency of transformation by getting the desired growth rate in the E. coli. To improve this calculation, it is also possible to take more care in counting the bacterial colonies on the agar plates after the experiment by using a microscope and more concentrated UV lights.
This would also explain why the control plates has less growth than those with the pGLO plasmid. The plate with the -pGLO had no growth because it had no LB broth or arabinose to stimulate growth. The plate labeled -pGLO LB/amp did not have the plasmid necessary for antibiotic resistance to ampicillin, and therefore the E. coli could not grow. However, the plate labeled +pGLO/amp/ara had both the plasmid necessary to encode for antibiotic resistance, as well as ambroise which will induce the bacterial cell to express the fluorescent trait and grow.
To make the environment within the the plate most suitable for cell growth it is then necessary to add the pGLO plasmid, the ampicillin, and the arabinose. It is plausible to believe that the E. coli cells will grow with the added presence of arabinose because this chemical is what begins to stimulate the E. coli growth with both the expression of the fluorescent trait, along with antibiotic resistance. For the transformation efficiency of the experiment it was determined that there were transformants/μg. Because it is common for the transformation efficiency calculations to result in higher numbers it is possible that there might have been an error while conducting the experiment. It is plausible to believe that too little of LB broth or arabinose was added to the agar plate. It is also possible that too much ampicillin was added.
While we obtained the results predicted, to obtain more accurate results, it would be suggested to take more accurate measurements of the time the plates were in ice or into the warm water, along with perhaps better, more detailed written observations of the transformation and control plates. It would also be beneficial to make sure that each plate contains very precise amount of LB broth and ampicillin to get the most accurate calculations of the efficiency of transformation by getting the desired growth rate in the E. coli. To improve this calculation, it is also possible to take more care in counting the bacterial colonies on the agar plates after the experiment by using a microscope and more concentrated UV lights.
