Thursday, March 6, 2014

PGLO Lab

Purpose
Thre purpose of this lab was to find the transformation efficiency of the plasmid in E. Coli in different mediums. The independent variables were the presence of plasmid and the type of medium. The different mediums were +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB. The dependent variable was the transformation efficiency. 

Introduction
Transformation efficiency is the total number of cells growing on the agar plate divided by the amount of DNA spread on the agar plate, and it shows approximately how many cells take on the the DNA of the plasmid. Plasmid was the DNA that was inserted into the E. Coli, and it was called pGLO. Heat shock is the procedure used to form holes in the plasma membrane of the E. Coli. 

Methods: First off, we labeled two micro test tubes with +pGLO and -pGLO. Then with a new pipet, we put 250 micro-liters of the transformation solutions which is CaCL 2 into both of the test tubes. Then after adding the transformation solution we had to put the tubes in a cup of ice.  We had to then go over to the starter plate and get a single colony of bacteria and put it into the +pGLO tube.  We had to spin the loop with the bacteria on it in the tube to get it off and make sure all of it stayed in the tube. Then, we repeated the same thing for -pGLO. Only for the +pGLO tube we put pGLO plasmid DNA into it. Then, we put the tubes back in the foam rack and put it in an incubator for 10 minutes. While we were waiting we labeled four LB agar jars:  +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, -pGLO LB. we put the tubes into a heart shock for 50 seconds, which was set at 42 degrees Celsius, then we had to put them on ice for 2 minutes. We took the tubes off the ice and with a sterile pipet, added 250 micro-liters  LB nutrient broth into both the the tubes and let them sit at room temperature for 10 minutes. Once again with a new pipet, we put 100 micro-liters  transformation and control suspensions onto the agar plates. Using a new sterile pipet for each plate, we spread the suspensions evenly around the surface. Then we stacked our plates and put them in a 37 degree celcius incubator overnight.  

Data
We took our data from the pGLO positive LB/Amp/Ara plate in order to determine the Transformation efficiency. This would tell us how many cells out of our total maintained the plasmid.
Using all of this data, we determined that there are 2.17*10^3 transformants per microgram. This means that approximately 2170 cells in each microgram take on the plasmid and its traits. The ones that take on the plasmid will have the ampicillin resistance and glow in the dark trait. 

Discussion
This lab was only possible because of the heat shock. When a heat shock occurs, it allows cellular membranes to pull apart just enough to let in a plasmid. This is how the pGLO plasmid entered into the E. Coli cells, allowing the traits to be adapted. When pGLO was not present, however, growth depended on the presence of ampicillin. With ampicillin in the growth medium, there was no growth of E. Coli cells. Without ampicillin, there is one large colony of E. Coli growing unimpeded.  But when pGLO is incorporated into the cell, E. Coli gains the trait of ampicillin resistance. The plasmid also allows the bacteria to glow in the dark, but only when supplied with sugar. This is why one plate, the one without sugar, has normal E. Coli colonies while the other, with sugar, was able to glow. The last two plates, with both pGLO and ampicillin, have sparse colonies compared to the unimpeded growth of the first. This is because the plasmid is not incorporated into every cell, causing not every cell to become ampicillin resistant. The transformation efficiency is calculated in order to tell us the number of transformants per microgram of DNA. Our transformation efficiency was in the range we expected, with 2.17*10^3 being a sensible number of transformants in each microgram of pGLO.


Conclusion: The conclusion we got was exactly what we were expecting it to be. The +pGLO LB/amp/ara plate had dots of bacteria basically covering the whole surface. Once we shined the ultraviolet light on it, it became green, so we knew we did it right. With all the combinations of substances in this one, is what made it glow. The +pGLO LB/amp had dots of bacteria as well, but didnt glow like the other one. The -pGLO LB/amp had no growth on it at all. And -pGLO LB was almost covered completely with bacteria. 




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