PGLO Transofmration

Section ETR6D 3/9/2012

Mohamed Sleem James Pione

Abstract To try and culture bacteria, and transform their DNA by combining their gene code with a plasmid, as well as trying to use certain indicators to see if they might become the ampicilin resistant. By avoiding Lys of the cell, and introducing the cell to a new enviornment, I was able to see how this transformation had an effect which it come to becoming ampicilin resistant.

The Introduction During this lab we will attempt to understand the transformation of bacteria, and the traits/characteristics of those bacteria. We will be tasked with the ability to perform a genetic transformation, and determine the degree of success in our attempt to genetically alter the organism. We will do this by introducing a plasmid to a bacteria, as well as the green fluorescent protein (GFP). We will also be moving one organism into another using a plasmid, which will help the bacteria adapt to its newly introduced environment. The pGLO inside the plasmid will work with the GFP, and produce an antibiotic ampeicillin. Methods and Material Tansformation procedure To start this procedure off we gathered four plates with a coat of apgar/Lb inside, labeling them: LB/amp +pGLO LB/amp ara +pGLO LB/amp pGLO LB pGLO.

We then continued by placing two test tubes one containing the label GLO+ and the other GLO-. We then placed a 500 microliter of the transformation solution into each of the tubes, as well as 250 micro liters of CaCl2 into each of the test tubes (this was done using a pippet). Now we added of pGLO into the tube that we had earlier labeled +pGLO, not touching the -pGLO, for it should not have the pGLO plasmid. We then incubate both testtubes in the solution for exactly 10 minutes. Followed by heat shocking the pGLO+ & - tubes in a heat shocked bath at 42C , while leaving it in to get heat shocked for 50 seconds, in the hot water bath. Now that the cell membrane has been weekend, it now has a short period of time to absorb the plasmid, so we place it in the ice bath for two minutes to try and stop any sort of lys (since it has a fragile membrane). Afterwards we added 250 microliters of LB nutrient to both test tubes, and one again placing it in the incubator for another 10 minute period. To

continue the process we plated 50 microliters from each of the test tubes matching the +test tube with the + agar plate, and the test tube with the agar plate. To conclude this process he held all four plates in the incubator for seven days, so that we could record our data at the end of the seven day period. Results

The Results as followed: +pGLO LB amp: A little natural resistance from contamination (more on this in the discussion). No E-Coli Colonies.

No Glow+pGLO LB amp/ara plate :

Glow under UV light Colonies growth present No Glow No Colony growth

-pGLO LB amp:

pGLO LB:

Colonies No Glow Discussion

+pGLO LB amp: While in the ideal world, we would not have any contimination, none of the bacteria would have survived. The fact that our agar plate had the bacteria which more circular or spherical may indcatethat it may have well be the streptococcus, contaminanted by one of the carriers or my lab partners. If there was no contamination nothing would have survived. +pGLO LB amp/ara plate : Successfully transformed, the plasmid entered the bacteria making the bacteria immune to the antibiotics. The pGLO is now detectible the the DNA using a UV light. This apart from the other experiment of not adding the plasmid to the plate, therefore transformation failed to occur. One could judge this by seeing If there are colonies alive, and if they were in fact ampicilin resistant there will be growth. To measure the success rate of how many Cells transformed I used the equation: Conclusions BY completing this experiment I have found that a transformation is possible under certain conditions, and even more obsticles are present in order to culture these bacteria cells. I have found that the plasmids were present in the E-coli cells that had survived, and thrived in their new enviornment, while the cells that failed to do so, died off. I did have some contamination and found that it already had the plasmid or genes necessary to survive in the enviornment that upheld ampicilin coated plate.

Citation

Campbell, Neil A., Lawrence G. Mitchell, Jane B. Reece. 1999. Biology, 8th Ed. Benjamin/Cummings Publ. Co., Inc. Menlo Park, CA. Pearson The Pearson custom library for the Biological Science-Symbiosis - Laboratory Manual General Biology II Brooklyn College Spring 2012.

Appendix

: Streptococcus pneumoniae more spherical or round, unlike the cone like E-Coli