Friday, March 7, 2014

pGLO E. Coli Lab

Purpose: In this experiment we injected the bacteria E.Coli with a fluorescent gene as well as a antibiotic resistant gene. We tested how organisms transfer specific genetic information via plasmids as well as through which media the antibiotic resistance and the fluorescence were best expressed. It’s a process in which we change a specific sequence of DNA in a gene in order for it to express a desired phenotype. This is called genetic transformation. 
We worked with the E. Coli bacterial plasmids which are simplified bacteria whose DNA is circular. This neat characteristic allows for the plasmid's DNA to be easily manipulated and transformed. We did so by icing the DNA, allowing the plasmid's membrane to weaken. We used a procedure called "heat shock" to denature the membrane, allow the anti-bacterial gene and the fluorescent gene to enter and integrate with the plasmid's DNA. Then we iced the plasmid again in order to put the membrane back together.

Brave Abe extracting the E.Coli and putting it into a test tube with pGlO

E. Coli on Ice (tube with pGLO and without pGLO)

"Heat shocking" the samples. The samples spent 50 seconds in the hot water.

We stored the bacteria in a 37 degrees Celsius (optimal temperature for bacteria to grow) for 24 hours.

       The gene that we mixed with the E. Coli is named pGLO. It has both information for anti-bacterial resistance (in this case, ampicillin) and the ability to fluoresce under ultraviolet light.
       We prepped a total of four petri dishes labeled LB (E. Coli by itself with no pGLO), LB/AMP (E. Coli with ampicillin with no pGLO),LB/AMP (E. Coli with ampicillin and pGLO is present), and LB/AMP/ARA (E. Coli with ampicillin with the sugar arabinose and pGLO is present). The contents of these preps will be discussed in detail in the "discussion" portion of this report.

Graphs and Data: We calculated the transformation efficiency for this lab. The transformation efficiency is a calculation that shows the amount of cells that have been transformed by the pGLO.
The transformation efficiency: 655.75
Our calculations are shown below:

We used the LB/AMP/ARA plate as it showed the most reaction to the pGLO to calculate transformation efficiency.

Discussion: Our lab was a great success. Every petri dish showed the results it should have as seen here.

As you can see, the LB- plate had unrestricted growth. The smears are the rampant E. Coli. There was no pGLO and no ampicillin to restrict growth. When looked at with the ultraviolet light on, there was no glow which indicates the absence of pGLO.

On the next petri dish labeled LB/AMP-, there was no visible growth of E. Coli. That is due to the ampicillin that killed the bacteria. Also, without pGLO, the plasmid couldn’t develop any resistance. Under the UV light, there was no fluorescent glow either. This showed that there was no contamination.

The third petri dish, LB/AMP+ there was significant growth. pGLO is present, which allowed for the bacteria to establish a resistance to the ampicillin. However, when compared to the LB plate, the cells look a lot more colonized and spacious rather than all smear-looking. This is because some of the bacteria got killed by the ampicillin before it could establish resistance to it. Also, when under the UV light, the E.Coli did not glow. This is the case because arabinose wasn’t present. Arabinose acts as a catalyst to expose the specific DNA sequence which controlled the glow of the E. Coli. Without the sugar, the E. Coli did not glow.

The fourth and final petri dish, LB/AMP/ARA+ was by far the most interesting to look at under UV light. Also, it showed the most change in its phenotype and genotype. With arabinose present, the DNA for fluorescence was able to be added through the pGLO. The ampicillin had a small effect on the bacteria, making it just as colonized. It probably killed some bacteria before it could establish resistance. If ampicillin wasn’t present, we can predict that the bacteria would have the appearance of LB- as well as a fluorescent and antibacterial property.

Conclusion: The plasmid we inserted into the E. Coli bacterium contained the gene (GFP), which codes for fluorescence and glowing in the dark. However this was only viewed in the LB/AMP/ARA+ because it was the only medium with arabinose sugar which supplied the bacterium with energy to glow in the presence of UV Light. Since, it did glow we know the plasmid was successfully integrated as previously mentioned. We can deduce that plasmids are a great way for biologists to isolate specific genes of their interest and integrate them into other organisms. This also raises the question of wether, humans in the near future can integrate plasmids with the more complicated eukaryotic cells. In either case integrating the more favorable genes probes an organism for survival, sort of like natural selection. Recently biologists discovered a gene that reduces the chances of diabetes by 2/3. Could the gene be isolated in a plasmid and then integrated into an organism? With this lab, we proved its possible.
References: Lab