Analysis of Lambda DNA by Gel Electrophoresis

Summary and Results 

Figure 1

Our gel successfully separated DNA fragments creating unique banding patterns for each restriction enzyme used. Fig 1 illustrates our final stained gel showing separation of DNA fragments and digital analysis of distance traveled on left.  We used the HindiIII bands (bottom well) as a molecular

weight ruler or marker as we knew the size of these fragments.  We then estimated the length of unknown DNA band sizes by constructing a a standard curve( figure 2) based upon the measurements obtained from the known DNA HindIII bands. Using Excel's trend line function, we found the relationship between fragment size and distance traveled to be

                fragment size in base pairs = 54892e^(-0.172*distance in mm)

This regression line has a high coefficient of determination of 0.9975 (between 0 and 1.0, used to describe how well a regression line fits a set of data).

Figure 2

Procedure

Gels are placed in electrophoresis chamber which is filled with buffer which helps current flow.

DNA digests are loaded into wells at negative end of the gel. 

From left: uncut lamda DNA, Pstl restriction digest, EcoRI restriction digest

HindiIII restriction digest

running the gels

the loading dye moved across the gel and can be observed easily

pouring the staining solution over the gel to help visualize the DNA bands.

destaining the gels before analysis

DNA Extraction Lab

We used strawberries as the source for our nucleic acids. Strawberries yield more DNA than any other fruit because they are octoploid - they have 8 copies of each chromosome. 

we prepared a solution containing detergent which disrupts the phospholipid membrane around the cells and the nucleus. Sodium chloride  disrupts histones that bind DNA. It also helps to 

keep the proteins dissolved in the aqueous layer so they don’t precipitate in the alcohol 

along with the DNA. 

the strawberry was pulverized by smashing it in a plastic bag with our detergent and salt solution to chemically break apart the cells.

 The solution was then filtered to get rid of strawberry skin and remaining chunks of flesh.

We added an equal volume of 70% ethanol. DNA is not soluble in isopropyl alcohol and  causes the DNA to precipitate and clump together into long strands.  These were captured by swirling a stick at the interface between the two layers. The DNA was transfered to a rube containing alcohol. 

Pglo lab

I was overflowing with excitement as I walked in to biology class. I couldn't wait to raise my glowing plate of bacteria in scientific triumph!

We had been so careful, so precise...

But it was all for naught.

Alas, my LB/Amp/Ara plate, like so many others before me, was utterly blank. There was not a SINGLE speck of glowing e. coli to give me any semblance of satisfaction. And thus, rather than expand upon the elusive causes behind my failures, I will use this blog post to explore some of the finer points of this otherwise fascinating lab. 

The Glowing Protein 

I wanted to learn more about the function and mechanism of the green fluorescent protein (GFP) responsible for the fluorescence of our e. coli specimens transformed with the pglo plasmid. GFP was discovered by Osamu Shimomura, Martin Chalfie, and Roger Tsien in the 1960's and 1970's. The first reported crystal structure of a GFP  in 1996.  Chalfie, Shimomura and Tsien shared the 2008 Nobel Prize in Chemistry for their discovery and development of the green fluorescent protein. In fact, I was lucky enough to meet Martin Chalfie at the Intel International Science and Engineering Fair in Los Angeles. 

GFP has a unique soda can shape. Inside the can is the chromophore (the part of a molecule responsible for 

its color). Thus, GFP is occasionally referred to as the “light in the can.”The chromophore produces the

 fluorescence. It consists of three peptides consisting of the residues serine, tyrosine, and glycine at positions 65-67 in the sequence. Although this simple amino acid sequence is commonly found, it does not generally result in fluorescence because the structure of the entire protein is necessary for the reaction. Upon formation of the polypeptide chain of GFP, the folding and other reactions occurs automatically and no activator is necessary for fluorescence. However, I still wanted to know exactly how this protein glowed. As usual, I found it was a lot more complicated that I would have thought.
"Fluorescence (indicated by a green glow surrounding the affected structural elements) occurs when oxidation of the tyrosine alpha-beta carbon bond by molecular oxygen extends electron conjugation of the imidazoline ring system to include the tyrosine phenyl ring and its para-oxygen substituent. The result is a highly conjugated pi-electron resonance system that largely accounts for the spectroscopic properties of the protein." (source http://www.olympusconfocal.com/java/fpfluorophores/gfpfluorophore/index.html). This is illustrated in the figure below.

A. The molecular structure of Aequorea green fluorescent protein as viewed from the top

B. A proposed mechanism for the series of post-translational modifications that converts the serine 65, tyrosine 66, glycine 67 tripeptide sequence into the fluorescent chromophore (Heim et al. 1994). 

Function

Apparently flourescent proteins are found in over 125 species. I couldn't help but to wonder what was the point? UV light from the sun is not strong enough to cause these proteins to light up and it's not as if animals are carrying around UV lights. 

GFP typically  functions in many bioluminescent organisms (which is quite different that florescence - Bioluminescence is a naturally occurring form of chemiluminescence where energy is released by a chemical reaction in the form of light emission.). GFP acts as a bioluminescence resonance energy transfer (BRET) acceptor. These convert the  blue emission of the bioluminescent protein into a longer wavelength green emission. OK, great. So what it the point of that?

We've finally come to the end of absolute answers. Biologists aren't really sure about the point of BRET molecules. One theory is the "Burglar-alarm" hypothesis in which the jelly fish will light up upon being attacked to attract secondary predators. The glowing jellyfish typically only light up after stimulation or stress.

Florescent proteins have other function in Anthozoans (stony corals). One study suggests they provide photoprotection to symbiotic photosynthetic algae living inside the corals. Another theory is that the range and patterns of coloring due to  fluorescent proteins may help reef fish identify different species of corals. 

My research indicated that there are numerous speculative hypotheses regarding the biological roles of fluorescent proteins. As one site put it "It is remarkable that, despite the critical role of coral reefs in supporting a wide diversity of ocean life and providing food for humans, so little research has been devoted to understanding the role of color and fluorescence in reef biology and ecology."

Experimental Design

A science fair judge (at least a good one) would not be so happy with this lab - namely its experimental design. At the end of the experiment (a successful one), the only thing we know for sure is that the transformed bacteria become ampicillin resistant while the untransformed bacteria are not ampicillin resistant. However, the interesting thing is the glowing part of the experiment is not well controlled because only 1 of the 4 plates has arabanose. For instance, what if the original bacteria actually glowed. We wouldn't know because we never but them on an ara plate unless they had been transformed. Were the creators of the lab trying to give us something to write about in our lab reports  or were they simply setting a bad example? 

Overall, the 2 -pglo plates functioned as the supposed  control and the +pglo were our variable group. The variable was the addition of pglo plasmids. It's fascinating to me, however, that we never tested out our original bacteria in the ARA or AMP plates. Perhaps they started out as amp resistant and glowing. Maybe our procedure of heating and cooling destroyed those characteristics. Who knows? The point however is that it wasn't that well controlled (though there is a cost factor to be considered, which I understand Mr. Wong)

Procedure

Rather than mindlessly plod through a procedure, I would rather understand why we did what did (which I'm sure most people didn't)

transformation solution (CaC12) - the positive charges of the Ca+2 ion neutralizes the negative DNA phosphates and negative charge of membrane phospholipids. 

Ice treatment - slows the fluidity of the cell membranes

Heat shock - increases permeability of the cell membrane

Applications

So who cares about a stupid glowing protein? It turns out a lot of people. GFP has a vast range of important applications. Rather than go through all those, I'm just going to look at one. The "Brainbow" mouse was an experiment in which each cell of a living mouse's brain was colored one of about 90 different colors. The colored neurons will help simply the complex tangle of neurons that make up the brain and nervous system: a sort of circuit diagram if you will. Use of the protein is important as these cells are not simply dyed different colors. They are randomly expressing different colors of florescent protein. See pretty pictures below: