The Light At The End Of The Tunnel

In the first lab of our second semester we will be performing genetic transformation, which is the process of inserting a gene into an organism to change the organism's trait. Genetic transformation is used in many aspects of daily life, for example in food. Many crops are increasingly being genetically modified to increase their ability to withstand viruses and to be produced as larger, healthier species. Transformation is also used to transform bacteria into helping the environment and to expand the process of gene therapy in humans. This new industry of genetic modification is growing faster and becoming a vital new part of life. It gives us the chance to further our understanding of life and help improve the quality of our own.

In our lab we will be transforming bacteria using a gene that codes for Green Fluorescent Protein. This gene is what causes a jellyfish to "glow in the dark". We will be learning to move genes from one organism to another using plasmid. Plasmid DNA contains genes for one or more traits that may be beneficial to bacterial survival. By using the process called "genetic engineering" we can insert genes coding for new traits into a plasmid.

In order to move the pGLO plasmid DNA through cell membrane we will, 1. use a transformation solution of CaCl2, 2. Use heat shock, and 3. provide nutrients and an incubation period so we can observe the newly acquired genes and assess our success at genetically modifying an organism.

DNA Chips-How the genes were affected

Results from our last lab involving microarrays were some of the samples had pink in them! This symbolizes that they represented lung cancer. The samples that showed up blue meant that they appeared in both normal and cancerous cells. The fourth gene was clear, which meant that it was not present in either cell.

DNA Chips- how genes affect disease

In this next lab that we are doing in class, we will be examining how to use micorarray analysis. If a person wanted to find changes in transcription in a specific tissue, they would use this process. One microarray can contain over 30,000 "spots of DNA" where each spot represents a different gene. The first step is to make a DNA chip, that is arranged in a pattern and that represents part of a genome. The chip could then be used to analyze complementary DNAs that were made from mRNA isolated from the same tissue. The two samples of tissue are dyed and then applied to the prepared "chip". You can determine the extent of transcrition by observing how each dyed sample adheres to its complement. Once you find this out computer analysis will reveal what similarities the two samples of tissue could have.  The analysis allows scientists and doctors to find problems in certain tissues much earlier than ever before.

We will be studying six genes in this lab:
C4BPA- helps initiate part of our immune system to kill pathogens
ODC1- codes for an enzyme in the polyamine biosyntheis pathway
FGG- encoded in this gene is a part of fibrinogen, a protein found in the blood, creates blood clots
HBG1- expressed in liver, spleen, and bone marrow
SIAT9- catalyzes the formation of a protein called GM3
CYP24- catalyze many reactions involved in drug metabolism and the making of cholesterol, steroids and other lipids

CSI- We've gotcha now

In this lab we began our identification of the murderer by first putting our tube with the restriction enzymes in an ice bath. Then we collected our evidence from the crime scene, in five test tubes. We had narrowed it down to five suspects! We got a sample of each suspects DNA and placed them in test tubes, then we added 10 ul of the enzyme mix into the samples and mixed well. Next, we placed the test tubes into an incubator at 37 degrees Celsius for 45 minutes. Mr. Chugh took out our test tubes at the correct time and got them ready for when we returned the next day.  

The next day when we came back we removed the tubes from the water bath and then we gentle tapped the tubes on the table to bring the liquid to the bottom of the tubes. Next, we added 5 ul of loading dye into the separate tubes, collecting the samples in the end of each test tube. We took the agarose gel and placed it in the electrophoresis apparatus. We  checked that the wells of the agarose gels were near the black negative electrode and the bottom was near the red... But we found that our positive and negative was switched! So, we replugged the electrophoresis to correct the error. The next thing we did was using a separate tip for each sample we loaded the DNA samples into the gel and after putting the lid over the chambers we turned on the power. When we returned the next day, we could finally compare our results! We found the real murderer and solved our simulated crime scene investigation.

CSI Lab- Recognizing the Culprit

In our next lab we will be comparing band patterns produced by restriction enzyme cleavage of DNA samples. Restriction enzymes are enzymes that move down the helix until it recognizes specific sequences of base pairs that tell the enzyme to stop moving. Then the enzyme will cut or chemically separate the DNA molecules, this spot is called a restriction site. When DNA is being looked at to compare, as if on a crime scene, the way you tell the difference between two different suspects is by looking at their DNA samples. Each persons DNA is "cut" differently by the restriction enzymes, in different fragments of size and length.The DNA can be observed by using agarose gel electrophoresis. Agarose Gel Electrophoresis separates DNA fragments by size when they are put in an "agarose gel slab" and then put into a separate tube with a buffer solution. The DNA fragments are negatively charged and when the agarose gel acts as a molecular sieve where only smaller DNA fragments will travel farther than larger ones. The fragments that are the same size will band together and we will see them when we finish our lab! 

In a crime scene investigation to determine whose DNA is found, the DNA must be examined and you must  look at its nucleotide sequence. Radioactive probes are what is used to locate, identify, and compare individuals DNA. DNA can be found form many different biological materials, such as, blood, hair and body tissues. the recognition plays a large part in today's crime scene investigations. In this lab we will learn the general bases for using DNA in modern times.   

Biofuels Results!

On the first day of our lab we began our experiment by filling five cuvettes with stop solution. Then we filled both two conical tubes labeled"Enzyme Reaction"and "Control" with the substrate (cellobiose). After pipetting 500 ul of solution from the "Enzyme Reaction" tube into the first cuvette we began our timer and at intervals of one, two, four, six, and eight minutes we added the substrate to the stop solution. Immediately we noticed that the cuvettes containing the stop solution had a color change from clear to a yellowish color. This was the artificial substrate making the glucose illuminate.

 On day two, we brought in store brought mushrooms. We weighed out 1 gram of mushroom and then ground up the mushroom with a mortar and pestle, for every 1 gram of mushroom extract we added 2 ml of extraction buffer. Next, using a centrifuge, we "pelleted" the solid particles by spinning at top speed for 2 minutes. After this we labeled our cuvettes from 1 until 6.  We obtained a conical tube and pipetted 3 ml of substrate into the tube, which we labeled "button" after the type of mushroom we used. As we did the day before, we added the extract (mushroom) at 1 minute, two, four, six and eight. Surprisingly the mushroom extract made the solution in the cuvettes have the same reaction, a change of color! 

Because the reactions were the same, they pose an idea. If the reactions with a natural extract were similar to a chemical than maybe we could use other organisms in nature as substitutes for chemicals. The idea of this lab was to show us how it may be possible to come up with new ways of obtaining resources that are less harmful to the environment.  

Biofuels Lab Introduction

Biofuels are groups of fuels that are produced from a source in nature that was recently living, versus fossil fuels which are made from sources that have been dead a long time. Biofuels are being increasingly used as alternative energy sources. There are four ways to produce biofuels, through cellosic ethanol,where a breakdown of cellulose to glucose followed by a fermentation step to ethanol. The biofuel industry uses cellulases in this method. Cellulases is an enzyme that converts the cellulose in plant cells to glucose and other sugars. An enzyme is a catalyst that speeds up the process of a chemical reaction. The enzyme is very helpful because it reduces the energy needed to make the reaction occur, therefore the chemical reaction can occur much faster. The next method is the breaking down of starches and sugars followed by a fermentation step to ethanol.  The third, involving biodiesels (fuels derived from oils) are found in recycled cooking oils or in plants that produce high levels of oils that are purified and burned in diesel engines. Scientists are always trying to find new ways to create biodiesels through modifying algae, yeast, and bacteria. the last method of creating biofuels are syngas. Syngas stand for synthetic gas, it is a mixture of carbon monoxide and hydrogen gases which becomes a burning biomass. Syngas can can be burned directly or chemically converted to be used as diesel. Biofuels are important to us because they are the answer to our problems as far as global warming. If we can find natural ways to make energy we can save our environment. Although scientists are working hard, we still have a long way to go, and if we do find an alternative there are still bound to be consequences.   

In our lab we will be exploring how biofuels work, and we will work towards understanding how biofuels could change the world we live in to be more efficient. We will have multiple test tubes that will contain a strong base and then we will be adding different amounts of cellobiase (an enzyme) and P-nitrate phenol at different time increments to see if fuel is created! On Day Two we will bring in mushrooms to grind up and add to our mixtures to see a new reaction.