Over the course of this lab, our class performed a transformation on bacteria, altering its DNA to cause it to fluoresce when exposed to UV light. Transformations, the process of taking in foreign DNA, are common procedures in the field of biotechnology, the exploration of life processes to develop new technologies. Biotechnology applies recombinant DNA technology, which is DNA made from two or more organisms, to improve organisms and solve issues. Scientists use plasmids, circular pieces of DNA, to “cut and paste” genes into other organisms. Researchers often use GFP, or green fluorescent protein, in their experiments and procedures. GFP naturally occurs in jellyfish causing them to glow green under UV light. Scientists worked with GFP when designing GloFish. Their original plan was to create a fish that could signal pollution in water, but they ended up developing a beautiful, glowing pet fish which is now sold throughout the U.S. We made …show more content…
coli pGLO DNA
42°C water bath
LB broth
Incubation rack
LB agar plate
LB agar and ampicillin plate
LB agar, ampicillin, and arabinose plate
UV light
Amphyl disinfectant
Procedure
Day 1:
Mix bacteria with CaCl2. Add DNA and incubate on ice for 10 min. The CaCl2 pokes hole in the cell wall so that DNA may enter.
Place the test tube in the hot water bath for 50 seconds and then back into the ice for at least. This makes the bacteria sick so that it will take in pGLO DNA.
Add LB broth which acts as food for the bacteria.
Place the test tube in an incubator so it can heal.
Day 2: Plate the bacteria.
Pipette 50 μL of the E. coli mixture onto each LB plate and spread evenly using a loop so that it can grow.
Incubate to help growth.
Day 3: GFP Expression
Shine UV light onto each plate to see if it is fluorescent.
Results
Number of Colonies:
+DNA
-DNA
LB
LB/AMP
- The most common method is with an agrobacterium. Since bacteria reproduce quickly, it’s easy to create the same
Use a test tube holder to put the test tube into a container of boiling water for 5 minutes, or until the solution changes color.
Introduction: The biological membranes are composed of phospholipid bilayers, each phospholipid with hydrophilic heads and hydrophobic tails, and proteins. This arrangement of the proteins and lipids produces a selectively permeable membrane. Many kinds of molecules surround or are contained within
The purpose of the PGLO lab was to be able to perform a procedure known as a genetic transformation. We used a procedure to transform bacteria with the gene that codes for a Green Fluorescent Protein (GFP). The actual source of the GFP gene that we used in this complicated experiment is the bioluminescent jellyfish Aequorea victoria. This protein causes the jellyfish to glow under a UV light that was provided in the dark. After the transformation procedure, the bacteria showed their newly acquired gene from a jellyfish and produced the fluorescent protein, which as a result, causes it to glow. If the bacteria glowed in the dark, that was the initial sign that the experiment was successful.
The experimental part of the lab consists of setting up the materials needed. A sample of E.coli and a solution of calcium chloride are first obtained and placed in different test tubes. 630µL of Calcium Chloride (CaCl2) are then removed from the test tube and inserted into the test tube containing E.coli cells (Alberte et al., 2012). The newly formed substance of Calcium Chloride and E.coli is then mixed and incubated in ice for 10 minutes, making the cells more competent. Two test tubes are obtained and labeled; the first test tube is labeled with pUC18 and the second one with “Lux” to represent the plasmids being used. These two test tubes are then incubated in ice. 3µl of the set plasmid are added to each of the two test tubes. The test tubes are tapped to guarantee the cells are well
Observation: no bugs were found except small, black, gnats were all close to the ground.
3. You test another new unknown bacterial sample, and find the G+C content is identical to one of the samples you have already identified, but the rRNA gene sequence contains one base that is different. What can you conclude: C.
In this experiment we were meant to observe the transferring of DNA. There are many ways in which DNA can be transferred into an organism, for example; transformation, transduction, and conjugation. In our experiment we used
Genetic transformation is the change caused by genes. This transformation includes the insertion of a gene into an organism, changing one of the organism’s traits. There are many other uses for genetic transformation including the altering of plant genes coding for frostbite, pests and spoilage resistance. It can also be used to digest oil spills and even alter in gene therapy to transform sick cells into healthy ones. This particular experiment will include the transformation of the bacteria with the GFP ( Green
Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.
1. The authors from this experiment were trying to gain information on how the diversity of gut microbes in humans evolved the CAZymes, to supply the body with its energy needs, from microbial populations living outside the body. 2. It is known that sulphated polysaccharides are the origin of carbon for heterotrophic bacteria that make the CAZymes in marine organisms. Also, these enzymes have been seen throughout human evolution to locate polysaccharides in terrestrial plants that have been eaten.
The purpose of the “chi-square test” was to see if our data was in an acceptable range of a specific ratio listed above. The chi-square test took into account the expected deviations in the F2 offspring’s alleles.
An association between enzyme production, gene copy number, and gene evolution was explored by conducting analysis of the salivary amylase enzyme, AMY1A gene copy number, and the ancestral starch consumption in Homo Sapiens (Tracey 2017, p.22). It was hypothesized that the relative amount of starch consumption was very high for my personal ancestral diet, thus my AMY1 diploid gene copy number in my genome and salivary amylase concentration would be significantly higher than the population mean. With a population of 28 subjects (n=28), individual saliva samples were collected and compared to a calibration curve to determine the approximate amylase concentration by analyzing absorbance values. Individual samples of buccal cheek cells were
This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.
7.When water bath is ready, put each test tube into the water bath. Wait 5 minutes.