Introduction:
Genetic transformation is when a part of a gene sequence is changed. This means taking one trait that is desired from part of one organism and then get it to be inserted into the gene of another organism. “Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species.”(Lorenz and Wackernagel 1994). The final result will hopefully the second organism expressing that wanted gene. However, not all cells are able to go through genetic transformation. The cells that are needed are called competent cells. Most of these cells are bacteria cells or plant cells due to their ability to take in new parts of a gene since they can share information with each other(especially bacteria. That is how resistant strands come about). There are 3 main ways to insert a new part of a gene into a competent cell. there is projectile bombardment, which takes a gene gun loaded with tungsten pellets coated with the desired DNA and fired at the cell. Another is electroporation which means taking cells and putting them in a medium containing foreign DNA and sending shocks through. And then the last method, which is the one used in this lab, is heat shock. The cells that want to be transformed are put into high and low temperatures to increase the permeability of the cell. This causes the foreign DNA to be taken up easier. A key point is how this will happen. The foreign DNA are in what are called plasmids. Plasmids can be found in many bacteria and are small rings of DNA with a limited number of genes. Plasmids are not essential for the survival of bacteria but can make life a lot easier for them(Steinbrecher 1998). This is how resistant strains are created when one has a resistance and that resistant strain gets shared with other bacteria. In this experiment we will be using Escherichia coli as the competent cell and pGLO as the foreign DNA. pGLO however needs arabinose to operate since it is a part of the operon. Also, the plasmid will have the gene for antibiotic resistance to ampicillin. This is we can take out the cells that did not take the pGLO gene. My hypothesis for this experiment is that the Escherichia coli will take up the plasmids and with the right medium can glow with the new gene expressed. This is important because this shows that we can incorporate different genes into bacteria. This is helpful because it can be made medicine like insulin which is really easy to make when using millions of bacteria or making a strain for plants to be resistant to certain pesticides.
Materials and Methods: In this experiment, there were two tubes labeled +pGLO and -pGLO. In each tube it has 250ul of transformation solution using a micropipette. This solution contains calcium chloride which makes it easier for bacteria to uptake. Kept tubes on ice. Then took a colony of bacteria(Escherichia coli) with a sterile loop and put it in both tubes. After both tubes have bacteria, took a sterile loop and put it in a tube with the pGLO plasmid DNA. Taking this loop, placing it into the tube +pGLO. There is no need to put it into the -pGLO as for this will serve as our control group. Leave both on ice for 10 minutes to incubate. After that 10 minutes, place both tubes into a 42 degree Celsius water bath for exactly 50 seconds. This is the heat shock treatment. Immediately after the 50 seconds, place back into the ice bath for an extra 2 minutes. Add in an extra 250 ul of LB nutrient broth to both tubes with a micropipette and incubate for another 10 minutes. After that, taking 4 plates with different mediums on them. First plate has just luria broth which is a nutrient agar plate. The second plate has LB and ampicillin. Both of these plates will have have the -pGLO bacteria on them. The third plate has the same as the second plate. And the fourth plate has LB, ampicillin, and arabinose. Plates three and four will have +pGLO bacteria on it. Using a sterile loop to put each of bacteria on respective plates. Then place in an incubator at 37 degree Celsius(optimum temperature for Escherichia coli to replicate) for 24 hours. The results will be ready by then because that is the minimum time to get enough replications to see the changes if any.
Results: This experiment too Escherichia coli and used heat shock to try and get the bacteria cells to uptake the plasmid with GFP and ampicillin resistance genes. After 24 hours, there were these results. The first plate with only the LB agar plate, had the -pGLO bacteria growing on them. The second plate with LB and the ampicillin had no bacteria growth on it. The third plate with LB and ampicillin ,but had the +pGLO bacteria on it did grow. The last plate with LB, ampicillin and arabinose also grew. Under a black light, none of them glowed besides the fourth plate.
Visual Representation:
Discussion: My hypothesis was that there will be growth on plate one three and four. There will be only glowing bacteria on plate four. There will not be any glowing on plate two. This had the right conditions to make the bacteria glow and also the bacteria had the uptake of the plasmids which are also required for the glowing to occur. The results did support my hypothesis. As from the results above and the data collected during the lab, there was only glowing on the fourth plate. It had the right sugar as the operon and the ampicillin resistance necessary for it. The third plate had everything but the sugar which means that the bacteria wouldn't glow. The second plate did not have the ampicillin resistance therefore it died. And the first plate with just the agar, let the bacteria grow naturally(control). There was a case with genetic transformation using enterobacter asburiae to produce ethanol from hemicellulose hydrolysates. They took E. asburiae JDR-1 with “pLOI555 or pLOI297, each containing the PET operon containing pyruvate decarboxylase (pdc) and alcohol dehydrogenase B (adhB) genes derived from Zymomonas mobilis, replaced mixed-acid fermentation with homoethanol fermentation. Deletion of the pyruvate formate lyase (pflB) gene further increased the ethanol yield, resulting in a stable E. asburiae E1(pLOI555) strain that efficiently utilized both xylose and methylglucuronoxylose in dilute acid hydrolysates of sweet gum xylan”(Bi et al. 2009). This just supports the results that were found in my experiment. Because there was a bacteria used and a plasmid that contained wanted gene. Then using the bacteria, it did what the scientists wanted to. In this experiment, we wanted the bacteria to glow. In theirs, they wanted to make ethanol. There are really no weaknesses in this experiment. The only way there could be any problems at all is if there was some type of human error or cross contamination which, again, is human error. There weren't any problems that arose during the course of the experiment.
Conclusion: This experiment was to show how genetic transformation worked. Taking a competent bacterial cell(in this case Escherichia coli) and put it with plasmids that contained the GFP, which is for the fluorescence, and ampicillin resistance. Using heat shock therapy, we made it so the bacteria took up the plasmids. Then using a control group and different plates, we tested each combination of bacteria and growth mediums. The bacteria only glowed on the plate with all the components necessary(specifically talking about the arabinose). Which is what was hypothesized.
Literature Cited:
Bi, Changhao, Xueli Zhang, Lonnie O. Ingram, and James F. Preston. "Genetic Engineering of Enterobacter asburiae Strain JDR-1 for Efficient Production of Ethanol from Hemicellulose Hydrolysates." ISI Web of Knowledge. Thomson Reuters, 15 Sept. 2009. Web. 1 Dec. 2009. <http://apps.isiknowledge.com/full_record.do?product=WOS&qid=7&SID=4BDb5CLL46jOj6F8cGe&search_mode=Refine&viewType=fullRecord&doc=2&page=1&log_event=no>.
"How to Test Genetic Transformation and Gene Splicing." ScienceRay. Triond, 4 Sept. 2009. Web. 1 Dec. 2009. <http://scienceray.com/biology/how-to-test-genetic-transformation-and-gene-splicing/>.
"How to Test Genetic Transformation and Gene Splicing." ScienceRay. Triond, 4 Sept. 2009. Web. 1 Dec. 2009. <http://scienceray.com/biology/how-to-test-genetic-transformation-and-gene-splicing/>.
Lorenz, MG, and W. Wackernagel. "Bacterial gene transfer by natural genetic transformation in the environment." PubMed. NCBI, Sept. 1994. Web. 1 Dec. 2009. <http://www.ncbi.nlm.nih.gov/pubmed/7968924>.
Steinbrecher, Ricarda. "What is Genetic Engineering?" Sfsu. A Magazine of Green Social Thought, July 1998. Web. 1 Dec. 2009. <http://online.sfsu.edu/~rone/GEessays/WhatisGE.html>.
