Abstract Some bacteria are able to go through transformation making new combinations of genes. Transformation is a way of gene variability in bacteria. This experiment is based on the transformation mechanism of bacteria and gene regulation. The bacteria used for the experiment was Escherichia coli and the genes introduces for the transformation were: gfp and bla by a pGLO™ plasmid. After the insertion of the target genes and growing the bacteria on specialized LB media, it could be seen that the transformants were positive for the gene expression.
The transformed E. coli on the media appeared fluorescent green under UV light. Introduction The bacteria used in this experiment is Escherichia coli which is not naturally competent. E. coli is a gram negative rod shaped bacteria and a facultative anaerobe. This bacteria forms part of the bacterial flora in the human intestine tract. The competence of a bacteria is based on its ability to take up naked DNA from the environment and incorporated on theirs, transformation. Alteration in the permeability of the membranes allows DNA to cross the cell envelope of E. oli. Since the outer membrane of the E. coli is mostly negatively charged and the DNA molecule also has a negative charge, then the addition of CaCl2 will neutralize the interaction so that the naked DNA molecule can enter the cell. (Microbe Library web) Another important factor on the competence of the bacteria is a procedure of alternating temperature between ice bucket and heat shocks. By the combination of this two procedures E. coli becomes competent. This procedure was first reported by Mandel and Higa. Singh 562) Even though it works it is only believed that CaCl2 helps DNA absorption to cell surface and the heat-shock step facilitates penetration of absorbed DNA into cell. (Panja 411) The main purpose of this experiment is to transform the bacteria to make it resistant to the antibiotic ampicillin. A secondary transformation is being made, and is to make the bacteria seem fluoresce green. The reason why the bacteria will fluoresce is because the gfp gene is being inserted under an ara promoter. The gfp gene encodes for the Green Fluorescent Protein (GFP).
The genes under the ara promoter will be expressed when the bacteria is in presence of the sugar Arabinose. When the transformed E. coli is in presence of Arabinose, the gfp will make the GFP and when the bacteria is placed under UV light it will fluoresce green. The gfp gene was found and extracted from a jellyfish, Aequorea victoria, and is being used as a visible reporter for gene expression. (Garcia-Cayuela 172) To introduce the gfp into the bacterial cell it was needed to be by a plasmid, as well as the gene to make the E. oli resistant to ampicillin, bla gene. The bla gene encodes for the protein beta lactamase which breaks down the ? -lactam ring in the structure of the ampicillin, therefore making it resistant to the antibiotic. Like already said to introduce this two genes to the E. coli it must be done through a plasmid. Both genes were introduced by the same one. In this case the one that was used was a pGLO™ plasmid. This is an engineered plasmid used as a vector to create genetically modified bacteria. This plasmid contains three specific genes: bla, gfp and araC.
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The ¬araC is a promoter region that regulates the expression of the gfp only under the presence of arabinose sugar. Materials and Methods In this experiment a pGLO™ transformation kit was used. First we needed two eppie tubes, one pGLO positive and the other pGLO negative. This two eppies were then moved to an ice bucket. During, one loopful of the pGLO plasmid was transfer to the pGLO+ tube. The other tube will be the pGLO-, the Escherichia coli without the plasmid. The two tubes were moved into an ice bucket and left there for 10 minutes. Then the tubes were put into a 42?
C water bath for 50 seconds and after back to the ice bucket for 2 minutes more. After the two minutes had passed, a 300 microliters aliquot of LB broth was added to the two test tubes. By adding the LB broth, the CaCl2 solution was also inserted in the tubes with the E. coli. Right after it the tubes were shook for ten minutes in a 37? C shaker. There were gather 4 petri plates, one with LB media, two with LB amp(ampicillin), and the last one with LB amp ara(arabinose sugar). After the 10 minutes each plate was given an aliquot of 100 microliters with one of the E. coli of the eppie tubes.
The LB plate and LB amp had the pGLO- and the other two plates, LB amp ara and LB amp, had the pGLO+. After this step it’s done the plates are prepared to be incubated at 37? C for two days and reveal the results of the induced transformation. LAB 9: TRANSFORMATION PROCEDURE Results The results for this experiment were a bit ambiguous but still recognizable and pretty clear. All of these plates were seen under UV light. In the LB plate pGLO- , after the incubation, there was found a lawn of Escherichia coli colonies that looked green because of the light. The LB amp plate with the pGLO- bacteria, the E. oli did not seem like it grow on it, the media just looked green. A count of 172 colonies that looked green, was found in the LB amp pGLO+ plate, this plate had ampicillin. In the LB ara amp media plate there were found 251 colonies of E. coli. In this plate the colonies looked fluorescent green under the UV light, the only plate. In a scale of growth from larger to smaller, the first in line would be the LB, then LB ara amp, proceeds LB amp (pGLO+), and last one LB amp (pGLO-). Table 1. 1 Results oftransformationof E. coli withpGLO plasmid mediapGLO+pGLO-color(under UV light)growth
LB -Yesgreenlawn of colonies LB amp-yesmedia look greenno growth LB ampyes-green172 colonies LB amp arayes-fluorescent green251 colonies -= n/a Discussion The results obtained in this experiment were as expected. The gfp should had been expressed under the presence of arabinose sugar and then under the UV light would fluoresce. The bla gene was expected to be expressed in the presence of ampicillin molecules. The LB pGLO- plate was a control plate meaning that this plate set a reference parameter to compare the results after the transformation. In this plate the growth of the E. oli was in a vast amount since this is a general media target for growth. In the LB amp pGLO- plate, the other control, the E. coli was not transformed with the plasmid, so in presence of the ampicillin the natural behavior of the bacteria is that is susceptible to it. In another hand, the plate of LB amp pGLO+ presented growth meaning that the bacteria took up the plasmid and was able to expressed the genes by an induce transformation. The result being that the transformed E. coli is now resistant to the ampicillin. The last plate, LB amp ara pGLO+, appeared with 251 fluorescent green colonies under the UV light.
The reason for it is that the bacteria took up the pGLO plasmid and when the E. coli was in the presence of arabinose and ampicillin, the bacteria could fluoresce green and be resistant to ampicillin which naturally the E. coli does not possess this genes. When this last plate is compared with the control plates it can be confirmed that the procedure done in this experiment was effective as hoped. The arabinose sugar is the intriguer that turns on the genes under the ara promoter. So when the gfp under this promoter turns on, all the other genes under the same promoter will expressed in the cell also.
No real noticeable source of error was found during the experiment since the results obtained were completely expected based in the information of the procedure. New studies are being made constantly and this transformation technique is widely used in the field of biotechnology. In the study of Plasmid DNA Transformation in Escherichia Coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation of CaCl2 Treated Cells, the experiment was based on how much quantitative is the difference between different variables possible to reach for the best optimum environment to exploit to the maximum the use of this technique.
These results suggest that a heat shock pulse of 30 sec at 42°C followed by a 10 min ice incubation step are ideal parameters to obtain maximum transformation efficiency, also suggest that post heat shock cold incubation step is also an important factor and enhances transformation of E. coli significantly (Singh 561) The relevance of this paper on the experiment performed and discussed previously is big. The results of Singh’s experiment helps our experiment in enhancing the correctness of our results and lowering the possible errors that can surge.
Also it can be a great reference of how to determine the optimum conditions of a specific bacteria which would contribute in other research fields. Citations Anh-Hue T. Tu. Transformation of Escherichia coli Made Competent by Calcium Chloride Protocol. Microbe Library. American Society of Microbiology. October 25, 2012. Web. November 10, 2012 Garcia-Cayuela, Tomas,. Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli. 172. Applied Genetics and Molecular Biotechnology.
Pdf Panja, Subrata,. Aich, Pulakesh,. Jana, Bimal,. Basu, Tarakdas. How does plasmid DNA penetrate cell membranes in arti? cial transformation process of Escherichia coli? 25(5): 411 Molecular Membrane Biology, August 2008. Pdf. Singh, Mahipal,. Yadav, Arpita. Ma, Xiaoling. Amoah, Eugene. Plasmid DNA Transformation in Escherichia Coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation of CaCl2 Treated Cells. Volume 6 Number 4, 2010. 561– 562 International Journal of Biotechnology and Biochemistry. Pdf.
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