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Vibrio fischeri is a type of gram negative bacteria that belongs to the Vibrionaceae, a large group of marine Î³-proteobacteria (Ruby et al., 2005). These organisms include a wide variety of species that engage in a multitude of pathogenic and beneficial interactions with animal tissue (Ruby et al., 2005). As shown in other Vibrio species, V. fischeri contains two chromosomes (Ruby et al., 2005). An interesting characteristic of the V. fischeri genome is its extremely low G+C content of its DNA. At a genome-wide value of 38.3%, it has the lowest G+C content out of the 27 species of Vibrionaceae (Ruby et al., 2005).
A marine bacterium, Vibrio fischeri is a symbiont that is responsible for bioluminescence in organs of certain fish and squids (Ruby et al., 2005). Vibrio fischeri accomplishes this by expressing the lux operon, which consists of a small chunk of genes found in several of the Vibrionaceae (Ruby et al., 2005). Luminescence is controlled via a diffusible compound called N-Acyl-homoserine lactone in the process of quorum sensing or autoinduction (Ruby et al., 2005). The light emitting reaction is catalyzed by luciferase (Ruby et al., 2005). Analysis of the pattern of light production in V. fischeri led to the conclusion that cells produce and release into the medium, such as squid cells, a diffusible inducing factor, which is called autoinducer (Ruby et al., 2005). This factor accumulates and triggers induction of luciferase when a particular concentration threshold is reached (Ruby et al., 2005).
The inducing factor was determined as 3-oxohexanoyl-L-homoserine lactone, or V. fischeri autoinducer-1 (Dunlap, 1999). After the inducing factor was chemically identified, a fragment of the V. fischeri chromosome was isolated and determined to allow E. coli the ability to produce and regulate production of light in a population density-responsive manner (Dunlap, 1999). This fragment contained genes coding for luminescence proteins and genes necessary for expression of those proteins, including a gene necessary for synthesis of the V. fischeri autoinducer by E.coli (Dunlap, 1999). The genes for luminescence proteins are organized into two divergent units, luxR and luxICDABEG, or the lux operon (Dunlap, 1999). The luxR and luxI genes are both regulatory; luxR specifies a protein required for cells to active lux operon transcription in response to VAI-1 while luxI allows for VAI-1 synthesis (Dunlap, 1999). The luxA and luxB genes code for the Î± and Î² subunits of luciferase respectively (Dunlap, 1999). The luxC, luxD, and luxE genes encode polypeptides of the fatty acid reductase complex, including reductase, acyl transferase, and acyl protein synthetase respectively (Dunlap, 1999). These polypeptides are required for synthesis and recycling of the aldehyde substrate for luciferase (Dunlap, 1999).
Quorum sensing allows bacterial cells to distinguish one habitat from another, thereby determining whether conditions are favorable for light production (Dunlap, 1999). Luminescence requires energy, both for the luminescence reaction and for synthesis of luciferase (Dunlap, 1999). Luminescence is mostly induced under conditions in which the survival or growth of V. fischeri is enhanced. Therefore, light production has a benefit for the bacteria, one that outweighs its actual energy cost, and a high level of light production may allow V. fischeri to adapt to its surroundings in the symbiotic state (Dunlap, 1999).
In this section of the experiment, chDNA from V. fischeri and plasmid DNA were isolated, repurified, and digested for later use in shotgun cloning of the lux operon. Shotgun cloning is the practice of randomly digesting a large piece of DNA into smaller pieces that can then be ligated into plasmids for transport to other organisms. The chromosomal DNA (chDNA) from the lux operon will be combined with the plasmid (PGEM) DNA to form a V. fischeri genomic library. This library can subsequently be used to transform E. coli, which will be screened for plasmids containing the lux operon. The purpose of the experiment is ultimately to make E.coli fluoresce using the lux operon.
III. MATERIALS AND METHODS
A. Isolation of chromosomal DNA from Vibrio fischeri
The pellet of an overnight culture of Vibrio fischeri which was centrifuged at 6000xg for ten minutes, was resuspended in 10ml of ice-cold TES buffer. The created suspension was then transferred into a 50ml Oak Ridge centrifuge tube, and an additional 2ml of TES buffer was added into the original container and then rinsed into the Oak Ridge tube.
One milliliter of lysozyme was added into the Oak Ridge centrifuge tube and mixed by inversion. After having the tube placed on ice for fifteen minutes, 65Î¼l of proteinase K was added. The Oak Ridge centrifuge tube was then incubated for ten minutes in a 55°C shaking water bath, at which point the cells were lysed by addition of 1365Î¼l of 20% sodium dodecy sulfate (SDS). The Oak Ridge tube was then placed back into the water bath for thirty minutes. An equal amount of phenol was added into the oak ridge tub and then additional phenol was added until the mass of the tube and its contents matched that of another tube in the centrifuge for balance. The centrifuge tube was mixed by inversion for about five minutes and then centrifuged for ten minutes at room temperature at 17000xg.
Fluid containing DNA from the aqueous layer was slowly drawn out of the centrifuge tube using the mouth end of a 5ml glass pipette. The collected DNA solution was transferred into a conical tube and placed on ice. Twice the volume of collected DNA was added as ethanol into the conical tube and then set to incubate on ice for ten minutes.
The DNA was wound onto a sterile glass rod and then placed into an incubator a 37°C for five minutes. The DNA was then transferred into a sterile FEP Oak Ridge centrifuge tube containing 15ml of sterile TE buffer followed by 150Î¼l of RNase A. The contents of the FEP Oak Ridge tube were then incubated for thirty minutes at 45°C.
B. Purification of Isolated chromosomal DNA from Vibrio fischeri
100Î¼l of proteinase K was added into the tube and then incubated for thirty minutes at 45°C. An equal volume, with respect to the previous contents of the tube, of phenol:chloroform was added into the tube. After gentle rocking of the FEP tube for five minutes, it was centrifuged for five minutes at room temperature at 17000xg. The mouth end of a glass pipette was used to transfer fluid from the top of the aqueous layer into a sterile FEP Oak Ridge tube.
An equal volume, with respect to the contents of the FEP Oak Ridge tube, of chloroform:isoamyl alcohol was added into the tube. After gentle rocking for five minutes, it was centrifuged for five minutes at room temperature at 17000xg. The mouth end of a 5ml glass pipette was used to transfer fluid from the top of the aqueous layer into a sterile 50ml conical tube.
1/10th of the volume, with respect to the contents of the conical tube, of 3M sodium acetate was added into the tube. The DNA was re-precipitated using twice the volume, with respect to the contents of the tube, of ice-cold 95% ethanol. After gentle mixing by inversion, the tube was incubated on ice for five to ten minutes. The DNA was wound onto a sterile glass rod and then placed in an upright position in an incubator set to 37°C until all the ethanol had evaporated. The DNA was then dissolved in 1ml of Te buffer.
C. Spectrophotometeric Analysis of chromosomal DNA from Vibrio fischeri
A spectrophotometer was set up to read absorbance and the wavelength was set to 260nm. 25Î¼l of DNA was diluted by addition of 475Î¼l of TE buffer. A cleaned disposable cuvette was filled with 1ml of TE buffer and placed into the blank slot in the chamber. Another cleaned disposable cuvette, was filled with the diluted DNA sample. After selecting the blank and zeroing the value, the sample's absorbance was recorded at 260nm. The wavelengths were changed to 280, 234, and 320nm. For each of these wavelengths, the absorbance was rezeroed using the blank and then the absorbance of the sample was measured.
Isolation of Chromosomal DNA from Vibrio fischeri:
The pellet obtained after centrifuging the culture of Vibrio fischeri was a dark yellow color with a distinct pungent odor. It was about the size of a nickel before it was resuspended in the TES buffer to about the consistency of cooking oil. The resuspended solution was the color of sand. Once SDS was added to the mixture, white, filamentous clouds of foam formed because the SDS started working. There was approximately 14 ml of solution before the phenol was added. After an equal volume of phenol was added, the total mass of the mixture was 65.60 g and a light yellow emulsion was formed after mixing. After it was centrifuged, several distinct layers formed in the tube. Above all the other layers was a layer of foam. At the bottom, there was a layer of phenol, which appeared cloudy. An aqueous, colorless layer formed above the phenol, which contained the DNA. White protein particles were floating in between the two liquid layers. 9 ml of fluid was obtained from the aqueous layer and 18 ml of Ethanol were added to that solution. It is possible that some of the white protein particles were taken up when the aqueous layer was being transferred to the conical tube. After the mixture was placed on ice, clumps of white strings formed as the DNA was precipitated. A relatively large "snotwad" of DNA about the size of a dime was obtained from the mixture. The "snotwad" of DNA was possibly large because of contamination from protein particles when the aqueous layer was being transferred. After the "snotwad" was incubated, the size of the sample decreased as the ethanol evaporated and the sample began to appear almost colorless.
Purification of Isolated Chromosomal DNA from Vibrio fischeri:
In purifying the isolated DNA, 100 µl of proteinase K was added instead of 75 µl because it was suspected that there was significant protein contamination in the sample due to the size of the "snotwad" of DNA that was obtained and the protein particles that were taken up when the aqueous layer was transferred from the centrifuge tube to the conical tube. The volume of the colorless mixture before the phenol: chloroform was added was approximately 15.25 ml. The total mass of the emulsion after the phenol: chloroform was added was 67.18 g. The two liquids formed separate layers and then combined into an emulsion that was colorless. Three layers were formed as a result of the centrifugation. A clear phenol: chloroform layer, was on the bottom, a thin layer of protein particles was in the middle, and a clear, aqueous layer, which contained the DNA, was on top. Once an equal amount of chloroform: isoamyl alcohol was added to the aqueous mixture, the total mass of the tube was 66.72 g. Upon mixing the contents of the tube, a brown flake was observed in the emulsion. When the tube was centrifuged, the brown flake broke against the side of the centrifuge tube and separated into smaller fragments. There were three layers that formed as a result of the centrifugation. The chloroform layer was on the bottom, followed by the thin layer of protein and dirt. The aqueous layer was again the top layer. However, the dirt fragments were scattered throughout the thin layer of protein particles and the bottom of the aqueous layer. Approximately 8.5 ml was obtained from the aqueous layer. 17 ml of ethanol and 850 µl of sodium acetate were added to the mixture. Clumps of white strings again formed as the DNA was precipitated. The "snotwad" collected on the sterile rod was significantly smaller in comparison to when it was collected in the isolation of the DNA. It was about the size of a small raisin. The "snotwad" became smaller when it was incubated as the ethanol evaporated and again became more colorless.
Spectrophotometric Analysis of Vibrio fischeri:
The purified DNA was diluted by a factor of 20. The total volume of the diluted solution was 500 µl so 25 µl of DNA were added to 475 µl of TE buffer. Table 1 shows the absorbencies obtained through spectrophotometric analysis of the DNA sample. The final concentration of chromosomal DNA collected in the sample was 1349 µg/ml. This value was determined by first converting the absorbency at 260 nm into the corrected absorbance at 260 nm. The absorbency was multiplied by the dilution factor of 20. This corrected absorbance was then used to determine the concentration of DNA in the sample collected. The value was multiplied by 50 µg/ml to convert AU into µg/ml.
The study was performed to isolate the chromosomal DNA of Vibrio fischeri, purify the DNA, and then determine the contents of the sample extracted to ensure that DNA was indeed extracted and purified. Based on the results of the spectrophotometric analysis of the DNA, the sample was indeed pure and contained an adequate amount of DNA. The absorption at 260 nm was taken to determine the concentration of nucleic acids in the solution. By converting this raw value to determine the final concentration of DNA in terms of µg/ml, the sample was found to have 1349 µg/ml of DNA, which is adequate to continue with the study and extract the lux operon for insertion into E. coli. The absorption at 280 nm was also taken to determine a crude measure of protein and RNA contamination in the solution. The ratio of absorbances at 260 nm to 280 nm was found to determine the level of protein and RNA contamination. For pure DNA, the ratio should be between 1.8 to 1.9. If the ratio was higher than 1.9, the sample was contaminated with RNA and if the ratio was less than 1.8, the sample was contaminated with protein. Table 1 indicates that the ratio was 1.87. Therefore, the sample was pure enough to proceed with the next step. For a better understanding of protein and phenol contamination, the absorbance at 234 nm was also taken. The ratio for Abs234 to Abs260 was used to determine the purity of the sample with respect to proteins. If the ratio was above 0.5, the sample was probably contaminated with proteins. According to Table 1, the 234 to 260 ratio was 0.453, which is less than 0.5, and the sample did not have significant protein contamination. The absorbance at 320 nm was also taken to determine the level of particulate contamination. If this value was greater than 5% of the 260nm reading, then particulates were present in the solution, or the cuvette was dirty. As a result, the reading taken would not be accurate. As shown in Table 1, the absorbance at 320 nm was 0 AU, suggesting that there was minimal particulate contamination in the solution.
Several reagents were required for this lab, including TES buffer, EDTA, lysozyme, proteinase K, SDS, phenol, chloroform, sodium chloride, sodium acetate, ethanol, RNase, and isoamyl alcohol. The TES buffer serves to keep the pH of the solution stable. The EDTA serves two purposes; the first is to chealete divalent magnesium cations, which help keep cell membrane of Vibrio fischeri intact this helps this helps speed up lysozyme action in destroying the cell wall. The second purpose of EDTA is to help inhibit DNase so that the dna is not destroyed. Proteinase K works with the SDS detergent, which dissolves the cell membrane and denatures proteins, to digest proteins and help purify the collected DNA. Phenol, chloroform, and isamyl alcohol are used to remove contamination from lipids, proteins, and sugars, and also to help with defoaming.