Restriction Digestion Lab Report

BIO 219
Group 1
Section 66
October 19, 2012

The extraction of purified DNA from A. fischeri by restriction digestion using
Sal I enzyme and pGEM for shotgun cloning

Introduction:

The ultimate goal of this experiment is to isolate the lux operon, a targeted piece of DNA that causes bioluminescence, from Aliivibrio fischeri and insert it into the DNA of Escherichia coli in order to make it glow. A. fischeri is a gram-negative bacteria which participates in a symbiotic relationship with many marine organisms (Perry et al., 2005). This particular bacterium has the feature of bioluminescence, which is regulated by a ~nine-kilobase fragment of their chromosomal DNA, the lux operon (Slock et al., 2000). The lux operon contains a group
of genes, lux I, lux R, lux C, lux D, lux A, lux B, and lux E, which code for proteins that regulate the processes of bioluminescence. The activity of the lux operon is regulated by a process known as quorum sensing, in which cell density controls gene expression (Greenberg et al., 2008). Transcription of the gene lux I produces the auto-inducer, N-acyl homoserine lactone, which diffuses out of the cell. As the cell density increases, more auto-inducers are produced. The inducer diffuses back into the cell and binds with the transcription factor of lux R initiating transcription of genes lux C, lux D, lux A, lux B, and lux E (Schaefer et al., 1996). The reaction that produces bioluminescence in A. fischeri involves luciferase, flavin mononucleotide (FMNH2), a long chain fatty aldehyde (RCOH), and excess oxygen (O2). The genes lux A and lux B produce the alpha and beta subunits of luciferase respectively. Lux D codes for Acyl-transferase, lux C produces Acyl-reductase, and lux E creates Acyl-protein synthetase. These three enzymes work together to produce the aldehyde necessary for the reaction. Luciferase contains the active site in which the substrates FMNH2, RCOH, and O2 bind and produces light (including biproducts RCOOH, water, and FMN), which allows the specified bacteria to luminesce (Alves et al., 2008).
In order to isolate the DNA from A. Fischeri, it was necessary to utilize spin columns. The negatively charged silica membrane in the columns allow the DNA to create a cationic bridge with the columns as a result of the lysis buffer that was added to the DNA. A resuspension buffer, proteinase K, and RNase was also added to denature the sample. During centrifugation, the bridge between the DNA and the membrane enabled excessive contents to be discarded. Addition of various solutions, including ethanol, wash buffer 1 (AW1), wash buffer 2 (AW2), and water, between centrifuging allowed ultimate re-naturing of the isolated DNA. After isolating the DNA, ultraviolet spectroscopy was used to determine the concentration and purity of the A. fischeri DNA sample. A spectrophotometer is a device that emits light through a sample and determines the absorbance of it. To obtain a precise absorbance reading of each sample, it was necessary to include a cuvette of DNA grade water that acted as a blank and had an absorbance reading of zero for each wavelength. The concentration of DNA in the sample was calculated by using the Beer-Lambert Law which states that absorbance is directly proportional to the extinction coefficient, times the concentration of the sample, times the light path length (A= εcl) (Ricci et al., 1994). Since DNA has the highest absorbance at 260 nm due to its nitrogenous bases, the reading at this wavelength was used as the absorbance, A, in the Beer-Lambert Law equation. The extinction coefficient, ε, was given as 0.020 μg/ml-cm, and the light path length was 1 cm. Solving for c determined the concentration. Assess the purity of each sample, the absorbance readings at each wavelength were recorded and compared to ideal values. Results from the spectrophotometer showed that the optimal 260nm wavelength had an absorbance of 4425ng. In addition to individual readings at each wavelength, it was necessary to calculate the ratio at 260nm/280nm and 260nm/230nm to determine possible protein and RNA contamination. The results from the 260nm/280nm readings were 1.28. The ratio for the 260nm/230nm shows a ratio of 1.
Restriction digestion was used to properly break up the isolated DNA, so that the target piece of DNA containing the lux operon and remaining fragments could be inserted into separate phagemid vectors, and transferred into the E. Coli bacteria. This process, also known as shotgun cloning, was used to create the genomic library of A. fischeri. An enzyme was required to cut the vector and digest the DNA into fragments of an appropriate size. Sal I, the chosen enzyme, cuts at a recognition site that is heavily populated with cytosine-guanine bonds. This is ideal since the DNA of A. fischeri has a low number of cytosine-guanine bonds, and the digestion with Sal I will result in larger strands of fragmented DNA. More importantly, the lux operon does not contain any cytosine-guanine bonds, so it will remain intact throughout the process.
To observe the effects of the restriction digestion, electrophoresis was performed.
A 0.8% agarose gel was used as the medium for the DNA solutions because it has a better separation of large DNA fragments due to the larger pores throughout the gel. The electrodes on each end of the apparatus allowed the fragments to migrate across the gel. The opposite end of the DNA-containing wells has a positively charged electrode that attracts the DNA, which is negatively charged due to its phosphate backbone. Electrophoresis confirmed whether or not the DNA samples were digested properly by observing the lengths of the fragments in accordance to their position on the gel after completion of the
technique.

Material and Methods:
Experiment Part 1: Extraction of A. fischeri DNA
1.5 microcentrifuge tubes containing an A. fischeri DNA pellet were used. Place into the tube 180 μl of Resuspension Buffer (Buffer ATL), containing detergent, and 20 μl of proteinase K enzyme. Mix the combination on the vortex machine. Place in a 56 degree Celsius bath for an hour, until the solution becomes clear. When the hour is up add 4 μl of RNase A (100 mg/ml). Then add 200 μl Lysis Buffer (Buffer AL), contains chaotropic salt (guanidinium chloride).Vortex for 30 seconds. Afterwards add 200 μl of 100% ethanol to the 1.5 centrifuge tubes. Vortex the microcentrifuge tubes for another 30 seconds. Micropipette the mixture (including any precipitate) into a DNeasy Mini spin column. Discard impure solution. Put the spin columns into the 2 ml collection tubes, and centrifuge at max speed for 1 minute. Remove the flow-through and collection tube.
Wash to remove the rest of the contamination and reassemble the split DNA into a whole strand is third task. Take the DNeasy Mini spin columns, centrifuge, and add 500 μl of Wash Buffer 1 (AW1). Position the spin columns in new collection tubes and run them in the centrifuge machine for another minute. Discard flow-through and collection tube. Place the spin columns 2 ml collection tube, and add 500 μl Wash Buffer 2 (AW2), which is comprised of ethanol. Centrifuge for 3 min at max speed. Discard flow-through and collection tube. Allow the alcohol to evaporate by placing the mini spin columns into new 2 ml collection tubes and spin dry with the column lid open in the centrifuge for 2 minutes before proceeding. Discard the microcentrifuge tube.
Place the spin columns into clean 1.5 ml microcentrifuge tubes. Place the tubes aside to incubate at room temperature for 2 minutes with the lid open on the column. The remaining ethanol should evaporate, if not the purity of the DNA will be impacted. After 2 minutes pipet 100 μl of DNA grade water directly onto the DNeasy membrane. Microcentrifuge for 2 minutes at max speed to complete elution. Once centrifuged, the DNA isolation and purification is complete. Label tubes and store at -20°C.
Experiment Part 2: Spectrophotometric Analysis of DNA Samples
First turn on the spectrophotometer. Vortex the 1.5 ml microcentrifuge tubes, containing the isolated chromosomal DNA, for 30 seconds. For each DNA sample micropipette into a fresh cuvette, careful not to touch the clear sides, 475 µl of DNA grade water and add 25 µl of the DNA solution to obtain a 1/20 dilution of the chromosomal DNA sample. With the leftover DNA store at -20° C. Create a blank, as a control, by pipetting 500 µl of DNA grade water into a clean cuvette, when tested it should be read as zero. Load each cuvette into the rotating cell holder of the spectrophotometer. The arrow on the cuvette should be facing the operator when in position to measure absorbance. Place the blank (water containing cuvette) in the spot marked as “B” and to distinguish each group member’s sample from one another, record the number of the cell everyone is inserted into. To work the spectrophotometers select ATC, and then set the mode to absorbance. The starting wavelength should then be programed to 260 nm. The first cuvette to be read is the blank, “B” position. After reading the absorbance for the blank continue the readings for the rest of the cuvettes by rotation, making sure to record the numbers displayed. To construct an absorbance spectrum take absorbance readings every 10 nm from 220 to 320 nm for each sample, always start with the reading of the blank, before continuing with the readings of the other cuvettes. Plot the data in a graph where wavelength is on the x axis and absorbance values are on the y axis, (Absorbance vs. Wavelength). The purity is found using the Beer-Lambert Law (A = εcl). To find the concentration of DNA in each sample, first correct the measured absorbance for the dilution. To do so calculate the absorbance ratios for A260/280, A260/230 and record the absorbance of 320 nm to ensure the quality and purity of each group members DNA sample. Then use the corrected absorbance to calculate the concentration of your chromosomal DNA sample by using the Beer-Lambert Law. Dump the cuvettes and contents. Thus ends the second part of the experiment.
Experiment Part 3: Restriction Digestion
Take eight sterile 1.5 ml microcentrifuge tubes and label them from A1 through G, then place them in a rack, in order. The volumes needed for 1.5 µg (tubes A1 and A2) and 0.5 µg (tubes B and C) of genomic DNA chosen from the sample group (the most pure group DNA sample), need to be determined along with the volumes of Sal I, H2O, and TE buffer. Once the values are found record and add to find the total volume. Add the assigned components to each tube using calculated volumes. The components are added in the following order; buffer, water, and DNA, pGEM/lambda/TE, then Sal 1. DNA and Sal 1 need to remain on ice. All the tubes received 2 µg of 10x buffer. Of the water that was pipetted, 12 µg is added to A1 and A2, 9.7 µg was added to B, 12.5 µg was added to D, 17 µg was added to E and G, and 15.5 µg was added to F. Of the DNA sample, 1.5 µg is added to A1 and A2, .5 µg was added to B and C, and D through G received 0 quantities. D and E receive 5 µg and 1 µg. F and G get 2 µg and 1 µg Lambda. C gets 9.7 µg TE. Cap and microcentrifuge for 30 seconds. Sal 1 is added last; add 10 units to A1 and A2 and 5 units to D and F. The total volume for tubes A1 and A2 are 40 µg, and tubes B through G are 20 µg. Place tubes A1-G, except tube C which goes into an ice bath, into a floating microcentrifuge tube holder and incubate at 37° C for 3-4 hours. Afterwards store at -20C. Create agarose gel that will be used to verify the chromosomal DNA has been properly digested. Produce a 250 ml Erlenmeyer flask containing 0.48 g pre-weighed agarose-LE. Add 60ml 1X TAE buffer to the flask and churn. Place an inverted 25-ml Erlenmeyer flask on top of the 250 ml flask and microwave to boil for 2-3 minutes, with occasional swirling in between, to insure that the agarose has completely dissolved.
Let cool to about 60° C and add the 6 µl of Gel Red. Lightly mix solution. Place the gel tray into the casting tray and gently add the agarose mixture into the casting tray. Insert a 12 tooth comb in the appropriate slots in the casting tray, to create wells. Let the gel solidify.
Experiment Part 4: Gel Electrophoresis
Take solidified gel with tray and place in electrophoresis chamber, making the wells face the black negative electrodes. Add 1X TAE buffer liquid to the chamber till it cover gel tray/wells. Take 10 microcentrifuge tubes and label accordingly; 8 tubes, ‘A1’gel’ through ‘G’gel’, 1 tube ‘λ std.’ and one tube ‘1 kb’. To the tubes add the following; from tubes ‘A1’gel to ‘G’gel’ add 5 µl of their corresponding digestion created in Part 3 (example: ‘A1’gel receives 5µl of A1 digestion). In tube ‘λ std.’ add 10 µl of λ, and in tube ‘1 kb’ add 10 µl of 1 kb, (these will be the ladders for the gel). Tubes ‘A1’gel’ through ‘G’gel’ will receive 5 µl of H2O, and all tubes will get 2 µl of loading dye. The total volume for each tube should equal 12 µl. after loading of components place all tubes in the microcentrifuge and spin for 30 seconds at max speed. Start the loading of the wells in order (‘λ std.’, then ‘A1’gel to ‘G’gel’, then ‘1 kb’) with the ladders being on the outsides. There should be 12 wells in gel, start the loading on the 2nd well, add the corresponding load volumes. Well 2: 10 µl of load volume, wells 3-4: 12 µl, wells 5, 7-11: 10 µl, and well 6: 4 µl. place lid on the electrophoresis and connect power, (voltage set to 120 V). Stop power when the bromphenol blue dye has covered ¾ of the gel. Remove lib to remove gel. Image gel by placing in Gel-Doc System Image Lab; select ‘new protocol’, ‘nucleic acid gels’, ‘gel red’, and run. Save image on Publication JPEG. Applications:
The purpose of the study was to see if bioluminescence is advantageous to bacteria by attracting fish. This study showed that zooplankton that feeds on the bioluminescent called Photobacterium leiognathi bacteria started to glow in digestion of fish. Bacteria are able to survive in these conditions because of the symbiotic relationship with their host having a safe environment and food. P. leiognathi strain was used and only ones that had the ability to glow in the dark after mutagenized with N- methyl- N – nitro- N – nitrosoguanidine. Luminescence was recorded a luminometer with the settings of a dark room. The way the study was preformed was that brine shrimp was isolated in a beaker with the bacteria. The brine shrimp would feed on the bacteria and results showed that the brine shrimp were able to glow after ten seconds with the bacteria as well as two and a half hours. Feeding rates and glowing are measured under IR illumination with a video camera. Next, the study showed that fish, Apogon annularis were placed in the dark room setting with the brine shrimp with the bioluminescence ability and easily spotted the glowing prey. The concluding results found in the study showed that any glowing prey were easier to spot and feed on than the non-glowing prey. Results were found using a camera recording all findings in the study. It was interesting to find that the bioluminescence was even found in the feces of the predator, which provided good evidence that the bioluminescence was able to withstand environments inside of the intestines of the prey (Zarubin et al., 2011)

In a second article, the lux operon of V. fischeri was examined in order to discover a way to create increased luminescence. The lux operon is a know succession of genes (luxABCDE and -G) in the V. fischeri genome that controls bioluminescence through a positive feedback pathway. This means when more 3-oxo-C6-HSL is present, the more the lux operon will be transcribed. One lux operon comes from an isolated strain from E. scolopes called ES114. This usually has a much dimmer luminescence compared to that of the V. fischeri. This strain was used in this study in order to identify regulators that affect the expression of the lux operon. To do so, transposon mutants were made by transferring pEVS170 to a vector containing the ES114 gene, and various other plasmids were made with mutant properties. These colonies were allowed to grow in petri dishes and then the luminescence of different colonies were observed and quantified. It could be seen that the luminescence in the usually dimmer ES114 gene was upregulated (more than 1,000 fold) in certain scenarios. Some of which included the insertion into arcA, arcB, and guaB genes. This study showed that not only can a V.fischeri cells’ environment (i.e surrounding cells and presence of autoinducers) affect the bioluminescence, but also regulatory pathways within the cell itself.(Lyell et al., 2010).

Lastly, in relation to the external regulatory pathways of bacterial cells, the quorum sensing ability of Staphylococcus aureus cells were investigated. Quorum sensing is supposed to be cell-density dependent and in this study other mannerisms of the regulatory pathway were investigated, such as diffusion rates and production of autoinducers. Specifically, individual S. aureus cells were isolated so that the effects of having a lone cells on self-induction to remain alive could be seen. Individual S.aureus cells were confined within a matrix and media could be added. The ability of AIP (the autoinducer) to build up around the cell was then evaluated using green fluorescent protein. The results of this experiment showed that quorum sensing can be induced when an S. aureus cell is in isolation because of the build up of the cell’s own extracellular AIP. The proposed mechanism to do so involved upregulation of the agr effector molecule RNA III. Cell viability is also increased due to quorum sensing which comes from this agr molecule. This experiment brings up the question of if lone cell luminescence induction can be considered quorum sensing, or is it more of a diffusion capability. It does however answer the question that quorum sensing does in fact help the viability of cells and may be a fact that can be applied in many medical and biological applications (Carnes et al., 2010).

Works Cited
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Carnes, E. C., Lopez, D. M., Donegan, N. P., Cheung, A., Gresham, H., Timmins, G. S., &
Brinker, C. J. (2010). Confinement-induced quorum sensing of individual staphylococcus aureus bacteria. Nature Chemical Biology, 6(1), 41-5. doi: http://dx.doi.org/10.1038/nchembio.264

Greenberg, EP, Schaefer, AL, Hanzelka, BL, and Eberhard, A. 1996. Quorum Sensing in Vibrio fischeri: Probing Autoinducer-LuxR Interactions with Autoinducer Analogs. Journal of Bacteriology (178): 2897-2901.

Lyell, N. L., Dunn, A. K., Bose, J. L., & Stabb, E. V. (2010). Bright mutants of vibrio fischeri
ES114 reveal conditions and regulators that control bioluminescence and expression of the lux operon. Journal of Bacteriology, 192(19), 5103-5114. doi: http://dx.doi.org/10.1128/JB.00524-10

Perry, LL, Bright, NG, Carroll, RJ, and Scott, MC. 2005. Molecular characterization
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