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A 1357.5 μg/ml pure sample of Vibrio fischeri chromosomal DNA, previously isolated, purified and quantified, was digested using Sal I. Upon digestion of the chDNA and the pGEM vector, ligation reactions with varied ratios of 1:1, 2:1, 3:1 and 4:1 were prepared. The four ligations varied in the amount of chDNA inserts present for ligation. To determine the success of both of the restriction digestion and the ligation reactions, the samples were electrophoresed on an agarose gel medium and visualized using the fluorescent properties of ethidium bromide under ultraviolet light. Based on the standard curve created by the lambda ladder, fragment sizes were determined for the bands seen on the gels. The range of fragment sizes appearing in the smear banding representative of fragmented chDNA inserts was 15.2 kb to 3.66 kb. The digested pGEM fragment was 3.48 kb in size, indicating a successful digestion. Also control samples showed that Sal I was digesting properly. The gel image that displayed the ligation samples contained pGEM and inserts before ligation, and those after ligation. The L1 through L4 ligation reactions resulted in bands that appeared to be 17.2 kb, 20.1 kb, 22.4 kb and 23.6 kb respectively, indicating presence of a pGEM vector ligated to a large chDNA insert. The ligation gel electrophoresis results show that all four ligation mixtures may have chDNA fragments ligated successfully to the linearized pGEM vector, but the highest likelihood of a vector with a lux operon insert is seen with the 2:1 and 3:1 ligation ratios. All four samples will be used for transformation of competent Escherichia coli cells in further experimentation.
Upon isolation of 1357.5 µg/ml of purified chromosomal DNA from the bioluminescent bacteria, V. fischeri, digestion of the DNA was conducted using the restriction enzyme, Sal I, for use in the ligation of the DNA to the chosen vector, pGEM, to transform into E. coli. It was hypothesized that after restriction digestion of chDNA and pGEM and ligation of the digested fragments, the samples would be prepared for transformation into E. coli. Restriction digestion was completed with using the restriction enzyme, Sal I. These enzymes are found naturally in most bacteria and have become integral in molecular biology experimentation (Williams, 2003). They are responsible for breaking the phosphodiester bonds that are present along the backbone of DNA, and these specific sequences, at which cleaveage occurs, are generally unique to the individual restriction enzyme (Pingoud et al, 2005). The restriction enzyme requires Mg2+ ions (Tock and Dryden, 2005). The cuts resulting from the restriction digestion leave both a 5' phosphate group and 3' hydroxyl group open and allow for the formation of phosphodiester bonds (Tock and Dryden, 2005). The digestion of the chDNA of V. fischeri allowed for fragmentation of the DNA which would be necessary for the recombination of the DNA with the selected pGEM vector. This type of digestion was also utilized to cut the pGEM plasmid vector.
To recombine the fragmented chromosomal DNA and the cleaved vector, a ligation reaction was carried out. Ligation primarily required an enzyme known as ligase to allow for the formation of covalent bonding (Lehman, 1974). Ligase is a naturally used enzyme that binds Okazaki fragments to one another in DNA replication (Lehman, 1974). While there are a multitude of different types of ligases, they all function in the same manner but may use different energy source cofactors (Wilkinson, 2001). Some require NAD+ while others utilize ATP (Day, 2001). T4 DNA ligase, isolated from T4 bacteriophage, is an ATP dependent ligase, as it is derived from a virus (Wilkinson, 2001). The formation of the phosphodiester bond using this ligase generally occurs in three steps. The T4 enzyme is activated by the production of an AMP-protein complex with phosphate group byproducts (Rossi et al¸ 1997). Following the formation of the AMP-protein complex, the nucleotide is moved to the 5' end of the cleaved DNA and an esterification reaction occurs allowing for the formation of the phosphodiester bond that reforms the backbone of the DNA structure (Rossi et al, 1997). With that, the AMP molecule is released and the ligation reaction is complete (Rossi et al¸1997).
The isolated and now digested V. fischeri chromosomal DNA was ligated into plasmid vector - pGEM-3Zf(+) used for transformation into E. coli. This vector was selected because it generally results in a high copy number (Vizcaíno et al, 1996). The pGEM vector has two polymerase promoters, T7 and SP6, which allow for transcription in two directions (Promega, 2007). It also contains both a resistance to ampicillin and contains the gene for the α-component of β-galactosidase which allows for blue-white screening to determine which plasmids contain inserts and are thus considered clones (Winfrey et al, 1997).
While the pGEM vector was selected based on its characteristics, the restriction enzyme used in restriction digestion was selected specifically for successful ligation and eventually lux positive cloned E. coli. Each restriction enzyme, as aforementioned, has a specific sequence at which it cleaves DNA. The sequence specific to Sal I is 5'-GTCGAC-3' at which the enzyme cleaves double stranded DNA on both strands (Rodicio et al¸1994).Since it is anticipated that the entire lux operon of V. fischeri will be successfully ligated to a pGEM vector and thus transformed into E. coli, the restriction enzyme was chosen to increase the likelihood of the successful transformation of a plasmid with the lux operon, which is approximately 9 kb in size (Meighan, 1988). Thus, to expect fragmentation of the chromosomal DNA to be large enough to include the entire functional lux operon, fewer cleavage sites must be found in the chromosomal DNA. With a mole percent of G + C of 40% in the V. fischeri genome, a cleavage site with mostly guanine and cytosine would result in fewer cuts at the Sal I cleavage site and produce larger chDNA fragments (Winfrey et al, 1997). With both the pGEM vector and chromosomal DNA cutting at the same sequences, the overhangs or sticky-ends will allow for the chDNA fragments and vector to be ligated together.
To determine the successfulness of restriction digestion and ligation, gel electrophoresis with an agarose gel was conducted. This technique is utilized to estimate the number of base pairs in a DNA fragment (Myers et al, 1976). The relationship between the size of the fragment of DNA and the distance traveled is inversely proportional (Myers et al, 1976). The DNA travels through the porous gel based on size. Ethidium bromide is utilized as the staining agent in gel electrophoresis as it interacts with the nucleic acids. The ethidium bromide is interspersed between the bases of DNA and binds to the bases; it fluoresces under ultraviolet light while attached to the DNA and can be documented using a photodocumentation system (Dvortsov, 2006). The agarose gel is prepared with small quantities of ethidium bromide to allow for visualization of the nucleic acids (DNA fragments) that run through the gel.
After restriction digestion, it was expected that the chromosomal DNA would be in the form of many various fragments with many different sizes and would appear on the gel in the form of a smear. The digested vector would appear larger than the undigested vector as the undigested vector can supercoil and move further down the gel. With both undigested and digested chromosomal DNA and vectors run, a comparison was drawn upon to determine whether or not the restriction digestion had been successful. Upon completion of ligation, it was expected that the unligated fragments would run further down the gel than the fragments that ligated to vectors. Additionally, vectors that ligated to themselves would also run further as they would be bereft of the insert and thus have fewer base pairs.
The gel images as observed after both restriction digestion and ligation were analyzed to determine if the samples could be utilized for transformation into E. coli. The first gel, as run after restriction digestion, showed the expected smear for the digested chromosomal DNA sample and digested vector sample as compared to the undigested vector. Fragment sizes for the DNA noted ranged from 15.2 kb to 3.66 kb. Nicked and supercoiled bands were seen for the vector and the sizes of these fragments were 4.05 kb and 1.08 kb, respectively. The gel images seen upon ligation indicated successful ligation as well. Of the four conducted ligation reactions, L1, L2 and L3 all indicated presence of some native pGEM vectors that self-ligated. These bands were about 4.85 kb in size. The L2 and L3 ligations are expected to have the highest likelihood of chDNA fragment ligated to a vector and a better chance of successful transformation. As the result of potential ligations with all four ligation reactions, all samples will be used for further experimentation.
Restriction digestion of V.fischeri Genomic DNA and pGEM Vector
Digestions were set up by calculating volumes using the known concentration of V. fischeri DNA and the total necessary volumes. Tube A, containing digested chDNA with Sal I, had 5 µl of 10X Tris Borate EDTA buffer (Tris base; boric acid; 0.5M EDTA, pH 8.0), 7.4 µl (10 μg) of V. fischeri, 32.6 µl of H2O and 5 µl (50U) of Sal I. Tube B, containing undigested chDNA, had 2 µl of 10X buffer, 0.74 µl (1 μg) of V. fischeri and 17.3 µl of H2O. Tube C with uncut chDNA contained 0.74 µl (1 μg) of V. fischeri and 19.3 µl of TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA).Vector digestion was set up in tubes D and E. Tube D with digested pGEM contained 2 µl of 10X buffer, 5 µl (1 μg) of pGEM, 11 µl of H20 and 2 µl of Sal I. Tube E served as the control for pGEM digestion and contained uncut pGEM. It had 2 µl of 10X buffer, 1 µl of pGEM and 17 µl of H2O. Tubes F and G were the lamda controls. Tube F contained digested lambda DNA and contained 2 µl of 10X buffer, 2 µl of lambda DNA, 15 µl of H20 and 1 µl of Sal I. Tube F with uncut lambda DNA contained 2 µl of 10X buffer, 1 µl of lambda DNA and 17 µl of H2O. All of the tubes were placed in a Beckman Coulter Avanti-JE 20.00 Centrifuge and microcentrifuged for 2 to 3 seconds, and then all but the C tube, which was placed on ice, were incubated in a 37° water bath for 60 minutes. After 30 minutes, all of the tubes were vortexed and microcentrifuged and placed back in the water bath for the remaining 30 minutes. 10µl (2 μg) of digest from tube A containing digested chDNA were transferred to Tube A' and 2 µl (1μg) of vector digest were transferred from tube D to Tube D' and 8 μl of water were also added to this tube. 2 µl of loading dye (bromphenol blue; xylene cyanol; sucrose; 20% SDS; 0.5M EDTA, pH 8.0) were added to the digested chDNA and digested pGEM tubes while 5 µl were added to all other samples serving as controls and the lamda ladder.
A 12 well, 0.8% agarose gel was cast using 0.48 grams of agarose and 60 ml 50X Tris Acetate EDTA buffer (Tris base; glacial acetic acid; 0.5 M EDTA, pH 8.0) and 4µl of ethidium bromide. The gel was run in TAE running buffer which contained 10μg/ml EtBr. The two outermost lanes containing samples (lanes 2 and 11) of the gel were loaded with 5 µl of lambda ladder. Lane 3 was loaded with 12 µl of the digested chDNA with Sal I and lane 7 was loaded with the same amount of the digested pGEM vector sample. Lanes 5 and 6 were both loaded with uncut chDNA but with varying amounts; lane 5 contained 2.5 µl and lane 6 contained 5 µl. Lanes 4, 8, 9, and 10 were loaded with 10 µl of undigested chDNA, undigested pGEM, digested lamda DNA, and undigested lamda DNA, respectively. The gel was run and photodocumented on the UVP Biodocit Photoimaging System transilluminator.
Ligation of V.fischeri genomic digest to pGEM vector
The Sal I digests of the vector and digested V. fischeri DNA were heated for 15 minutes at 65°C. Four ligation tubes, L1, L2, L3 and L4 were set up. L1 had an insert to vector ratio of 1:1, and contained 2 µl (0.1 μg) of digested vector, 2 µl (0.3 μg) of genomic digest, 23 µl of H2O, and 3 µl of 10X buffer. L2 had an insert to vector ratio of 2:1, with 2 µl (0.1 μg) of digested vector, 4 µl (0.6 μg) of genomic digest, 21 µl of H2O, and 3 µl of 10X buffer. L3 had an insert to vector ratio of 3:1, with 2 µl (0.1 μg) of digested vector, 6 µl (0.9 μg) of genomic digest, 19 µl of H20, and 3 µl of 10X buffer. L4 had an insert to vector ratio of 4:1, with 2 µl (0.1 μg) of digested vector, 8 µl (1.2 μg) of genomic digest, 17 µl of H20, and 3 µl of 10X buffer. The ligation tubes were all vortexed and microcentrifuged for 2 to 3 seconds. 5 µl of the corresponding ligation mixtures were transferred to the tubes, L1/T0, L2/T0, L3/T0 and L4/T0, and they were microcentrifuged for 2 to 3 seconds. 1 µl of T4 DNA ligase was added to each ligation reaction (L1-L4 tubes). The ligation reactions were then incubated at 10-12°C overnight.
A 12 well, 0.8% agarose gel was cast in the same way as during restriction digestion using the same reagents. The gel was run in TAE running buffer with the addition of 10μg/ml EtBr. 5 µl of each ligation mixture was transferred to the tubes, L1/Tend, L2/Tend, L3/Tend and L4/Tend, and they were microcentrifuged for 2 to 3 seconds. 1 µl of the loading dye was added to each T0 and Tend tube and they were pulsed for 2 to 3 seconds in the microcentrifuge. Hind III digest of the lambda DNA was heated in a 65°C water bath for 2 to 3 minutes and 5 µl of it was added into wells 2 and 11. The entire contents (6µl) of each of the ligation tubes were loaded to wells 3-10 as follows: well 3: L1/T0, well 4: L1/Tend, well 5: L2/T0, well 6: L2/Tend, well 7: L3/T0, well 8: L3/Tend, well 9: L4/T0, and well 10: L4/Tend. Also 6µl of native living pGEM was loaded into well 12. The gel was electrophoresed at 120V and after completion, a UV transilluminator was utilized to visualize the gel and a photograph was taken.
Restriction digestion of Vibrio fischeri Genomic DNA and pGEM Vector
To create a genomic library of the 1357.5 µg/ml chromosomal DNA isolated and purified from V. fischeri, restriction digestion was conducted with Sal I, a clear colorless restriction enzyme. During restriction digestion setup, Tube A' contained the digested V. fischeri genomic DNA with Sal I. Tubes B and C containing undigested chDNA acted as controls to check for endonuclease contamination and to check if Sal I was digesting properly. Tube D' contained the pGEM vector that would be digested by Sal I while tube E acted as the control to show how undigested pGEM vector would appear. Tube F contained the lambda DNA that would be digested by Sal I to ensure that the enzyme was working properly and tube G acted as the control to show how undigested lambda DNA would appear.
The gel image was documented by a UV transilluminator as seen in Figure 1. The lambda ladders are represented by lane 2 and lane 11; their digestion by Hind III resulted in the formation of 7 bands of 23.1 kb, 9.42 kb, 6.56 kb, 4.36 kb, 2.32 kb, 2.03 kb, and 0.56 kb. By plotting the distance traveled by the lambda ladder fragments versus the log of the fragments' sizes (kb), the standard curve was obtained as shown in Figure 2. It showed a negative correlation in the size of the fragments with increasing distance traveled in millimeters. The linear trend line equation was y =0.0221x + 1.6687 while the R² value was 0.9548. The equation was used to calculate the size in kilobases of the bands obtained in lanes 3-10 by plugging in the distance traveled in millimeters as the x-value. The corresponding size (kb) of each band in every lane is shown in Table 1.
Lane 1: x
Lane 2: lamda ladder cut with Hind III
Lane 3: chDNA digested with Sal I
Lane 4: chDNA undigested
Lane 5: 2.5 μl chDNA uncut
Lane 6: 5 μl chDNA uncut
Lane 7: pGEM cut with Sal I
Lane 8: pGEM uncut
Lane 9: lamda DNA cut with Sal I
Lane 10: lamda DNA uncut
Lane 11: lamda ladder cut with Hind III
Lane 12: X
C:\Users\Kiran\AppData\Local\Microsoft\Windows\Temporary Internet Files\Low\Content.IE5\E6K0Y71Q\UVP01382.JPG
Figure 1. Sal I digestion at 37°C of V. fischeri chDNA, pGEM vector and lambda DNA on 0.8% agarose gel. The lambda ladders represented in lane 2 and lane 11, formed seven distinct bands. The digested chDNA in lane 3 resulted in the formation of smear banding with six fragments of 15.2 kb, 10.1 kb, 7.86 kb, 6.09 kb, 4.49 kb, and 3.66 kb and the digested pGEM in lane 7 formed one thin, distinct band of 3.48 kb.
Figure 2. Standard Curve of V. fischeri and pGEM vector restriction digestion representing a negative correlation in fragment size (kb) to distance traveled in millimeters.
Table 1. Gel Electrophoresis results of the V. fischeri chDNA and pGEM vector restriction digestion, presenting the size in kb of each fragment band.
Lane 3: digested chDNA
Lane 4: undigested chDNA
Lane 5: 2.5 μl uncut chDNA
Lane 6: 5 μl uncut chDNA
Lane 7: digested pGEm
Lane 8: undigested pGEM
Lane 9: digested lambda DNA
Lane 10: undigested lambda DNA
The digested chDNA in lane 3 resulted in the formation of smear banding with six fragments of 15.2 kb, 10.1 kb, 7.86 kb, 6.09 kb, 4.49 kb, and 3.66 kb. The range of chDNA fragments was 15.2 kb to 3.66 kb while the average chDNA fragment size was 6.44 kb. The control for the undigested chDNA in lane 4 resulted in one slightly smeared band at 14.5 kb. Lanes 5 and 6 contained 2.5 μl and 5 μl of uncut chromosomal DNA. The 2.5 μl sample resulted in the formation of one less intense band of 13.1 kb, while the lane with the 5 μl sample displayed a thicker, brighter band of 13.7 kb. The digested pGEM vector in lane 7 resulted in the formation of one thin, distinct band of 3.48 kb. The expected size of the linearized pGEM vector is 3.2 kb but 3.48 kb is close enough to the expected value (Winfrey et al, 1997). The control for the undigested pGEM vector in lane 8 displayed a less intense, thick band at 1.80 kb and a barely distinguishable band at 4.05 kb. The lanes with lambda DNA serve as a control for the functioning of restriction enzyme, Sal I. Digested lambda DNA resulted in the formation of a thick, bright band of 17.2 kb and a less fluorescent band of 13.9 kb; undigested lambda DNA resulted in the formation of a single band of 14.7 kb. As demonstrated by Figure 1, restriction digestion of chDNA and pGEM vector was successful.
Ligation of V.fischeri genomic digest to pGEM vector
The ligation reactions were assembled with different insert to vector ratios (molar ratios)- 1:1, 2:1, 3:1 and 4:1- to increase the chances of the vector fragment and the chDNA fragment joining together in a phosphodiester bond. The digested vector volume remained constant as the digested chDNA volume increased in each successive ratio in the L1, L2, L3 and L4 tubes, respectively. The L1/T0 - L4/T0 served as controls to show what banding should look like before T4 ligase was added. to the original L1 -L4 tubes since the L1/To - L4/To tubes would not receive any DNA ligase. Prior to the addition of the genomic digest, the L1 tube had not been heated to 65°C which would negatively affect its ligase activity. Besides the 1:1 sample, the rest of the samples contained heated ligase.
The lambda DNA ladders were loaded into lane 2 and lanes 11 while the unligated and the ligated chDNA samples were loaded alternately. The gel image was documented by a UV transilluminator as seen in Figure 3.The digestion of the lambda ladder by Hind III would ideally result in the formation of seven bands but according to this gel the lambda ladder resulted in an indistinguishable smear. The standard curve for ligation was created by plotting the distance traveled in millimeters by the lambda DNA fragments with the size (kb) of the fragments. As a result of the paucity of distinct visible bands of the lambda ladder, the sample lambda ladder was employed to generate the standard curve in Figure 4. The fragment size (kb) was negatively correlated with distance traveled (mm) down the gel. The trend line provided the equation y= -0.0229x + 1.7622 while the R2 value was 0.9647. The equation was used to calculate the size (kb) of the bands by plugging in the distance traveled in millimeters as the x- value. The corresponding size (kb) of each band in every lane is shown in Table 2.
L1/T0 in lane 3 resulted in two bands of 18.1 kb and 5.99 kb and the 18.1 kb band was much thicker and brighter than the 5.99 kb band because it represented the chromosomal DNA insert while the less fluorescent band represented the pGEM vector. Since the insert is expected to be larger than the vector, the formation of the brighter, thicker band and the shorter, lighter band coincides with the expected outcome. This result was mirrored for the remaining samples without the ligase added to them. The L2/T0 in lane 5 displayed two bands of 18.1 kb and 6.67 kb. L3/T0 in lane 7 resulted in two bands of 20.1 kb and 6.67 kb while L4/T0 in lane 9 displayed two bands of 7.47 kb and 23.6 kb. The L1/Tend in lane 4 displayed two bands of 17.2 kb and 3.54 kb. The 17.2 kb band was less intense and the 3.54 kb band which represented the self-ligated pGEM vector was even fainter. L2/Tend in lane 6 resulted in two bands of 20.1 kb and 4.14 kb and the L3/Tend sample in lane 8 displayed two bands of 22.4 kb and 4.14 kb. L4/Tend in lane 10 resulted in two bands of 23.6 kb and 4.85 kb.
Lane 12: native living pGEM
Lane 11: lamda ladder cut with Hind III
Lane 10: L4/Tend
Lane 9: L4/T0
Lane 8: L3/Tend
Lane 7: L3/T0
Lane 6: L2/Tend
Lane 5: L2/T0
Lane 4: L1/Tend
Lane 2: lamda ladder cut with Hind III
Lane 3: L1/T0
Lane 1: x
C:\Users\Kiran\AppData\Local\Microsoft\Windows\Temporary Internet Files\Low\Content.IE5\LGBIW5XR\UVP01396.JPGFigure 3. Gel electrophoresis picture of the V. Fischeri chDNA and pGEM vector ligation with T4 ligase. The lambda DNA ladder inserted into lane 2 and 11 displays an indistinguishable smear. The T0 lanes bereft of T4 ligase yielded two bands; one representing the insert and the other the vector. The Tend lanes contain ligase and display insert ligated to pGEM and self-ligated vector.
Figure 4: Standard curve of Vibrio fischeri chDNA and pGEM vector ligation shows a negative correlation in the fragment size (kb) with increasing distance traveled in millimeters of the restriction fragment.
Table 2. Gel Electrophoresis results of the V. fischeri chDNA and pGEM vector ligation, presenting the size in kb of each fragment band.
Lane 3: L1/T0
Lane 4: L1/Tend
Lane 5: L2/T0
Lane 6: L2/Tend
Lane 7: L3/T0
Lane 8: L3/Tend
Lane 9: L4/T0
Lane 10: L4/Tend
Lane 12: native living pGEM
Lane 12 consisted of native pGEM in its supercoiled form with a single band of 4.85 kb which was used as a comparison for the self-ligation vector. It was also noted that the 17.2 kb band in L1/Tend was further down the gel than the 18.1 kb band in the L1/T0 lane. However, the 20.1 kb and 22.4 kb bands in L2/Tend and L3/Tend were slightly higher than the 18.1 kb and 20.1 kb bands in L2/T0 and L3/T0, respectively. The 23.6 kb band in the L4/Tend lane was the same length as the band in the L4/T0 lane. Moreover the 4.14 kb, 4.14 kb, and 4.85 kb bands in L2/Tend, L3/Tend, and L4/Tend, respectively, became progressively lighter until the 4.85 kb band in L4/Tend was barely visible. These bands were similar in size to the supercoiled control, native pGEM, which had a size of 4.85 kb indicating presence of supercoiled pGEM in these samples as well. Also these bands were less intense than the bands representing pGEM in the T0 lanes. In L3/Tend, the band at 22.4 kb was the most prominent band amongst the ligation samples.
A pure concentration of 1357.5μg/ml of V. fischeri chromosomal DNA was isolated to create a genomic library. Restriction digestion of chDNA and plasmid vector pGEMTM-3Zf(+) using the enzyme Sal I was conducted. Using the genomic digest and the vector, four ligation set-ups were prepared with varying insert to vector ratios to assure that T4 ligase allowed for ligation and cloning to occur. Successful restriction digestion and ligation was observed on an agarose gel medium. The entire genomic library will be cloned and transferred into the host organism, E. coli for identification of the pGEM vectors that have been ligated with the lux operon.
Restriction digestion of V. fischeri chDNA was conducted successfully as seen by the corresponding smeared banding pattern on the restriction digestion gel, which was the result of chromosomal DNA and the enzyme, Sal I. The smeared banding pattern indicates that numerous chDNA fragments were present and that Sal I successfully cleaved the DNA. Based on the standard curve derived from the lambda ladder, the sizes of the fragments of all other bands were calculated. Fragments with sizes ranging from 15.2 kb to 3.66 kb were observed. The three control lanes containing undigested chDNA confirmed successful digestion. The lane with just V. fischeri DNA and 10X buffer and no Sal I displayed a single, lightly smeared band. This showed that there was little to no endonuclease activity in the DNA sample, water or restriction buffer (Winfrey et al, 1997). The other two undigested chDNA samples also did not contain Sal I; of the two samples run in different lanes, one was loaded with a quantity of 2.5 µl while the other was 5.0µl. The resulting fragment sizes were determined to be 13.1 kb and 13.7 kb, respectively. The lane containing 2.5 μl displayed a much fainter band than the lane containing 5 µl showing that fluorescence is dependent on the concentration of DNA. A greater quantity of DNA would absorb more ethidium bromide and result in more fluorescence and brighter banding.
Ethidium bromide intercalates into the nucleic acids in DNA and this facilitates its ability to fluoresce (Olmsted and Kearns, 1977). It absorbs ultraviolet light at a wavelength of 302 nm and fluoresces at 506 nm (Winfrey et al, 1997). Fluorescence is dependent on the proton transfer rate upon binding to double stranded DNA, because as ethidium bromide intercalates among base pairs a water molecule is removed from ethidium (Olmsted and Kearns, 1977). As there is a reduction in the transfer rate of the excited proton an increase in fluorescence is observed (Olmsted and Kearns, 1977). The amount of fluorescence is directly related to the amount of DNA present, and this can allow for relative quantification (Winfrey et al, 1997).
Digestion of plasmid vector pGEM was also successful. The lane containing pGEM and Sal I displayed a single band with a size of 3.48 kb, confirming cleavage by the enzyme and presence of a linear pGEM vector. The lane containing uncut pGEM served as a control displaying the approximate size of the uncut vector. A nicked band of 4.05 kb and a supercoiled band of 1.80 kb were observed. Linear DNA consists of cleavage of phoshodiester bonds in both DNA strands, while nicked DNA is undigested as it only has cleavage of a phosphodiester bond in one strand (Webb and Ebeler, 2003). Nicked DNA is more relaxed and cannot supercoil; as a result, it is not as tightly packed it cannot travel as far down the agarose gel as supercoiled DNA (Webb and Ebeler, 2003).
Two control lanes containing lambda DNA were run as controls for vector digest and to determine if Sal I was cutting properly. The two restriction sites on lambda DNA recognized by Sal I were at 32745 and 33244 base pairs (Winfrey et al, 1997). The lambda DNA and Sal I displayed a thick, bright band of 14.5 kb and a much fainter band of 11.8 kb, while uncut lambda DNA displayed a much thinner band of 12.4 kb. The thickness of the cut lambda DNA band was an indication that Sal I did cut the DNA into fragments and they were just in close proximity, since a faint band could be distinguished just under the thick band.
Sal I recognizes the six base pair sequence of GTCGAC on the V. fischeri DNA and cuts at these site to produce restriction fragments (Almashanu et al, 1996). The V. fischeri genome has a very low G+C content so Sal I would cut less frequently (Ruby et al, 2005). The GTCGAC sequence is not present in the lux genes and therefore it increases the chances of having a fragment with the complete operon (Almashanu et al, 1996). The lux operon is located on a 9 kb Sal I fragment so it is possible to be cloned in entirety (Engebrecht et al, 1983). These cleavage sites are fairly spaced out through the genome to allow for cloning of the lux operon (Almashanu et al, 1996). The restriction digestion of chDNA and pGEM with Sal I produces sticky ends which can temporarily bind together and allow for T4 ligase catalyzes a connection between them (Almashanu et al, 1996).
In order to assure successful ligation of chDNA and pGEM, four ligation set-ups were conducted with varying insert DNA to vector ratios. For the L1 ligation with a 1 to 1, insert to vector, ratio it was assumed that to have 0.3 μg of genomic digest, 2 μl were used. This was based on the fact that during restriction digestion, 10 μg of V. fischeri chromosomal DNA was used to get a total volume of 50 μl. Therefore based on a proportion, to get 0.3 μg of genomic digest, 1.5 μl were used. However this value was raised to 2 μl in order to confirm that the reaction went to completion. The plasmid vector, pGEM, was about 3.48kb while it was known that the lux operon containing insert was 9 kb (Engebrecht et al, 1983). Based on this difference in size, to promote the ligation of one vector to one insert, a larger amount of insert must be utilized. Therefore to maximize ligation, three other insert to vector ratios were used (2:1, 3:1, and 4:1). Increasing the ratios any further would not be beneficial due to concatemer formation as inserts would ligate to themselves (Dugaiczyk et al, 1976).
Photodocumentation of the ligation gel, showed that all four ligation set-ups likely had successful ligations that could potentially contain the lux operon. The L1/T0 - L4/T0 lanes were an indication of the reaction before ligation occurred and the L1/Tend - L4/Tend lanes contained the samples that had been exposed to the T4 ligase for ligation. The lambda ladder was not clearly visible on the gel photo as distinct measurable fragments and so the standard curve was made using the data from the sample ladder and utilized for the calculation of the fragment sizes. With a standard curve developed from a sample ladder and not from the one produced on the ligation gel image, the values for fragment size likely will not correspond to the actual fragment sizes. The L1/T0 lane displayed a prominent band of 18.1 kb and a lighter band of 5.99 kb, indicative of separate unligated insert and vector. The L1/Tend lane had very faint bands of 17.2 kb and 3.54 kb, displaying poorer chances for ligation. This band should have been slightly above the 18.1 kb band to indicate successful ligation since the genomic insert and pGEM vector would be of a greater size and travel a shorter distance down the gel medium. However since the size of the self-ligated vector decreased to 3.54 kb, some of the insert may have ligated to the vector. The L2/T0 lane had bands of 18.1 kb and 6.67 kb while the L2/Tend lane did have a band with a size of 20.1 kb, slightly above the 18.1 kb showing that ligation did occur. Ligation also occurred in the L3 set-up which had bands of 20.1 kb and 6.67 kb in lane 7 (the T0 lane) and had a very fluorescent band of 22.4 kb in the L3/Tend lane. The L4 set-up could possibly have ligation but chances are not as high as for those in the L2 and especially L3 set-ups. The L4/T0 lane displayed banding of lengths 23.6 kb and 7.47 kb, while the L4/Tend lane also had a band of 23.6 kb and one of 4.85 kb. This could result in the insert remaining as itself indicative of separate unligated insert and vector but since the pGEM size was smaller in the Tend ligation as compared to the T0, some ligation could have occurred. The expected size of the undigested pGEM vector, at approximately 3.54 kb, is less than the calculated fragment sizes from the gel.
The L1, L2 and L3 all had light banding patterns at around the same location as the control which contained native living pGEM. This control of 4.85 kb was used to compare if any of the vector had self-ligated with the help of T4 DNA ligase. The L2/Tend and L3/Tend lanes both displayed bands that did not travel as far as the insert bands in their respective T0 lanes confirming ligation and chances for having clones containing the lux genes. The L3 lane had the thickest band slightly above the 20.1 kb band with little unligated sample further down the lane, exhibiting the most success of having V. fischeri clones and getting a clone of the lux operon. The digestion of this extracted DNA with Sal I along with the cleavage of pGEM displayed successful restriction digestion. Also, visualization of ligation on the agarose gel and the suggest having clones of genomic DNA indicate a greater probability of L2 and L3 to contain the clone for the lux operon.
Ligation is one of the most important factors in successfully creating a genomic library. Improvements that can be made include varying temperature and adjusting pH. Determining the optimal temperature for ligase while making sure already ligated DNA fragments are not compromised into multiple fragments is essential (Dugaiczyk et al, 1976). Temperature is also dependent on the concentration and size of DNA fragments. Temperatures for optimal chances of ligation increase as the concentration increases and as DNA fragment size decreases (Dugaiczyk et al, 1976). Another factor affecting T4 ligase is pH, which needs to be in the range of 6.0 and 7.8 with a most favorable pH of 7.2 (Murray et al, 1979). To counteract variation ligation reaction buffers are needed, the most common being Tris-HCl. For best ligation results, a high concentration of DNA inserts, ATP, and enzyme are required (Murray et al, 1979).
Successful restriction digestion was conducted of the V. fischeri chromosomal DNA as seen by the smeared banding on the agarose gel, and of plasmid vector pGEM of approximately 3.48 kb band visualized on the gel. The controls confirmed that the samples were free from endonuclease contaminants and that Sal I was digesting properly. Four ligation set-ups were conducted with insert to vector ratios of 1:1, 2:1, 3:1 and 4:1 to improve chances of successful genomic digest to vector ligation. The 1:1 ligation did not indicate recombinant plasmid according to fragment calculations as the fragment representing recombinant pGEM was smaller than the insert itself. The 4:1 ligation also indicated a lower success rate of ligation because the band representative of the recombinant plasmid for the 4:1 sample was also close in size to the insert alone. Successful ligation was observed in the 2:1 and 3:1 ratio samples. The corresponding bands were intense when viewed under UV light and followed the banding pattern to indicate ligation. They will likely have a greater number of genomic clones and greater chances of containing the lux operon. Transformation of the ligated chDNA and pGEM into the host organism, E. coli, will allow for the identification of the presence of the lux operon by screening for bioluminescence through the genomic library. This will allow for ultimately re-isolating the lux genes and performing restriction mapping to determine the orientation of the lux operon within the pGEM vector.
While shotgun cloning is a feasible way of developing a genomic library, it is not the sole method for production of a DNA library. In a study conducted by the Department of Animal and Human Biology at the University of Rome, La Sapienza, the FIASCO method was utilized to isolate and formulate a genomic library of the DNA of Atherina boyeri (Milana et al, 2009). The FIASCO method, or fast isolation by AFLP of sequences containing repeats, allowed for the formation of three partial genomic libraries. These three partial libraries, di-, tri- and tetra nucleotidyl, specific to the sequences of interest that the experimenters were looking to extract and analyze, were determined with biotin labeling probes (Milana et al, 2009). The repeating nucleotide sequences that were found and effectively labeled by the probes were AC, AG, AAT, AAC, GATA and CACC. These fragments were amplified using polymerase chain reaction and were then utilized to isolate eleven different linked loci from the A. boyeri genome. The microsatellite loci were compared to those of various species and were considered the first isolated loci from the family Atherinidae (Milana et al, 2009). This experimentation varies from shotgun cloning as it requires a previous knowledge of the specific sequences that need to be labeled for extraction and formation of a multitude of partial genomic libraries. The shotgun cloning conducted with V.fischeri is far more randomized, and contains the entire genome as opposed to just small, partial sequences of the genome.
In another study, published in Systematic and Applied Microbiology, an entirely novel method of formation of cloned DNA is presented. This method utilized the production of a fosmid library with the genomic DNA of Spirochaeta thermophila by shearing the DNA into approximately 40 kb fragments (Angelov et al, 2009). The fosmid library was propagated within E. coli cells and isolated for transformation into Thermus thermophilus where the xynalase activity of the cells were tested as a screen for the functionality of the transformed components of the genomic library of S. thermophila (Angelov et al, 2009). The screening for xynalase activity showed that when transformed into E. coli and T. thermophilus, more genes allowing for the functionality of xynalase were found in the T. thermophilus cells than in the E. coli cells (Angelov et al, 2009). The screening process is seemingly similar to the ampicillin resistance screening in experimentation. The ampicillin resistance is only conferred in cells that have acquired the necessary genetic information to digest the ampicillin that is in the growth medium on which the bacteria are plated. In the case of the experiment discussed, the functionality of xynalase will only be seen in the cells that have properly conferred components of the S. thermophila genome.
The formation of a cDNA library was utilized by researchers in Japan to determine the target sequences of specific microRNAs in Caenorhabditis elegans. miRNAs generally bind to the untranslated regions at the 3' end of mRNAs, but the function of the sequences to which these miRNAs bind is still unknown (Andachi, 2008). To determine the functions of these sequences, a genomic library was produced with the use of denaturing agents to prevent any obstructions that might stop the reverse transcription of mRNAs to form the cDNA library (Andachi, 2008). The produced cDNA fragments were amplified with polymerase chain reaction with biotin-tagging primers corresponding to the miRNAs (Andachi, 2008). The miRNA-corresponding sequences were cloned and isolated. The researchers determined the miRNA sequence lin-4 and its target sequence lin-14 first (Andachi, 2008). To determine the efficacy of this method of targeting certain clones, subsequent experiments were conducted to determine the presence of lin-14. The frequency of isolation of lin-14 was 78.9% at its peak, indicating this method is highly efficient for isolating target sequences. This same method was then applied to another miRNA, let-7, with resulted in determination of a new target gene, K10C3.4 (Andachi, 2008). The formation of a cDNA library and use for screening of a target sequence is again more specific than the formation of a library via shotgun cloning as executed in the case of V. fischeri and the lux operon. The specific sequences were able to be isolated from the library for further genetic studies. With new methods for formation of genomic libraries still being deciphered and tested for efficacy, other methods may be used in the future for extraction and transformation of the lux operon from V. fischeri into host cells.