Identification of Unknown Plasmid Code Named 681A18 By Cody Latham Plasmids are small circular, doublestranded DNA molecules commonly found in bacteria that are separate from the chromosomal DNA found in a cell. Generally they encode genes that are not required for basic life or cell replication, but instead encode for "accessory genes." These genes may be beneficial to the survival of the cell like a resistance to an antibiotic, or the ability to survive in extreme conditions like high temperatures. Plasmids replicate separately from a cells chromosomal DNA and a cell may have just one copy of the plasmid or may have hundreds of copies. Additionally bacteria use plasmid DNA to share acquired traits and mutations between organisms. In biotechnology plasmids are often engineered to carry genes that have been taken from one organism and insert and express them in another organism. This was most notably first done in 1978 when researchers with Genentech isolated the insulin producing gene in humans and were able to express it in E. coli providing a way to mass produce insulin for sale to diabetics worldwide. In this experiment I was given an unknown plasmid and was asked to determine which one of three potential plasmids I had. I did two digests of the unknown plasmid. A single digest with one restriction enzyme, and a double digest that was done with two restriction enzymes paired together. The restriction enzymes are used here to cut the DNA of the plasmid at certain known sequences of nucleotides separating the circular plasmid DNA into linear fragments of DNA of varying sizes. Gel electrophoresis was then used to separate these DNA fragments based on their size in base pairs (bp). DNA being negatively charged will move towards the positive side of the gel electrophoresis chamber when a current is ran through a buffer in the chamber, and the agarose gel provides the resistance necessary to separate the fragments of DNA. The smaller DNA fragments will travel further through the gel in an electric current than the larger fragments of DNA will in the same amount of time. Along with my digested plasmid DNA, a DNA marker or ladder was also ran simultaneously. This marker contains linear fragments of DNA of specific known lengths of base pairs. This allows for a direct comparison of my DNA fragments with those of known sizes in the marker to help determine the size of my fragments of DNA. The restriction enzymes that I chose to use will give me different enough sizes of DNA fragments for each of the three potential plasmids that I could have, so I can then easily identify which plasmid that I do have based solely on the sizes of DNA fragments present. Accomplishing this took every skill and technique that I had learned in the past four months as well as allowed me to try a few new
techniques and some changes that I felt would give me the best results that I could ask for. Materials and Methods In order to run a pair digests I first had to figure out what restriction enzymes I would use as well as which buffers to pair them with. For that I enlisted the help of the internet. DNA Learning Center's 1 website provided the genomes for the plasmids pamp, pkan, and pblu. I was then able to use New England Biolabs 2 website to do a virtual digest of each of the plasmids with a variety of different restriction enzymes and choose which ones I wanted to use for both my single and double digest of the unknown plasmid. Finally I used Fermantas' 3 website to find out which buffer to use with each of the my chosen enzymes. Now I had the enzyme BglI paired with the buffer O chosen for my single digest and had the enzymes PstI and PvuI paired with the buffer R chosen for my double digest. I needed to have 350ng of my unknown plasmid DNA code named 681A18 for each restriction digest, but since my plasmid was already in a solution, I used the NanoDrop to find out the concentration. With a concentration of 141.0 ng/µl, I calculated that I would need 2.482µL -which I rounded off to 2.5µL- of the solution to have 350ng of the plasmid DNA. The last thing I needed to do was to label the four microcentrifuge tubes I would be using for the digests. Two would be the controls, one for each of the buffers 'O' and 'R', and two for the actual restriction digests 'B' and 'P'. Restriction Enzyme activity (%) Buffers B G O R PstI 50-100 50-100 100 100 PvuI 0-20 20-50 50-100 100 BglI 0-20 50-100 100 100 Table 1: The percentage of activity a particular restriction enzyme has within a given buffer. Highlighted values are the recommended buffer. Taken from the Fermentas 3 website. Each of the four microcentrifuge tubes was loaded 2.5µL of the plasmid DNA solution and 2µL of the determined buffer. Tubes 'O' and 'B' got Fermentas buffer O and tubes 'R' and 'P' got Fermentas buffer R. Tube 'B' was then loaded with 1µL of the enzyme BglI and tube 'P' was loaded with 1µL each of the enzymes PstI and PvuI (all three also from Fermentas). All four tubes were then bought to a total volume of 20µL with dh 2 O. The four tubes were then placed in a 37 C water bath for about two and a half hours. After incubation, the tubes were frozen to be stored until the following week when they would be ran on an agarose gel. A 1X TAE solution would be needed several times to complete the project so a 200mL 20X stock solution was made to dilute from as needed. This 20X TAE solution was 800mM Tris-acetate (combined Tris base and acetic acid) and 20mM EDTA. With Tris having a formula weight of 121.14g/mol and EDTA a formula weight of 372.25g/mol, I calculated that for 200mL of 800mM concentration I needed 19.382g of the Tris base and for 200mL of 20mM concentration I needed 1.489g of EDTA. The Tris base was dissolved in a beaker containing approximately 150mL of dh 2 O. Added to the beaker was 4.6mL of glacial acetic acid followed by the EDTA. After all was dissolved the total volume was brought up to 200mL with dh 2 O. The 20X TAE solution was then taken to a calibrated ph meter and was determined to have a ph of 8.37.
A 50mL 0.8% agarose gel was prepared by diluting 2.5mL of my 20X TAE stock with 47.5mL of dh 2 O and combining that with 0.4g of agarose. This mixture was then microwaved on high for about one minute until the agarose was completely melted and then poured into a prepared gel tray set with a 6-tooth gel comb and allowed to solidify. As the gel was setting up, 500mL of 1X TAE was made by taking 25mL of my 20X stock solution and bringing it to a total volume of 500mL in a graduated cylinder. Once the agarose gel was set up it was put into an electrophoresis chamber and covered with the 1X TAE solution. The four previously prepared digests now had added to them 4µL each of the 6X loading dye. 20µL of each digest as well as 6µL of the Fermentas O'GeneRuler marker were loaded into the wells of the gel. The gel was then ran at 130V until the yellow dye band from the loading dye was about ½ inch from the bottom of the gel. The remainder of the prepared 500mL of 1x TAE solution (approximately 265mL) was combined with 15µL of Ethidium Bromide in a square plastic container. The ran agarose gel was stained in this solution for 30-35 minutes for the DNA bands to become visible under UV light and photographed. Results and Conclusions Figure 1 is the picture taken of the agarose gel that I ran my digested plasmid through. Lane 1 contains the marker and more detailed information on each of the DNA bands is found in Table 4. Lanes 3 and 5 contain the digested plasmid DNA. Lane 3 has three bands of DNA with band 1 being between 3000 and 3500bp, band 2 between 1000 and 1500bp, and band 3 with fewer than 250bp. Lane 5 also has three bands of DNA. Band 1 is between 2000 Figure 1: Picture of agarose gel taken under UV light. Lane 1: Marker DNA fragments. Lane 2: Control 'O'. Lane 3: Plasmid digested with BglI. Lane 4: Control 'R'. Lane 5: Plasmid digested with PstI and PvuI. Lane 6: Blank and 2500bp, band 2 is between 1000 and 1500bp, and band 3 between 750 and 1000bp. Although bands 2 in both lanes are between 1000 and 1500bp in length, clearly the band in lane 3 is smaller as it traveled a little bit further through the gel. During the early part of the experiment I chose which restriction enzymes I wanted to digest my unknown plasmid with and Table 2 shows the sizes of fragments of DNA that I would get for each of the three potential plasmids. Table 3 is the estimation of the DNA fragment size seen in Figure 1 based on the relationship between the bands of DNA in lanes 3 and 5 with the known bands of the marker DNA in lane 1. Also Table 3 has the distance (mm) that each band of DNA traveled through the agarose gel that can be used with the graph of the standard curve in Figure 2.
Distance Traveled (mm) Fragment sizes (bp) from digestion with BglI pamp 158-1118-3263 pkan 261-794-3139 pblu 1576-1740-2121 Fragment sizes (bp) from digestion with PstI and PvuI pamp 896-1314-2329 pkan 545-572-923-2154 pblu 125-160-197-453-480-726-1316-1980 Table 2: (Above) Virtual digest done on the three potential plasmids taken from the New England Biolabs 2 website. Table 3: (Below) Estimation of size made on the unknown plasmid DNA based on the relation of the bands of DNA with the marker DNA as well as the distance (mm) each band traveled through the agarose gel. DNA fragments from unknown plasmid 681A18 Digest with BglI Digest with PstI&PvuI Estimated size (bp) Distance traveled Estimated size (bp) Distance traveled (mm) (mm) 3000-3500 22 2000-2500 25.5 1000-1500 31.5 1000-1500 30 <250 43.5 750-1000 33 Analyzing the data in these figures and table shown above and below I can only come up with one conclusion. The bands of DNA in lane 3 are clearly in line with that of the expected sizes of DNA fragments from the plasmid pamp shown in Table 2. Lane 5 also matches up directly with pamp on Table 2, all three DNA bands. Therefore, plasmid code named 681A18 is in fact the plasmid pamp. 45 40 35 30 25 20 15 10 5 0 Standard Curve for Figure 1 y = -7.661ln(x) + 84.301 R² = 0.9959 0 2000 4000 6000 8000 10000 DNA Fragment Length (bp) Figure 2: (Above) Standard curve graph from the DNA marker. Values used are taken from Table 4. The R 2 value of 0.9959 indicates a very reliable trendline. Table 4: (Below) Data taken from the marker lane of the agarose gel and used to graph a standard curve (see Figure 2). Numbers in bold are labeled in Figure 1. Marker Lane (lane 1) DNA fragment size (bp) Distance Traveled (mm) 10000 15 8000 16 6000 17.5 5000 19 4000 20.5 3500 21.5 3000 22.5 2500 24 2000 26 1500 28.5 1000 32 750 34 500 37.5 250 41 References 1 Website 2 Website DNA Learning Center. Internet: <http://www.dnalc.org/resources/plasmids.html>
3 Website New England Biolabs. Internet: <http://tools.neb.com/nebcutter2/> Fermentas. Internet: <http://www.fermentas.com/en/tools/doubledigest>