Subcloning procedures were performed to obtain a recombined plasmid that presented a favored gene. A double restriction digest was completed to attain an isolated and purified vector/plasmid DNA and insert. The digested vector and insert were purified from a 1% agarose gel. The ligation of an insert containing ampicillin resistance, and a vector capable of independent replication were recombined and transformed into competent bacteria, such as E. coli. To determine the presence of the recombinant DNA from the transformed bacterial cells the recombinant gene was screened in gel electrophoresis with another restriction digest enzyme.

Introduction:

Subcloning is a technique used to produce recombinant DNA. A DNA fragment that contains a gene of interest is inserted into a vector/ plasmid DNA which can replicate independently of chromosomal DNA to produce recombined DNA. Subcloning experiments can be useful for finding treatments to various illnesses, such as diabetes. Diabetes is a lifelong disease marked by high blood sugar levels; it can be caused by too little insulin, resistance to insulin, or both (Ari S. Eckman). In experimental trials, animal insulin has been modified for human use; the insulin gene is inserted into a suitable vector, the E. coli bacterial cell, to produce insulin that is chemically identical to human insulin. This has been achieved using Recombinant DNA technology (Recombinant DNA Technology in the Synthesis of Human Insulin). In order for DNA to recombine, isolation, purification, quantification, digestion, electrophoresis, ligation, transformation, and screening must for all intents and purposes be performed. Isolation and purification of the vector and insert of choice use the alkaline lysis method, the cell wall is opened and plasmid DNA is released, RNA and protein that may contaminate the insert and vector DNA are removed. The amount of DNA that was isolated is determined by quantification. Digesting the DNA sequences with restriction enzymes allows for the excision of insert and vector DNA at proper sites, which help determine the purity of the DNA samples with the use of agarose gel electrophoresis. Ligation of the insert to the vector is performed and then transformed into competent cells and grown on plates containing a selectable marker. Screening determines if the procedures mentioned were successful; the recombinant gene is screened by isolating DNA by doing a restriction digest (Schramm). In this particular experiment, the vector should take in the insert gene containing ampicillin resistance, and bacterial growth should be seen in the presence of ampicillin, and this recombinant DNA will be able to undergo isolation.

Experimental Procedure:

DNA was isolated and purified from a bacterial culture of E. coli with a vector/plasmid DNA sequence from another bacterial culture of E. coli containing ampicillin resistance, as an insert. To isolate the DNA from the E. coli cells an alkaline lysis, with SDS detergent, was used along with COLD lysis solution. The lysate was incubated at room temperature for 3 minutes and then centrifuged, then washed with wash buffer. The DNA was eluted with water, and underwent centrifugation twice and was then used for quantification. Two vectors and two inserts were used to increase the chances of obtaining purified vector and insert. For quantification, 5 tubes were used, 1 blank (water), 2 separate tubes each containing inserts, and 2 separate tubes containing vector. The blank contained 1000ul of water, each of the 4 tubes contained 5ul of vector DNA and insert DNA and 995ul of water. A spectrophotometer was used to determine the concentration of DNA, and the absorbance. Calculation of the volume of vector and insert was done to obtain 5ug of DNA to be digested by restriction endonucleases Xba1 and BamHI.

A 1% agarose gel was used to load the vector and insert DNA; electrophoresis was done by running the gel for an hour at 120V. Vector1 and insert1 were excised out of the gel as they were highest in concentration of DNA, and then weighed. Three volumes of DNA binding buffer was added to every volume of gel slice and incubated at 50oC for 15 minutes. An original gel slice volume of isopropanol was added and mixed by repeated pipetting. The samples were spun, and the filtrate was discarded. Wash buffer was put in and centrifuged and the filtrate was removed. Centrifugation, elution with 20ul water, and another round on centrifugation was done.

Next, ligation of the insert to the vector was done by joining 100ng of vector with no insert (a1:0 ratio), with an equal molar concentration of insert (a 1:1 ratio), and then 3 times the concentration of insert with the vector (a 1:3 ratio). To calculate the volume of the vector and inserts, the molar ratio of insert to vector was determined. We changed the volumes to 10ul of deionized water and added ligation buffer and mixed. T4 DNA ligase was added and mixed, then incubated at room temperature for 15 minutes. Later, 5ul of the ligation mixture was transferred into a microfuge tube. Competent cells were added to the DNA and gently mixed by pipetting up and down. The mixture was incubated on ice for 30 minutes and heat shocked for 2 minutes at 37oC, and then cooled on ice for 5 minutes. Luria broth was added, followed by incubation of the cells for 1 hour, 37oC. These mixtures were added to and spread on the appropriate plates. The plates were then incubated at 37oC overnight.

Screening and Purification were performed on the recombined DNA. Bacterial culture was pelleted, the supernatant was removed. This step was repeated with COLD lysis buffer, followed by constant vortexing. The lysate was incubated at room temperature for 3 minutes, and then centrifuged. Wash buffer was added to the tube, followed by centrifugation, removal of the filtrate, decant, and then centrifuged again. The sample was eluted with 30ul of water and centrifuged. Then, DNA quantification was performed. Restriction digest, with the enzyme HindIII, of the quantified recombinant DNA, with a master mix of BSA, Restriction buffer enzyme buffer, and restriction enzyme A, in one microfuge tube was performed. Four more tubes were filled with 3ul of DNA each, and 4ul of H2O, and 3ul of master mix into each of the 4 tubes. These tubes were spun for 1 minute, and incubated at 37oC for 30 minutes. Lastly, 2ul of 5xdyes was added to each tube, so they can be used in electrophoresis, in 1% agarose gel.

Results:

After isolation and purification of the vector and insert DNA, DNA quantification was performed to obtain the concentration of both samples. These concentrations can be seen in table1. To gain an understanding of what our actual restriction digest should look like after its gel purified, we performed a virtual digest with the restriction endonucleases Xba1 and BamH1, seen in figures 1a and 1b. Our actual gel purification showed more than one fragment for vector and insert, they were cut in multiple sites, as seen in figure2. Then ligation and transformation were performed using T4 DNA ligase to ligate the insert to the vector and transform them into competent cells. As shown in figure3, there was growth on all of the plates except the 1:0 ratio plate. Conversely, growth should not have occurred in the negative control. Then isolation with HindIII enzyme and purification with gel electrophoresis was performed to screen for recombinants that were ligated during transformation. As seen in figure4, one band was obtained for lane 4, and two bands were obtained for lanes 5, 6, 7.

Discussion:

After going over the results, it is evident that the recombination of DNA did occur, however instances of contamination have been detected. Based on our results, in quantification, the obtained concentrations of vector DNA were insufficient and the concentrations of insert DNA were abundant. The absorbances of the vectors were in between the given ratios of 1.8 and 2.0, indicating sufficient presence of DNA. Whereas the inserts absorbances were above the given ratios indicating that these samples are contaminated with RNA. Since our DNA samples were impure they were replaced with purified vector and insert samples. This was followed by gel purification of the vector and insert, which illustrated two bands for the vector and three bands for the insert. This suggests that our DNA was not linear and was cut at multiple sites, our insert DNA was shown with three fragments and our vector DNA was shown with two fragments. These samples were replaced with double digested and gel purified samples which were ligated with T4 DNA ligase and transformed into competent cells. The transformation was done to make the cells competent, or capable of acquiring a plasmid that contained amp-resistant gene. Four out of five of the ampicillin containing plates these cells were spread on showed bacterial growth. Growth should not have been seen on the negative control, this indicated that the water was contaminated. The 1:0 plate did not show growth which indicated that the vector DNA was digested by the endonucleases. Growth on the positive control indicated that the competent cells could take up DNA. Then an agarose gel electrophoresis of HindIII digest was run, which showed one band for each DNA sample indicating that digestion occurred at the 4.4 kb fragment. In lanes #5, #6, #7 an additional band was present indicating protein contamination. The fragment in lane #4 indicated that there was only vector DNA present and no recombinant DNA, two fragments seen for lanes #5, #6, #7 which indicated that both vector and insert DNA were present and recombinant DNA (Bryan).

Conclusion:

Even though the recombinant DNA was obtained, it was impure. Review of the results show there were contamination issues that led to impurities. The contamination of DNA could have resulted from improper isolation and purification of protein. Inaccurate amounts of buffer could have resulted from incorrect use of the micropipetter to obtain set amounts of lysis and wash buffer. Nonetheless, in the end the DNA was recombined as seen in the results of the screening procedure with HindIII, two DNA fragments were present in the gel which indicates that the recombinant sample was digested by the enzyme and the desired sequence was present, since the enzyme cut at two sites. Regardless of outcome of this experiment, the methods and procedures carried out served as practice in laboratory technique.

Results Figures:

Table1. DNA Quantification of Isolated and Purified DNA

DNA samples

Concentration (ug/ul)

Absorbance (260nm:280 nm)

Vector 1

0.092

1.86

Vector 2

0.095

1.87

Insert 1

0.297

2.5

Insert 2

2.155

2.3

These results show the concentrations and absorbance ratios of insert and vector samples. The vectors are in between the given ratios of 1.8 and 2.0, indicating sufficient presence of DNA. Whereas the inserts absorbencies are above the given ratios indicating that these samples are contaminated with RNA.

Figure1. Virtual Digest

Insert DNA b. Vector DNA

http://tools.neb.com/NEBcutter2/TMP/757ac9d0-.4d7d492d8d9d8.gel.gifhttp://tools.neb.com/NEBcutter2/TMP/757ac9d0-.4d7d488c4e236.gel.gif

The figures above illustrate a virtual digest of what our actual restriction digest should look like. Both insert and vector DNA were cut with enzymes BamHI and Xba1, in a 1% agarose gel. The presence of two DNA fragments, following the scale of a 1Kb ladder; indicate that both vector and insert were cut at two sites for proper isolation.

Figure2. Gel Electrophoresis for Restriction Digested Vector and Insert

Gel 23.jpg Lanes 1(mark) 2 3 4 5 6 7 8 9 10 11 12

Gel purification of the restriction digested, by enzymes XbaI and BamHI, vector and insert ran in 1% agarose gel electrophoresis, following a 1Kb marker scale. The insert and vector are present in lanes #7 which is the vector and lane #8 which is the insert. The size of the vector is 4.5Kb, there was not enough sample to calculate insert size. Lane 7 has two fragments and lane 8 has 3 fragments, which suggests that restriction endonucleases cut at multiple sites.

Figure3. Transformation of Recombinant DNA in LB plates with Ampicillin.

PMlbra.JPG

The recombination of the AMPR gene was confirmed, by placing ligated and transformed recombined DNA into cultures of bacteria, in LB and ampicillin media on the plates. Growth is seen in the Positive control, the Negative control, the 1:1 plate, and the 1:3 plate. No growth on the 1:0 indicates the presence of digested vector. Growth on the negative control indicates contamination of water.

Figure4. Screening for Recombinants

Gel 24.jpg (1Kbmark) 2 3 4 5 6 7 8 9 10 11 12

Gel purification of the restriction digested recombinant DNA, by the enzyme HindIII; vector ran in 1% agarose gel electrophoresis, following a 1Kb marker scale. The four DNA samples are present in lanes 4, 5, 6, 7. The DNA sample in lane 4 contains one fragment indicating that DNA was not cut by HindIII. The DNA samples lane 5, 6, 7 indicate that the DNA was cut at two sites; however they contain fragments above 4.4Kb which can be a sign of protein contamination.