DNA and Restriction Enzyme Interaction
:Identification Of An Unknown Plasmid
The fundamental purpose of these experiments is to research the interaction between DNA molecules and restriction enzymes. Two different experiments are performed. In the first experiment, two restriction enzymes, Hind III and Bam H1 interact with an unknown plasmid and the resulting DNA fragments are separated through electrophoresis. Lambda sample is used to create a standard curve, and through it, the sizes of DNA fragments are determined. An uncut sample is used as a negative control, and is compared with cut samples to verify the enzyme activities. The size of the unknown sample is determined to be 4,200 base pairs through the single cut sample. In the double cut sample, the sizes of fragments are determined to be 2,350 and 1,850, which concludes that the unknown sample is pKAN. In the transformation experiment, TE, pAMP, pKAN, and two unknowns are put in five different tubes. E-coli is used as the host bacterium. The TE serves as a negative control because is does not contain DNA. The pAMP and pKAN serve as positive controls and colonize in ampicilin and kanamycin media respectively. The orange-labeled unknown shows identical phenotype with pKAN thus its genotype confirmed to be pKAN. The green-labeled unknown shows identical phenotype with pAMP thus its genotype confirmed to be pAMP.
Modern genetic engineering began in 1973 when Herbert Boyer and Stanley Cohen used enzymes to cut a bacteria plasmid – ring of “extra” DNA found outside the nucleus in many single-celled organisms; the relaxed control plasmid was used in experiments because it produced a lot more copies of plasmid than stringent control plasmid1 – and insert another strand of DNA in the gap2. Both bits of DNA were from the same type of bacteria, but this milestone, the invention of recombinant DNA technology, offered a window into the previously impossible — the mixing of traits between totally dissimilar organisms. To prove that this was possible, Cohen and Boyer used the same process to put a bit of frog DNA into bacteria2. In 1990, a young child with an extremely poor immune system received genetic therapy. Some of her white blood cells were genetically manipulated and re-introduced into her bloodstream. These new cells have taken over for the original, weak white cells, and her immune system now works properly2. Although relatively few people have had their cells genetically altered, these advances have made the prospect of mainstream genetic medicine seem more likely.
DNA electrophoreses is a process in which a DNA strand is cut by a restriction enzyme at a certain point so it can be measured and compared with other DNA2. Electrophoresis begins with the pouring of the gel. The gel is an auger poured into a water bath. Combs are then placed in the forming gel to produce wells where the samples are then placed. The gel is placed in the electrophoresis apparatus and loaded with the samples. The different samples are loaded into the wells formed in the gel. The DNA samples are loaded using a measuring instrument called a micropipeter. There is then an electric current passed through the gel to separate the DNA fragments. The current separates the DNA segments by moving the smaller ones farther than the large ones. Every DNA sample separates differently. This way they can be compared with others. It is highly unlikely that two samples will have the same results4.
For about 50 years, antibiotics have been the answer to many bacterial infections. Antibiotics are chemical substances that are secreted by living things. Doctors prescribed these medicines to cure many diseases. During World War II, it treated one of the biggest killers during wartime – infected wounds3. It was the beginning of the antibiotic era. But just when antibiotics were being mass produced, bacteria started to evolve and became resistant to these medicines3.
Bacterium uses the Restriction enzyme as a form of defense mechanism3. Restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. It is used to defend against bacterial viruses called bacteriophages, or phages. A phage infects a bacterium by inserting its DNA into the bacterial cells so that it might be replicated. The Restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. In the bacterial cell, Restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensable tools of recombinant DNA technology2.
Restriction enzymes recognize certain site (base pair) of an invading foreign phase DNA molecules. If the hypothesis is true, then certain restriction enzymes such as Hind III and Bam H1 will digest certain site of DNA molecules when exposed with foreign phages like pAMP – ampicilin plasmid – and pKAN – kanamycin plasmid. Using given information by the plasmid map cut sizes on p.142 in the Lab manual, if the certain phage is pAMP (4539 base pairs), Hind III will cut the pAMP DNA at the specific site of 1904 base pairs from the origin and Bam H1 will cut the site of 1120 base pairs. If only Hind III is used, the circular DNA will be cut once and turn it into linear DNA leaving the 4539 of base pairs unchanged. If Hind III and Bam H1 are used simultaneously, the DNA molecules will be cut into two pieces with base pairs of 705 (1904 1120) and 3834 (4539 705) respectively. If the certain phage is pKAN (4207 base pairs), Hind III will cut the pAMP DNA at the site of 2292 base pairs from the origin and Bam H1 will cut the site of 417 base pairs. If only Hind III is used, the circular DNA will be cut once and turn it into linear DNA leaving the 4207 of base pairs unchanged. If Hind III and Bam H1 are used simultaneously, the DNA molecules will be cut into two pieces with base pairs of 1875 (2292 417) and 2332 (4207 1875) respectively. Using pAMP and pKAN as positive controls, unknown plasmids can be identified. An uncut sample is used as a negative control, which will then be compared with restriction enzyme cut samples. The uncut sample might produce several bands. These bands, however, are not probably due to fragments from restriction enzyme digestion, but due to different degrees of coil of the double helix structure of the DNA. A standard sample Lambda will be used to create a standard curve because it will produce seven different fragments that are cut by Hind III.
If certain plasmid successfully transforms a bacterium, the bacterium will have the specific characteristics that the plasmid DNA will code. If the DNA has resistance gene for certain antibiotic, the transformed bacterium will survive in the antibiotic medium. If this hypothesis is true, then when certain plasmids such as pAMP and pKAN are injected in certain host bacterium such as E-coli under proper transformation conditions, the bacterium will be transformed and have characteristics that the DNA codes. Transformation is a very sensitive process; therefore proper plasmid, host bacterium, and medium are critical. Luria Bertani (LB) medium is a non-selective nutrient rich medium that suggests a standard growth environment. If pAMP, which has resistance gene for ampicilin, transforms E-coli, E-coli will survive and colonize in ampicilin medium, but will not survive in other antibiotic media such as kanamycin. If pKAN, which has resistance gene for kanamycin, transforms E-coli, E-coli will survive and colonize in kanamycin medium, but will not survive in other antibiotic media such as ampicilin. TE that does not contain any DNA serves as negative control and will not colonize in any antibiotic media. pKAN and pAMP serve as positive control and will colonize in KAN and AMP respectively.
Materials and Methods
Pour 0.8% Agarose Gel into electrophoresis tray and wait 30 to 40 minutes to allow the gel to solidify after making sure there is no leak. Four DNA samples are used including three treatments. First sample U is the uncut sample plasmid in its natural state. Second sample S is the single cut sample by the HindIII restriction enzyme. Third sample D is the double cut sample by both HindIII and Bam H1 restriction enzymes. The Lambda sample is cut by HindIII into many pieces, which is used to create the standard curve. This sample must be kept on ice. Place each sample carefully over the well, and make sure the power switch is turned off. After lining sample successfully, cover the lid and turn the power on, don t forget to match the color of cords: red-to-red and black-to-black. For about 30 to 45 minutes, allow the samples move across until right before the first one reaches the last red band. Examine DNA fragments after the TA dyes DNA using Ethidium Bromide and move the gel to the UV transilluminator. The TA provides photograph of the gel. Measure and analyze the distances between fragments and create the standard curve. Using figure3 on p.142 of Lab manual, identify the unknown plasmid.
For a transformation to happen, five steps are necessary. 1. preincubation 2. incubation 3. heat shock 4. recovery 5. plating. BS 111l prep staff have done the preincubation step. 5ul of five different samples in microfuge tubes are provided. The samples include TE, pAMP, pKAN, and two different unknowns. TE does not contain DNA. pAMP is resistant to antibiotic Ampicilin while pKAN is resistant to Kanamycyn. 100ul of competent cells are put in each tube and incubated on ice for thirty minutes. During the critical heat shock process, the tubes are heat shocked precisely for 45 seconds in 42oC water bath and transferred back to ice for two minutes. After adding 0.9ml of LB broth in each tube, place the tubes in 37oC water bath for 60 minutes for recovery. Transfer 100ul of each sample to LB broth, AMP, and KAN plates respectively making sure to use a new tip for each DNA sample on pipettor. LB broth is the standard growth medium while AMP and KAN are antibiotic media. Using hockey stick, perform confluency streaking and incubate for 24 hours at 37oC. Measure and analyze the numbers of bacterial colony and identify the unknown samples.
As shown in Figure 1, the negative contol uncut sample shows three bands. The standard sample, Lambda shows seven different bands. The experiments, single and double cut samples show one and two bands respectively. The size of seven different framents of the standard sample Lambda is shown in .
Distance migrated (mm) 12.5 13 14.5 16 17.5 22.5 24.5
Size of standard DNA fragments in base pairs 23,130 9,416 6,557 4,361 2,322 2,027 564
Using the data from , the standard curve is created and is shown in figure 2. The numbers of base pairs of the samples are determined through the standard curve. The uncut sample migrated 12, 14, and 17mm that correspond to base pair number of 17000, 10000, and 4200 respectively. The single cut sample migrated 17mm that corresponds to base pair number of 4200. The double cut sample migrated 19 and 20.5mm that correspond to base pair number of 2350 and 1850 respectively.
Sample Growth on LB Growth on AMP (number of colonies) Growth on KAN (number of colonies) Ampicilin Result Kanamycin Result Comment
TE Lawn 0 0 Negative Negative (-) control
pAMP Lawn 1256 0 Positive Negative (+) control
pKAN Lawn 0 416 Negative Positive (+) control
Unknown (orange) Lawn 0 440 Negative Positive PKAN
Unknown (green) Lawn 1168 0 Positive Negative PAMP
In the standard growth medium, LB, all the samples show lawn as shown in the . The negative control TE, which does not contain DNA show no growth except LB. A positive control pAMP shows 1256 colonies in the ampicilin medium. Another positive control pKAN shows 416 colonies in the kanamycin medium. The orange-labeled unknown shows 1169 colonies in the kanamycin medium. The green-labeled unknown shows 440 colonies in the ampicilin medium.
Electrophoresis utilizes electric attraction to move DNA fragments, therefore, the bigger and heavier DNA fragment, in other words fragment with more base pairs travels relatively slower than fragments with fewer base pairs in given time. The numbers of base pairs in this lab are estimated from the standard curve created by using the fragments Lambda sample. The negative control uncut sample shows three bands that are distinguished from cut samples in their locations as expected. It shows that other samples are fragmented by the restriction enzymes. The single cut sample is measured to have 4200 base pairs that are highly close to pKAN s expected number of base pairs of 4207. The double cut sample is measured to have 2350 and 1850 that also are incredibly close to pKAN s expected numbers of 2332 and 1875. Shown high resemblance of their number of base pairs, it is evident that the given sample is pKAN.
In transformation experiment, negative control TE fails to survive in any antibiotic media as expected. The positive control pAMP and pKAN survive and colonize in AMP and KAN respectively as expected. The orange-labeled unknown shows similar number of colonies, or phenotype, with pKAN in kanamycin, therefore its genotype is proven to be pKAN. The green-labeled unknown shows identical phenotype with pAMP, therefore its genotype is proven to be pAMP.
The main problem that I faced during these experiments was the clarity of DNA fragments bands. They were too small that I could not tell if some of them were actual bands or not. This can be a critical problem, since this kind of experiment requires highly sophisticated measurements. Since Ethidium Bromide uses the charge of DNA molecules to stain them, one can use this property of DNA to design a computerized instrument that allows more accurate and precise measurements in future.
Also in future, utilization of the DNA – Restriction enzyme interaction will be a lively research field through out the world. Although gene therapy is still experimental, in other ways genetic research already is changing how medicine is practiced. This is because of the genetically engineered drugs that are now available through biotechnology5. Take, for example, the treatment of diabetes. In the past, the only way to get insulin for diabetics was to process it from pigs and cattle. Then researchers learn how to make insulin by cloning the human gene that carries the instructions for making insulin.4 Cloning and other techniques of genetic engineering are having many positive results. Genetic engineering is helping increase the supply of medical products and lower their costs. It results in new drugs being created. Another benefit of genetically engineered materials is their purity. This is important, since there have been cases in the past where medical products processed from animals or human donors carried disease.5
Minneapolis, MN; Lerner Publications, 1997
3. Lewis, Ricki. The rise of antibiotic-resistant infections
FDA Consumer. Sept. 1995. p. 11-15.
Brookfield, CT; Milbrook Press, 1995, General DNA