Identification of Unknown Plasmid

I. Title

Identification of an Unknown Plasmid

In this experiment, we determined the phenotypic capability of an unknown plasmid along with its size. With the use of gel electrophoresis, we analyzed the gel photograph by using a standard DNA marker, Lambda HindIII, and came to a conclusion based on our results.

II. Abstract

Two experiments were done to identify an unknown plasmid. The success of these experiments came from the use of modern day technology involving gel electrophoresis. First, bacterial transformation to E. Coli DH5 was performed on our unknown plasmid along with two known plasmids, pAMP and pKAN, and a negative control TE, a buffer without DNA. By performing confluency streaking of bacteria in plates containing antibiotics, we were able to examine the recombinant DNA of the bacteria. After incubation of the plates, we analyzed the samples and found that our unknown plasmid reacted positively on the LB/AMP plate. There were a total growth of three colonies on the LB/AMP plate and a negative result on the LB/KAN plate. With this data along with the positive reaction of pAMP on the LB/AMP plate, we came to the conclusion that our unknown plasmid was pAMP. In our next experiment, we analyzed the DNA via gel electrophoresis. First, we had to treat our unknown plasmid. Three treatments were performed: Uncut (U), single cut (S) with HindIII, and double cut (D) with HindIII and Bam H1. The gel was then stained with Ethidium Bromide, often used in chromatography, in order for us to view the gel under UV light. A photograph of the result was then printed out. This allowed us to determine the migration of each sample along with the number of base pairs in each fragment. Standard fragments of DNA were used to determine the size of our unknown plasmid, which at this point was pAMP. With the use of both pKAN and pAMP plasmid maps, we were able to solidify our conclusion that the unknown plasmid was pAMP.

III. Introduction

The advancements in biotechnology have expanded the limits of research. In our experiments, recombinant DNA allowed us to isolate DNA for the transportation to other foreign organisms so we could investigate the effects of the DNA on the foreign organism. Recombinant DNA is used in many ways that aid humans and their everyday lives. The extracting of DNA from one genome and transporting it into another, has helped such health issues as diabetes by the making of insulin for diabetics. Not only has the use of recombinant DNA been essential for health issues, but it has also advanced modern day agriculture through the process of pest control and the hormone injection of farm animals. Though some consider this a separate health issue, it has helped with the improvement of growth regulation in animals (1). With the improvements in technology and recombinant DNA, research has expanded to the lengths of cloning and genetic alteration.

In order for DNA to be transported from one organism to another, the use of restriction enzymes is needed (2). These enzymes distinguish foreign DNA, and degradation, or cutting, begins. An organism, or vector, is then needed to transfer the specific fragments from its host organism to another. To further understand recombinant DNA, we were given known samples of DNA, pAMP and pKAN, which are plasmids that carry an antibiotic resistance gene, and an unknown sample. pAMP carries an ampicillin resistance gene that produces a beta-lactamase protein which disables ampicillin within the cell; meaning plasmids containing this gene would grow on an AMP plate consisting of a nutrient-rich medium, LB (LB/AMP). pKAN carries a kanamycin resistance gene that produces a phosphotransferase protein which blocks kanamycin's ability to bind to a ribosome, also meaning that a pKAN plasmid in bacteria would grow on LB/KAN plates. Plasmids are another form of a vector, and we used the two to replicate DNA and analyze the unknown plasmid through a series of experiments.

Transformation is one of three bacterial mechanisms of DNA recombination through the transfer of genetic material in which free DNA of a genotype is taken in through the cell surface of bacteria of another and is integrated into the foreign cell chromosome. The other two bacterial mechanisms, conjugation and transduction, are options with certain circumstances, but can be inconsistent under normal conditions. Because of this, transformation is the ideal and most commonly used mechanism when transferring naked DNA. In our first experiment we plated three samples of DNA, pAMP, pKAN, and an unknown, along with a TE buffer without DNA onto the LB, LB/AMP and LB/KAN plates. We hypothesized that a positive growth of our unknown on LB/AMP would mean that our unknown was pAMP, but if the growth were on the LB/KAN then our unknown was pKAN.

After transformation is done, phenotypic changes can be viewed by way of gel electrophoresis. Phosphate groups making up the backbone of the double helix in DNA contain negatively charged oxygen, making electrophoresis a primary way of sorting the DNA by size. Restriction enzymes, HindIII and Bam H1 were treated to our unknown DNA. Three treatments were done to better analyze our samples: Uncut, single cut, and double cut. This was a necessary procedure because it helped to determine the sizes of the cut and uncut plasmid samples compared to a standard lambda marker. The samples of DNA were then placed in a well containing 0.8% agarose gel, which is ideal for efficiently separating fragments. After the DNA fragments were separated by electrophoresis, the change in fragments were observed by the staining of ethidium bromide (EtBr) and viewed under UV light. After viewing the photograph of our gel, we had enough data to begin concluding on whether our unknown plasmid was pAMP or pKAN. Plasmid maps of pAMP and pKAN were given, informing us the number of base pairs each fragment contained when cut with a particular enzyme. After viewing the fragments of the three samples, we compared the data to the standard, lambda DNA fragments. For the lambda fragments, the number of base pairs for each fragment was given. With this data we were able to make a prediction on our unknown plasmid by comparing the length of migration of the standard fragments to the uncut, single cut, and double cut samples.

IV. Materials and Methods

Our research on recombinant