Identification of Mystery Plasmids

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Identification of Mystery Plasmids
Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used make many copies of a particular gene. The aim of this experiment was to identify three mystery plasmids based upon their characteristics; such as size, antibiotic resistance, lacZ profile and conjugative properties. The results obtained showed that plasmid number2 was the pDSK519 plasmid and its size was 26229.58 Bp. Plasmid pDSK519 also was found to be resistant to kanamycin only and it possessed the LacZ gene. pDSK519 had the oriT gene but it did not have the tra gene. Plasmid number 22 was found to be pHSG298 as it had a size of 9767.08 Bp and it was found to be resistant to kanamycin only. It also possessed the LacZ gene and conjugation results showed that it did not possess the oriT gene the tra gene. Plasmid number 12 was found to be pRP1:: Tn501 as it was resistance to all three antibiotics. It was also found out that this plasmid did not possess the LacZ gene as evidenced by the white colonies. pRP1:: Tn501 had oriT gene as well as the tra gene as shown in the conjugation results. The use of plasmids in very beneficial towards humans and these types of experiments contribute greatly towards this. Introduction

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA (Chandrasegaran, 2008). In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms. Plasmid size varies from 1 to over 200 kilo base pairs (Russell and Sambrook, 2001). Plasmids can be considered to be independent life-forms that tend to form symbiotic relationships with their hosts, as they endow their hosts with useful packages of DNA to assist mutual survival in times of severe stress. For example, plasmids can convey antibiotic resistance to host bacteria, who may then survive along with their life-saving guests who are carried along into future host generations. Moreover, plasmids can be considered to be mobile genetic elements that can move between genomes since they are often associated with conjugation, a mechanism of horizontal gene transfer (Barkay and Smets, 2005).

Conjugation, or mating, is a process of genetic transfer that requires cell-to-cell contact (Barkay and Smets, 2005). The genetic instructions for conjugation are encoded on a double-stranded, circular piece of DNA. The circular DNA exists in the bacterial cell entirely separate from the much larger bacterial chromosome. This is known as the conjugative plasmid or the fertility factor (Snyder et al., 1997). Cells that possess it are donor or male cells, and those that lack a conjugative plasmid are recipient or female cells (Snyder et al., 1997).

There are multiple genes involved in the process of conjugation. Some of the genes code for a surface structure found on donor cells, the sex pilus (Snyder et al., 1997). This is a threadlike tube made of protein. The sex pilus recognizes a specific attachment site on a recipient cell. When the donor cell comes near a recipient, the sex pilus attaches to the specific site and begins to retract, pulling the two cells together (Snyder et al., 1997). As the two cells draw close, their connection stabilizes and their outer membranes fuse together to allow the transfer of DNA from one cell to the other (Snyder et al., 1997). Only one of the two strands of DNA making up the plasmid passes through the fused membranes into the recipient cell. Thus, DNA synthesis must occur in both donor and recipient to replace the missing strand in each (Snyder et al., 1997). The genes encoding the enzymes responsible for this part of the conjugative process are also found on the plasmid. Once passage and synthesis are successfully completed, both donor and recipient cells contain a whole double-stranded, circular, conjugative plasmid...
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