There is a large number of species of microbes found on the human body. This bacterial organism are found in the skin, mouth, or nose. This lab consisted of the collection of skin bacterial organisms and amplification of the 16s rRNA to construct a small molecular phylogeny of the human body microbiome, or the community of microorganisms that reside in the epithelia of humans. This information could only be acquired through processes such as DNA extraction, amplification of specific genetic target by the polymerase chain reaction (PCR), agarose gel electrophoresis, restriction enzyme digestion, cloning of DNA fragments into plasmid vector, transformation and blue/white clone colony screening. Through the phylogenetic tree analysis
This lab consisted in the amplification of 16s rRNA gene sequence, PCR based sequencing of forearm bacterial components. The 16s gene is a small subunit of the ribosome with 1500 nucleotides (George, 2013). This 16s rRNA gene sequence has emerged as a preferred technique when analyzing mycobacteria that are isolated (Abbott, 2007). It’s very important because it’s large enough to provide novel pathogens, noncultured bacteria and it can also be used to accurately measure time of evolution, because it has rapidly and slowly evolving regions (George, 2013). It is present in all cells, it has the same function in all cells and it is conserved enough in sequence and structure and accurately aligned. Most importantly there is a large database of aligned sequences available (Abbott, 2007). Skin bacteria also known as skin micro biota are small bacterial organism located in the superficial layer of the epidermis, these bacteria are around 1,000 species from 19 phyla (Todar, 2009). This is a long process that required several steps. The 16s phylogeny gene sequencing methods, included PCR purification and amplification, cloning, ligation, plasmid preparation and sequencing methods. The rRNA genes are amplified via the polymerase chain reaction using primers 27F and 1492R, amplified products are cloned into a plasmid vector, E. coli cells are transformed with the recombinant vectors then individual colonies of transformed E. coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.
There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted...