BISC209/S11: Assignment 209 Lab6

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Wellesley College-BISC 209 Microbiology -Spring 2011

Summary of Theory behind Molecular Tools for Culture-Independent Identification of Bacteria

Due at the beginning of Lab 7
Write a brief summary of the theory behind the following techniques that we used to identify our bacterial species by molecular tools:
Genomic DNA isolation,
Polymerase chain amplification of part of the 16s rRNA gene,
Use of the Zero Blunt® TOPO® PCR Cloning Kit to create a library of unique plasmid vectors with different bacterial 16S rRNA gene inserts,
Transformation and selection of One Shot® TOP10 Competent E. coli Cells that allowed us to select and separate our 16S rRNA genes for sequencing,
DNA sequencing by chain termination, sometimes called Sanger sequencing, (not 454 pyrosequencing)

NOTE: WRITE THIS AS A SINGLE NARRATIVE (including required elements in whatever feels best; include appropriate transitions); DO NOT STRUCTURE AS BULLETED POINTS!

You have already used each of these molecular tools and written about all of them as a Material and Methods section, but you haven't yet been required to explain the theory behind how each of them accomplishes each of the crucial steps toward our goal of identifying unknown bacteria by genus and species name from DNA sequencing. One of the problems in using sophisticated molecular tools is that you can have a very successful lab day, yet it can be mostly "hands on, brain off". Since much of what you have been doing is pipeting, mixing, and incubating of miniscule quantities of liquid reagents that come in kits, it is easy to lose sight of what is actually happening in those tubes or spin columns at each stage. The problem of "doing without knowing" is exacerbated by kit manufacturers who make their reagents "proprietary". That prevents us from knowing exactly what's in them, making it even harder to follow the chemical or physical reactions.

Despite our use of such proprietary kits, it is possible to understand how it all works. All of these tools were discovered by scientists who published their findings. You don't, however, probably need to go to primary literature (Sanger's original paper, for example) to find out how Sanger sequencing works. There are good animations of Sanger sequencing, transformation, pcr, etc. prepared by the Dolan DNA center at [| http://www.dnalc.org/resources/animations/]. Pay particular attention to the difference between a polymerase chain reaction and the Sanger sequencing reactions described. Note that the type of cloning described in the Dolan animations is organismal cloning---not what we are doing. We are doing molecular cloning. A good animation that describes our type of plasmid cloning is found at : | http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html. Wikipedia is also a great place to start to find out some of what you need to know for this assignment. Although it won't be difficult to find out the principles behind Sanger sequencing, polymerase chain reaction, plasmid cloning, making cells chemically competent for transformation, genomic DNA isolation (which pretty much uses the principle of differential solubility of DNA in ethanol), why we picked the 16S rRNA gene for sequencing to differentiate our bacterial species, etc., it will be challenging to condense each tool to essentials in your summary. Being able to distill and write a broad outline, while understanding the specifics, will be important when you describe your experimental design in your final paper.

The users' manuals for the Zero Blunt® TOPO® PCR Cloning Kit might be helpful in getting a better understanding of the specifics of our cloning. You can download it as a pdf file from the manufacturer, Invitrogen's web site at: [1]

Another good source of information is the background information found in this wiki. Be careful about inadvertent plagiarism.

Remember that this summary should be not more than a couple of pages double spaced (or 1.5 spacing). If you are really good at picking out essentials and being concise, you might be able to adequately explain these molecular tools in a page.

The goal of this assignment is to make sure that you have a clear understanding of the biological and chemical basis of these common molecular tools and an appreciation of the complexity of the genetic engineering that went into the creation of our cloning vector and the genetically modified strain of E. coli we transformed.

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