June 16-18, 2013
I have been postponing this account of my experiments because it’s hard to explain microbiology to nonscientists, especially as a non-scientist myself! But one of the goals of my internship is not only to see if this is possible, but also to explore whether or to what degree disciplines need to have their own distinct language. The languages that are unique to each discipline in academia prevent outsiders from engaging with disciplines. A philosopher should be able to explain her time in a microbiology lab to a non-scientist even if she has not mastered the technical microbiology vocabulary that a ‘true’ scientist would insist on using. Or so I hope….
So I am going to attempt to explain to you my experiments in the lab in three different posts. The first post, which you are reading now, will give you the background necessary to understand the main idea of the experiments that I have conducted over the past two months. The second post will explain my experiments that designed the DNA fragments and then the third post will explain the experiments that analyzed these fragments. However, please keep in mind I am still struggling to explain these experiments in everyday language, and that I am jumping into the deep end of microbiology with no experience in the shallow end.
I am working with E. coli, which is a type of bacteria that lives in your gut. For bacteria cells there is DNA stored in both the plasmids and the chromosome inside of the cell. Here is a quick explanation of plasmids and chromosomes:
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms. Plasmids are considered transferable genetic elements, or “replicons”, capable of autonomous replication within a suitable host.
(that’s not very simple, is it?)
A chromosome is an organized structure of DNA and protein that is found in cells. A chromosome is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.
The major general differences include:
Plasmids have much less base pairs than chromosomes
Plasmids are rarely organized by chaperone proteins
Plasmids are easily transferred
Plasmids usually contain non-essential genes
Plasmids function can be lost or gained without harming the organism
Plasmids are usually found in “lower” organisms
After understanding the functions of the plasmids and the chromosome, the question becomes how can we change the base pairs of these fragments of DNA in both the plasmids and the chromosome? The set of base pairs that make up the strand of DNA is called the sequence of the DNA, and these base pairs are the building blocks to the DNA. They are designated by specific hydrogen bonding and they are abbreviated as: A,T,G and C. The A and the T base pairs bond together and the G and the C base pairs bond together to form the sequence of DNA in both the plasmids and the chromosome.
In the lab, by a PCR, it is possible to unwind these strands of DNA and ‘insert’ new regulatory elements into the plasmids and the chromosome. A PCR is a method based on a cycle of heating and cooling the DNA fragments to replicate the DNA with the desired fragment embedded into the strands of DNA. In these reactions you use primers, short fragments of DNA, that contain sequences that are complementary to the region along the strand of DNA that you are trying to amplify (wiki). Amplification is a term used in the process of DNA replication and refers to artificially increasing the number of copies of a DNA fragment through the replication of the fragment. By heating and cooling the DNA you can melt the DNA, open or unwind the double helix and adhere your desired fragment on to the DNA fragment. After the PCR you can check to see if your fragment was properly inserted. The fragment of DNA has GFP, which is fluorescent. This means that after the PCR you can, with the aid of a black light, check to see if your fragment was inserted into either the plasmids or the chromosome.
After replicating the DNA with the desired fragment inserted into the chromosome you send this fragment off to a sequencing lab where they will tell you the order of the base paris in your fragment. This can be checked with theoretical data of what the sequence should be according to our knowledge of how base pairs bond with one another.
Now that you have an idea of how you can insert regulatory elements into fragments of DNA you should check out my post called: ‘The last week in the lab, part II” to see how these experiments actually went in the lab!