For some reason, I can never get more than six or seven hours of sleep. That said, I groggily dragged myself out of bed this morning to meet Dennis and Ali at the Ratty—the cafeteria that just opened this morning.
Then, I went of to my morning lab session. We began the first part of our three day lab in which we will create a recombinant plasmid, grow bacterial cultures containing the recombinant DNA, and then test whether the desired plasmid is actually contained in the cultures.
Today, we did the first step, creating the recombinant plasmid. To do this, we first did a restriction digest of BamhI and HindIII on two separate samples of plasmid DNA, pAMP and pKAN. Next, we incubated this mixture for thirty minutes. Meanwhile, we prepared the agarose gel. When our sample was finished incubating, we added loading dye to the samples, and loaded the samples into the gel. We then ran electrophoresis for about thirty minutes. When the gel was finished running, we took it into the UV photography machine, and captured the DNA's movement through the gel. This was done in order to be certain that the restriction digest had continued to completion. Next, we put the remaining samples of the restriction digest into a sixty five degree heater in order to kill the BamHI and HindIII. Finally, we combined the pKAN and pAMP, and mixed in DNA ligase so that the fragments will combine.
Also, last weeks lab reports were passed back and I received check pluses in both practical applications and theoretical understanding. This was especially pleasing because I've been having a lot of trouble understanding the theory behind DNA manipulation.
In the afternoon session, we went over an article we had been assigned to read which dealt with epigenetics—the link between genes and environment. This was particularly fascinating. Often in science, you hear the debate between nature and nurture. Are some people born more intelligent, more athletic or healthier? Or do environmental factors such as nutrition or a mother's education level determine this? After reading the article, a clear answer resounds—both.
Phenotypic differences between individuals are often caused by differences in the genetic code of individuals. However, genes exist that are not polymorphic—genes that have only one possible arrangement. How does one account for phenotypic differences here? The answer lies in gene regulation. Environmental factors can determine the amount of methylation in genes. For instance, ninety nine percent of non-smokers show partial to complete methylation of the CYP1A1, while only thirty three percent of heavy smokers show methylation. Thus, smoking increases the expression of this gene. Also, drug resistance, which is coded for on MDR1 gene, varies between people. However, there is no polymorphism of this gene. The amount of methylation of this gene determines drug resistance.
Small portions of RNA called micro RNA (miRNA) act as gene regulators. miRNA bonds to mRNA, which carries the information for protein translation, creating dsRNA. However, genes code for the automatic destruction of dsRNA. Thus, by creating small pieces of RNA, the cell is able to stop the production of certain proteins. The amount of miRNA shows a clear correlation with the level of gene expression. Thus, levels drug resistance are also determined by miRNA. Interestingly, miRNA was only discovered in 2007, so the study of this RNA and its regulation is very young. Who knows what discoveries will come next.
Once class was over, I back to my dorm room to catch up on my sleep. Then, it was on to meeting with Ms. Kent, dinner at a different cafeteria that just opened up, and finally studying in the P5 lounge.
Tomorrow, I will be transforming the genetic make-up of E. coli.
Until then,
Joseph Young