July 16-18, 2013
** you should read ‘The last week in the lab, part I’ before reading this post !!
This post is a summary of the experiments that I conducted throughout the last two months in the microbiology lab at ENS-Cachan. The main idea of my research was to ‘insert’ a promoter into a fragment of chromosomal DNA and then analyze how the promoter affects the growth rate of the cell during replication. A little confusing right? Well, I will try to explain.
A promoter is a regulatory element that initiates the transcription of a particular gene. Transcription is the first step to gene expression, where the DNA gets converted into RNA, and gene expression is when the DNA of a cell is ‘interpreted’ and replicated. Again, very complicated but the main idea here is that promoters can be inserted into chromosomal DNA through a PCR. My first month and a half in the lab I attempted to insert a promoter called rrnBP1 long into the chromosomal DNA of E. coli bacteria. To do this I first had to insert this promoter, by PCR, into a plasmid called pkk and then into a plasmid pdoc before attempting to insert it into the chromosome. The promoter rrnBP1 long was never successfully inserted into the first plasmid, and so after a month we tried a different promoter called phage t7 P5.
To insert the P5 promoter into the chromosome we went through the same steps. We successfully inserted P5 into both plasmids and then the chromosome by PCR. We inserted the P5 promoter into the chromosome at three different positions of the chromosome. The three positions were: the origin of replication (ori), the terminus of replication (ter) and a position on the left side of the chromosome. The origin of replication is the particular sequence of the DNA where the cell begins to copy its genetic information and multiply. Once this begins the cell continues to replicate its DNA throughout the cell until it reaches the terminus of replication. For bacteria the genome, or the entirety of the cell’s genetic information, is constructed in a circle, so the terminus is 180 degrees from the origin. If you go here you will see a picture of where the ori. and ter. are for an E. coli cell.
After the P5 promoter was successfully inserted into the three positions we sent the fragments of DNA to be sequenced at a sequencing lab. The reason that we are testing the different positions is because we are interested in the activity of this promoter at each different position. This will tell us if the cell is uniform throughout its structure, and it will show us if there is a difference between the activity of the promoter at each of the different positions of the chromosome.
Once we sent the fragments of DNA to be sequenced we waited to hear back from the sequencing lab if the base pairs of our DNA fragments were in the order that we expected. The results from the sequencing lab were not as we had hoped. These ‘poor results’ for the sequencing of the DNA fragment means that the machine that analyzed the base pairs of the DNA fragments did not report that our fragments had the sequence of base pairs that we had expected from our theoretical data.
However these results did not stop our experiments. Due to outside pressures, Bianca needed the results of these experiments to be collected and analyzed by the end of the week. She needed to include the results of the activity of the phage t7 P5 promoter in her next grant proposal, and so, correct sequence or not, we continued with the experiments. Bianca explained that there could have been a problem with the machine that sequenced the DNA, and so we hoped that our experiments would still work.
I still don’t know how Bianca could tell whether it was a problem with the machine at the sequencing lab or a problem with one of the 20 other aspects of the experiment. I am also confused about the progression of these experiments. I spent a month trying to insert the rrnBP1 long promoter into the plamids DNA, which never went successfully, and then I moved on to try the phage t7 P5 promoter, which although we thought went successfully we now have data from the sequencing lab telling us otherwise. So how do we know when an experiment is successful? How did Bianca know when to give up trying to insert the rrnBP1 long promoter? What values or assumptions guided her decisions? When does it actually matter if you receive bad results from an experiment? And what does ‘bad results’ even mean? Does it really only mean that the results produced were not as you had predicted from theoretical data?
All of these questions make me wonder why this is considered a ‘scientific’ process. There are values embedded in every result that we get back from a machine, and at every step of these experiments. Maybe we need to re-think the idea that science offers factual data and concrete answers.
So to summarize, the majority of my time in the lab I worked on inserting either the rrnBP1 long promoter or the phage t7 P5 promoter into the plasmids and then the chromosome of the E. coli bacteria cells. This was done through PCRs as well as other reactions that are necessary so that the PCR will work. After we had inserted the phage t7 P5 promoter into the chromosome, we sent the fragments of DNA to be sequenced. After hearing back from the sequencing lab we began with the experiments that gave us information about the activity of this promoter at the different positions in the chromosome.
Anyways, now that you have an idea of how I inserted regulatory elements, the promoters, into fragments of DNA you should check out my post called: ‘The last week in the lab, part III” to see how these fragments were used in experiments that produced data for us to analyze!
-axio