Degree project in Biology, Master of Science (2 years), 2021
Examensarbete i biologi 45 hp till magisterexamen, Uppsala universitet, 2021 Biology Education Center and Department of Cell and Molecular Biology Supervisor: Prof. Leif Kirsebom
Does Ms1 gene expression contribute to the high Ms1 RNA level in the stationary phase?
Shuowen Cao
Mycobacteria include a large number of pathogens, known to cause serious diseases in mammals. Mycobacterium tuberculosis is the most famous one, among them which causes over 1 million deaths every year. However, its close relative Mycobacterium marinum is an opportunistic pathogen that is safe for healthy people and therefore suitable as a model system. Ms1 RNA (Mycobacterium smegmatis small RNA 1) is a recently characterized small non-coding RNA. A non-coding RNA does not produce any proteins, but it usually functions as a regulator in biological processes. It appears in some Mycobacterium species and expresses at a high level in response to stress conditions such as starvation, heat shock, acidic environment, ethanol, etc. Despite its essential regulatory function, there is still much to learn about Ms1 RNA. After inoculating a fresh flask of medium with a small number of mycobacteria, the mycobacteria try their best to take in the nutrients in the medium and replicate as much as they can. This period is called the exponential phase because the number of the mycobacteria in the flask increases exponentially. The large amounts of mycobacteria take up the living space and decrease the accessibility of the nutrients in the flask. As living organisms, they also need to excrete. The large occupancy of the mycobacteria, limited nutrients, and accumulation of toxins slow down
mycobacterial replication. During this period, the total number of mycobacteria in the flask almost remains the same, so it is called the stationary phase. The stationary phase stress is a combination of many stresses.
Previous studies show a much higher Ms1 RNA level in the stationary phase compared with the exponential phase counterparts. Since the Ms1 RNA in the cells is dynamic, we do not know whether the higher Ms1 RNA level is because of the higher yield of the new Ms1 RNA or the slower degradation of old Ms1 RNA. In this project, we made a construct that can be delivered into the mycobacterial cells and allows the expression of the reporter gene under the control of the regulatory region of Ms1 gene. As a result, the Ms1 gene
expression levels were quantified and analysed. An analogy should make it easier to understand. A cell is like a swimming pool that has a tap (Ms1 gene) to pour in freshwater (newly synthesized Ms1 RNA) and a drain (enzymes to degrade RNA) to discharge waste (digested Ms1 RNA). In normal conditions, both the tap and the drain are on, so we only know that the volume of water (total Ms1 RNA levels) in the pool is larger in the afternoon (stationary phase) than in the morning (exponential phase) but cannot know why. In this project, we made a construct like a plumber to enter the pool and install a special tap (only the regulatory region of Ms1 RNA) releasing small balls (reporter gene products that can be measured easily) so we can measure the amounts of the balls released from the special tap in a certain period by collecting and counting the balls before and after that period.
The results show the expression level of Ms1 RNA in the stationary phase is higher than in the exponential phase. By analogy, the special tap does release more balls in the afternoon than in the morning. As for the drain, we cannot say whether it discharges more or less water in the afternoon compared with the condition in the morning since we did not quantify that. To conclude, Ms1 gene expression does contribute to the high Ms1 RNA level in the stationary phase.
