Team:Xiamen city/Design
Design
Staying up late is an unavoidable topic for most people. A long period of sleep deprivation can cause a lot of damage to the body, including various metabolic diseases and even premature death1. Recent research has found that sleep deprivation will induce the production of a mass of superoxide compounds (ROS) in the intestinal tract, leading to a large number of damages to the body and causing sudden death of model animals2. The synthetic live bacteria first came up into our mind because of its characteristics: high-available, low-cost and able to be mass producing3-5. The final goal of this project is to construct an engineered bacteria which will scavenge ROS in gut quickly and efficiently.
Expectation
-The expectation of ROS-scavenging gut bacteria
To achieve our goal of designing the ROS-scavenging gut bacteria, we selected two classical enzymes-- superoxide dismutase (SOD) and catalase(CAT)6, which are capable of effectively degrading ROS, for overexpression and purification in Escherichia coli BL21 (DE3). Secondly, we constructed an experimental system to detect ROS and hydrogen peroxide degradation in vitro, and verified the activity of ROS degradation by these two enzymes. Then, we selected probiotics--Escherichia coli Nissle 1917 for genetic manipulation7, and co-expressed two validated ROS-degrading enzymes in the bacteria. By monitoring ROS consumption, the ability of the engineered strain to degrade ROS was verified. Further work, we expressed the two enzymes in fusion and displayed them on the cell membrane8, which increased the efficiency of the enzymes in contact with the substrate, hoping to further improve the activity and rate of degradation of ROS by the engineered bacteria.
Princicle explaination
The design principle of ROS-scavengeing intestinal bacteria is to overexpress the enzyme that can eliminate ROS in probiotics, so as to realize the purpose of clear ROS of intestinal tract in situ.
How This Works: Superoxide dismutase produced by Escherichia coli Nissle 1917 catalyzes ROS into hydrogen peroxide, while downstream catalase rapidly converts hydrogen peroxide into water and oxygen, achieving the goal of eliminating toxicity. By detecting the consumption of ROS, we verified that the engineered bacteria could degrade ROS effectively.
Further Plan
In order to eliminate ROS of the intestinal tract more efficiently and rapidly, we further designed the fusion expression of SOD and CAT with membrane proteins (Ag43)9 of E. coli, so that the enzymes responsible for superoxide degradation were displayed on the surface of the cell membrane. This strategy can further promote the contact of enzymes and substrates and accelerate the process of ROS degradation by engineered bacteria theoretically. We have constructed plasmids for this purpose and will further test this hypothesis.
Process
In order to achieve the goal, we use several types of equipment and techniques.
First, when we prepare the project, we have to learn enough knowledge about how to use the equipment in the right process to manipulate the process. On the first day, we learned the operating steps. For example, whenever we touch something in the lab, we needed to make sure that we wear gloves because some ingredients we touched during the research may be toxic and corrosive. Moreover, we learned how to use the centrifugal machine, PCR, Aseptic operation table, Electronic balance, and so on.
Second, we operated several experiments to get the result. For example, we configured the PCR, then we produced the gel and did the gel electrophoresis. Moreover, we collected the data we got from the gel electrophoresis and then measured the concentration of DNA. After it, we used EcoRI to cut our plasmids and genes. Then we needed to connect the plasmid, etc.
For the result of our research, we analyzed the data and came up with the conclusion of our research. Also, during the process, we faced several problems. For example, the result of imperfect gel meant that we couldn't get the result from the gel electrophoresis.
During the process, I learned some techniques and could use this equipment. For example, I learned how to use the spectrometer. This machine is particularly important because many of our data such as measuring the DNA concentration was collected from it. Also, because it's expensive and accurate, even tiny negligence can cause it to break. Therefore, having the right way to use it was important. First, before we use it, we need to make sure that the equipment is clean and tidy, Second, before we put the sample, we needed to clean the surface of the machine three times and make the first result to be the control group. Also, the amount of samples was only a drop. Third, after we got the data we wanted, we needed to clean the equipment as the first step and put everything back to the right place.
For the flaw of our research, I think the main issue was that we don't have the previous experience of working in the lab. As a result, our conclusion and data may have a big gap to the ideal result. Moreover, during the process, we didn't follow the rule of the lab every moment. As a result, it was not only harmful to the result of our experiment but also harmful to our health. Furthermore, we didn't have enough time to operate the result more times because of the limitation of our project.
To solve the following problem, I have some suggestions. For example, we can communicate with team members and our teachers to get the improvement of our research. Also, we can continue the research alone after the project to make sure that our result is right. Because we have previous experience, we can operate the test more properly and rapidly. Therefore, we will be able to fix the problem we faced during the camp.
Reference
1Davies, S. K. et al. Effect of sleep deprivation on the human metabolome. Proc Natl Acad Sci U S A 111, 10761-10766, doi:10.1073/pnas.1402663111 (2014).
2Vaccaro, A. et al. Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut. Cell 181, 1307-1328 e1315, doi:10.1016/j.cell.2020.04.049 (2020).
3Isabella, V. M. et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857-864, doi:10.1038/nbt.4222 (2018).
4Satchell, K. J. F. Engineered Bacteria for Cholera Prophylaxis. Cell Host Microbe 24, 192-194, doi:10.1016/j.chom.2018.07.013 (2018).
5McCarty, N. S. & Ledesma-Amaro, R. Synthetic Biology Tools to Engineer Microbial Communities for Biotechnology. Trends Biotechnol. 37, 181-197, doi:10.1016/j.tibtech.2018.11.002 (2019).
6Xu, J., Duan, X., Yang, J., Beeching, J. R. & Zhang, P. Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots. Plant Physiol. 161, 1517-1528, doi:10.1104/pp.112.212803 (2013).
7Scaldaferri, F. et al. Role and mechanisms of action of Escherichia coli Nissle 1917 in the maintenance of remission in ulcerative colitis patients: An update. World J Gastroenterol 22, 5505-5511, doi:10.3748/wjg.v22.i24.5505 (2016).
8Schuurmann, J., Quehl, P., Festel, G. & Jose, J. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application. Appl. Microbiol. Biotechnol. 98, 8031-8046, doi:10.1007/s00253-014-5897-y (2014).
9Huang, F. Y. et al. Bacterial surface display of endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis. Int. J. Cancer 134, 1981-1990, doi:10.1002/ijc.28511 (2014).
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