Team:Shanghai HS United/Design
Design
Overview
Breast milk contains numerous substances that newborns need such as nutrients, hormones, and antibodies. Some babies get jaundice, diarrhea, liver damage, and kidney problems since they can’t assimilate the lactose in the breast milk or formula with lactose. This disease is called galactosemia caused by an inborn error in their galactose metabolism. This disease is not as rare as people normally think. There is one Type I galactosemia patient among every 6000- 10000 people. To avoid this problem, some vitamin and mineral supplements are added in patients’ diets instead of galactose and lactose. There is no doubt that it is inconvenient. More importantly, the dietary treatment proves to be ineffective in preventing patients from some serious long-term complications such as the damages in patients’ cognitive and reproduction ability. Our team is quite interested in seeking some novel treatment options. We are inspired by countless microorganisms in human intestines since gut flora is important for health. In detail, the gut flora interacts with carbohydrates and converts them into short-chain fatty acid. About 95% of the short-chain fatty acids in our bodies are acetic acid, propionic acid, and butyric acid. Among them, butyric acid is the most beneficial one. Based on the information presented above, we came up with the plan using engineering probiotics to degrade and digest the galactose in galactosemia patients. The galactose is used as a carbon source to generate butyrate acid as xenobiotic compounds in our intestines, which can potentially promote health.
Expectation of our research
By accomplishing this research, our team is seeking to utilize the engineered probiotics to help patients digest the galactose that they can’t digest normally. This goal can be achieved with the help of numerous techniques that synthetic biologists normally use. By transferring the probiotics with engineered plasmids into patients’ bodies, our team is seeking to restrain or even eradicate galactosemia symptoms. If our group turn this idea into reality, millions of patients’ life will be greatly changed. They can have a relatively normal life, get rid of multiple complications associated with galactosemia, and enjoy drinking milk again. Taking a broad view, our research can further demonstrate the efficacy of using engineering probiotics to cure some illnesses.
Core
Throughout the research, our team has acquired a wide range of experimental skills. These skills include PCR, overlap PCR, prepare culture medium (LB and M9), culture the bacteria, make gel and run agarose gel electrophoresis, perform plasmid extraction, gel extraction, enzyme cutting, transform the plasmids into DH5a competent cells, measure the consumption of galactose and the amount of butyric acid.
In order to reach the final result, we did some improvements to the experiment. There are four aspects.
Use galactose instead of lactose when we test (three kinds of culture medium are listed below: 100% glucose, 100% galactose, 50% galactose & 50% glucose).
Add a new type of engineering bacteria into the final experiment—the 1917 intestinal bacteria without plasmid 1 or plasmid 2—to be a negative control compared with 2 other intestinal bacteria.
Shorten the sample interval from 4h to 2h to acquire more correct rates. (0,2,4,6,8).
The measured value has changed from 2 aspects to 4: bacteria concentration glucose concentration, galactose concentration, and butyric acid concentration.
Future plan
We cannot still create plasmid 4, maybe the reason is the length of butyrate acid synthesis gene cluster (8kb). So, the final result showed is about the comparison between plasmid 2&3. We will find a solution for the creation of plasmid 4 in the future. However, as plasmid 4 is a syncretic product of 2 and 3, adding 2 and 3 into a single bacterium is the same as using plasmid 4 directly.
Just as the final result showed, we did not get the correct concentration of butyric acid. The alternative product we got instead of butyric acid was ethanoic acid due to the failure to overexpress the butyric acid synthesis pathway gene. However, the results for plasmid 2 is promising as the lac-Y gene enables the bacteria to use up the galactose firstly. Due to the covid-19 pandemic, we did not have time to further revise our methods and create a better working of plasmid 3. The improvement we should make in the future is to construct a plasmid to overexpress the gene for butyric acid synthesis.
Reference:
Karadag, N., Zenciroglu, A., Eminoglu, F., Dilli, D., Karagol, B., Kundak, A., . . . Okumus, N. (2013). Literature Review and Outcome of Classic Galactosemia Diagnosed in the Neonatal Period. Clinical Laboratory, 59(09 10/2013). doi:10.7754/clin.lab.2013.121235
Coelho, A. I., Rubio-Gozalbo, M. E., Vicente, J. B., & Rivera, I. (2017). Sweet and sour: An update on classic galactosemia. Journal of Inherited Metabolic Disease, 40(3), 325-342. doi:10.1007/s10545-017-0029-3
Morrison, D. J., & Preston, T. (2016). Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes, 7(3), 189-200. doi:10.1080/19490976.2015.1134082
Vergara, D. M. (2017). Ácidos grasos de cadena corta (ácido butírico) y patologías intestinales. Nutrición Hospitalaria, 34(4). doi:10.20960/nh.1573
Welling, L., Bernstein, L. E., Berry, G. T., Burlina, A. B., Eyskens, F., Gautschi, M., . . . Bosch, A. M. (2016). International clinical guideline for the management of classical galactosemia: Diagnosis, treatment, and follow-up. Journal of Inherited Metabolic Disease, 40(2), 171-176. doi:10.1007/s10545-016-9990-5
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