Team:NCKU Tainan/Contribution
Overview
In the process of developing our project MenTAUR, we have also attempted new experiments, designs, and methods that can inspire and benefit other iGEM teams. In our oxidative stress sensing system experiments, we collected data that prove paraquat as a more suitable inducer that more closely resembles oxidative stress in the intestine. As the inventor of the microfluidic chip, we have also listed the step-by-step development methods, experiment process, and other tips to improve its function. We hope that our efforts and contributions can assist future teams in their projects to solve the urgent problems our world currently faces today.
Oxidative Stress @PQ
Hydrogen peroxide (H2O2) was changed to paraquat (PQ) as the inducer for the oxidative stress sensing system for the following reasons:
>Paraquat: a better inducer for the oxidative stress sensing system
1. PQ produces superoxide radical (O2¯·) catalyzed by NADPH-cytochrome P450 reductase.[e] Then, O2¯· is converted into hydrogen peroxide (H2O2) by the SOD enzyme system [f] or into hydroxyl radical (OH¯·) by the HWR enzyme system[f][g]. The pathway is shown in (Fig.3).
2. Because chronic stress-induced depression (CSID) is related to a variety of abnormal changes in oxidative stress,[h] a single kind of oxidative stress molecule cannot specifically mimic the changes in the human body. Therefore, PQ was chosen as the inducer in our project to more closely simulate oxidative stress in the intestine.
3. In our oxidative stress assay, we compared the strength of the oxidative stress sensing system when induced by PQ compared to when it is induced by hydrogen peroxide (H2O2). As shown in figure 4, sfGFP expression is greater when induced by PQ .(Fig.4)
Microfluidics
Flow Chart
Fabrication of Microfluidic Chip
1. Place the PDMS mixture inside a vacuum chamber as long as possible.
Based on our own experience with a total PDMS value of 15gr on a circular container (diameter: 9cm), we put the solution in the vacuum chamber for at least 30minutes (you do not have to pay attention very much to this progress, or even better if you leave it running for an extended duration). But your mileage might vary: a more robust vacuum pump, more extensive surface area of the container used, and less solution mixed will make this progress faster.
2. Make sure the silicon wafer is clean!
It is a good idea to use high-pressure nitrogen to clean any excess particles that may exist on the silicon wafer. However, if you do not have access to high-pressure nitrogen, high pressured air can be an alternative[1]. If there is any visible excess PDMS residue from the last iteration, it is a good idea to scrape it using any sharp object such as a cutter, then blow the wafer with high-pressure nitrogen or air. Close the lid as soon as possible! to prevent any foreign particles from landing on the silicon wafer.
3. Hold the PDMS container on your dominant hand, maintain a very close distance to the silicon wafer, and pour it slowly!
Based on our own experience, it is better to hold PDMS container on your dominant hand and the silicon wafer on your less dominant hand (rather than placing the wafer on top of a table); this is to maintain a better flow of PDMS solution due to its high viscosity, which can be a little tricky to control. Then, pour the mixture slowly and maintain a very close distance without touching the silicon wafer, which will reduce the chance of bubble occurrence.
4. You have to pay attention during the second vacuum chamber process!
After pouring the PDMS mixture into the silicon wafer, there will be micro air bubble formation exist on the wafer (even that you already did your best to prevent bubble development, the perfect zero bubble condition is almost impossible to be reached), you have to perform the second vacuum chamber process, and you have to pay attention in this process (take a look every 15-20 seconds).
There will be several possibilities that will happen during this process, such as :
i. The air bubble propagates up normally, as shown in Video x. (This condition is the best case scenario)
ii. The air bubbles propagate uncontrollably, as shown in Figure 1. (This condition can be catastrophic or a very good implication).
a. If you only fabricate one chip, then it is disastrous because you need to wipe all PDMS that had spread all over the place.
b. If you cut the entire silicon wafer, the PDMS will spread very evenly after turning off the vacuum pump.
Microfluidic Chip Assembly
Our team was honoured to interview the general manager of Chen En Food Product Enterprise, Mr Vincent, as Chen En is one of the largest bubble suppliers in both local and global markets. Before the online interview session began, we signed a Memorandum of Understanding (MOU) with them. As a giant bubble supplier, they provided advice on preparing our bubble with the engineered bacteria inside. In addition, they also gave us valuable market data to further our business model.
References
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- Zhang, G., Brokx, S., & Weiner, J. H. (2005). Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature Biotechnology, 24(1), 100–104.
- Passmore, I. J., Letertre, M., Preston, M. D., Bianconi, I., Harrison, M. A., Nasher, F., … Dawson, L. F. (2018). Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria. PLoS pathogens, 14(9), e1007191.
- InterPro EMBL-EBI. “4-Hydroxy-Tetrahydrodipicolinate Synthase, DapA (IPR005263) < InterPro < EMBL-EBI.” Ebi.Ac.Uk, 2019, www.ebi.ac.uk/interpro/entry/IPR005263. Accessed 5 July 2019.
- Merlin, C., Masters, M., McAteer, S., & Coulson, A. (2003). Why Is Carbonic Anhydrase Essential to Escherichia coli? Journal of Bacteriology, 185(21), 6415–6424.
- Hashimoto, M., & Kato, J.-I. (2003). Indispensability of the Escherichia coli Carbonic Anhydrases YadF and CynT in Cell Proliferation at a Low CO2 Partial Pressure. Bioscience, Biotechnology, and Biochemistry, 67(4), 919–922.
- Coralli, C., Maja Cemazar, Chryso Kanthou, Tozer, G. M., & Dachs, G. U. (2001). Limitations of the Reporter Green Fluorescent Protein under Simulated Tumor Conditions. Cancer Research, 61(12), 4784–4790.
