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Revision as of 14:39, 19 October 2021
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
Dr. Po-See Chen 5/28
Director, Division of General Psychiatry, National Cheng Kung University Hospital
Professor, Institute of Behavioral Medicine, National Cheng Kung University
We met with Dr. Po-See Chen online to obtain further knowledge about depression. He provided us with valuable insight and information regarding the characteristics of depression and the relation of depression with the gut. We met him during the early stages of our project, and he gave us a lot of valuable advice regarding the behavior of patients, such as self-diagnosis which is very dangerous among people who suffer from depression. By conducting this meeting, we can understand more regarding the nature of depression to continue with our project. He also gave advice regarding our education activities, encouraging us to better spread awareness regarding depression, which you can find in our education page.
Professor Yu-Cheng Lin
Professor, Department of Engineering Science, National Cheng Kung University
Our first hardware idea was to detect human stress levels through saliva, we planned to extract cortisol which is the biomarker of stress, by detecting cortisol levels throughout a couple of days, we will be able to determine someone’s depression level. We made several discussions with professor Yu-Cheng Lin due to the fact that he has the real experience of extracting the different substances from saliva and detect the concentration of several substances through the paper-based electrochemical device. We wanted to continue and make this concept a reality, but suddenly covid-19 strikes Taiwan, so we were not able to go into any lab and we finally suspend the idea then search for another alternative.
Professor Chung-Hsien Wu
Professor, Department of Computer Science, National Cheng Kung University
Due to the fact that covid-19 in Taiwan escalated very quickly, we were thinking to find another way to build something even that we are not present physically. Then, we had an idea to made an artificial intelligence that can automatically detect depression using sound, and because none of our team members is from the Department of computer science we asked for guidance from Prof. Chung-Hsien Wu in developing the artificial intelligence.
Professor Chien-Hsiang Chang
Professor, Department of chemical engineering, National Cheng Kung University
Before settling with our current procedure of manufacturing Menbels, we wanted to make sure that our procedure and knowledge are sufficient for us to meet the requirements and make our system feasible to work. And professor Chien-Hsiang Chang has experience in making alginate layers to wrap several substances, Prof. Chang also ensures us that our plan is feasible and isn’t very hard to perform.
Microfluidic Experiment:
Based on our own experience, our the final volume of microfluidic chamber + outflow tube of 50 microliters; we got the best retention rate result when BSA (Bovine Serum Albumin) is injected with 1 ml/h flow rate for 6 minutes or 0.1ml, these are because:
1. During microfluidic assembly (glass plate and the chip) using the oxygen plasma chamber[8], the oxygen plasma itself reduced the hydrophobicity of both the channel and glass plate. However, long baking progress makes the channel turn back into hydrophobic, making the chip useless if we directly use the chip without pre-application of BSA.
2. The BSA solution that we used has a concentration of 3.85 wt% (1gr BSA + 25ml DI water), and BSA acts as a primer that can let the inner channel walls become hydrophilic[9].
iGEM 3-D Printed Logo
Besides fabricating microfluidic channels, we did a mini side project which is 3-D printing iGEM logo using an Epoxy-based 3-D printer. We created iGEM logo using Autocadand our,and product can be seen in Fig. 3
References
- Saito, Y., Sato, T., Nomoto, K., & Tsuji, H. (2018). Identification of phenol- and p-Cresol-producing intestinal bacteria by using media supplemented with tyrosine and its metabolites. FEMS Microbiology Ecology, 94(9).
- 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.
