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

Fig. 1. Flow chart of Microfluidic Chip Fabrication

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

Fig. 3. iGEM 2021 Logo using Epoxy-based 3D printer

References

  1. 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).
  2. 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.
  3. 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.
  4. 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.
  5. Merlin, C., Masters, M., McAteer, S., & Coulson, A. (2003). Why Is Carbonic Anhydrase Essential to Escherichia coli? Journal of Bacteriology, 185(21), 6415–6424.
  6. 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.
  7. 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.

e-mail : igem.ncku.tainan@gmail.com
phone : 06-2757575