Preface

We have reached a partnership relationship with CSMU_Taiwan since June. Throughout the entire season of the iGEM Competition 2022, we have successfully helped one another in various aspects.

Project

We kept in touch frequently, had online sub-team meetings with CSMU_Taiwan every month and have helped each other in different aspects of our project. Through this, both teams have managed a smoother journey throughout the competition preparation.

YouTube program

We invited CSMU_Taiwan to shoot a short video introducing the contents of their project. We established a YouTube channel- Taiwan iGEM and uploaded videos about synthetic biology and our projects to enrich popular science resources in the Chinese-speaking world.

Questionnaire

In order to gain a deeper understanding of diabetes patients’ needs, we conducted a questionnaire. In order to run this event smoothly, our partner CSMU_Taiwan (Chung Shan Medical University is a medical university located in Taiwan, and easier to get in touch with people with diabetes) assisted us in contacting the patients. With CSMU_Taiwan, we were able to have more respondents.

Podcast program

On June 30th, we were invited by CSMU_Taiwan to record the podcast together. Through the process of recording the podcast, both of us got to know each other's projects and teams much better. After recording the podcast, we exchanged opinions on Wet Lab and Human Practice, through this opinion exchange we learned a lot of experiences with each other.

Dry lab

LDH enzymatic assay

Throughout the past research, the operation of GDH enzymatic assay was trying to quantify the concentration of PQQ. However, in the following experiments, a more efficient and sensitive PQQ quantitative method was established. It indicated the GDH enzymatic assay was not as important as before, due to the precipitation of electron transfer (PMS) which makes insufficient sensitivity. But the LDH enzymatic assay proposed by CSMU_Taiwan was seen to be a good way to go further for dry lab and modeling. It’s because one of the main functions of PQQ in vivo is to facilitate the conversion of pyruvate to lactate, which makes huge benefits to the human body. Although the process of the enzymatic assay of PQQ-bound LDH was published, it still had some shortages, such as the model for predicting the result needed to be refined.

Design of Microfluidic System

After CSMU_Taiwan proposed a concept of a microfluidic system, we gave them some suggestions for their designs. The second version of our blueprint used a Z-shape channel with dead ends to separate CHO cells and antibodies. However, we were concerned that this design couldn’t do cell retention, meaning the cells would accumulate at the dead end and block the channel. In order to tackle this problem, we suggested that tangential flow filtration might be a proper solution. This advice made it a crucial factor for them to complete the design of our bioreactor.

Wet Lab

Solution to electroporation obstacle

We chose to use electroporation to transfer the plasmid into the Bacillus subtilis natto. The first time, we failed due to some potential problems. The potential problem obstructed us for a long time until we met with CSMU_Taiwan. CSMU_Taiwan said, “The electric current passes through the electroporation cuvettes, and bacteria will be arced by the remaining electrolyte so that the transformation success rate will be plunged.” After that, we suspected the plasmid extracted from stored bacteria still had electrolytes left over. We dialyzed DNA with ethanol and nitrocellulose filter to wash out the electrolyte and did the transformation again, and the result was successful.

Reporter Gene Choice

When CSMU_Taiwan exchanged ideas about construct design with us, we reminded them that Green Fluorescent Protein (GFP) expression could deteriorate over time. Also, GFP-tagged cells are prone to death owing to the cytotoxicity of GFP, causing obstacles when CSMU_Taiwan conducts experiments and undermining their results. Therefore, CSMU_Taiwan tried to find if there are other reporter genes suitable for their construction. Fortunately, they replaced GFP with Red Fluorescent Protein (RFP) and solved this concern.