Dr. Ng is the mother of NCKU-iGEM team. Since the establishment of NCKU-iGEM 2016, Dr. Ng have brought synthetic biology as well as iGEM to the flashlight of NCKU campus. Dr. Ng always inspires team members by asking us some critical questions without telling us the answer directly. She gave us advice from every aspect. She initially gave us many critical suggestions during the brainstorming. Except for teaching us knowledge about synthetic biology, she always reminds us to think more about the real application of our project.

Various useful advice was given by her. She emphasized the advantage of protein production by E. coli, she also suggested us to study more on high cell density fermentation. Without her timely advice, our team cannot improve our project.

Our PI, Dr. Huang work as an assistant professor under the institute of Basic Medical Sciences of National Cheng Kung Univercity. His major field are Bacteriology, Bacteria Genetics, and Molecular Biology.

We invited Dr. Huang to attend our first presentation. During our first presentation, he provided us a lot of useful feedback and we could feel the intention of Dr. Huang. So, our team think that Dr. Huang could guide us for all our project and we wanted to invite him as our PI. Luckily, we have succeed!

From Dr. Huang's first day as our PI, he did a lots for us. Firstly, he provided us all the resources that we needed or could make us convenient. Secondly, he gave us many ideas when we have met difficulties or junction in our progress whether it is dry lab or wet lab. We are really appreciate to what he have told and taught to us.

This year, our project is related to carbon sequestration. Therefore, we grab the chance to discuss our idea with Professor Bieng-Zih Hsieh. Professor Bieng-Zih Hsieh is an associate professor from the Department of Resources Engineering. One of his research topics is about CO2 sequestration, which is a topic that related to our project, the difference is he employs a physical method in this aspect, while we use a biological method.

At first, we define our project as carbon capture. However, he introduced the idea of carbon utilization to us as he thought it is more suitable for our project. Professor Hsieh introduced some carbon capture or utilization strategies that implemented in the industry currently and suggest us to do more research on those methods. He also remind us to compare our system with other biological approach to figure out our strength and weakness.

Prof. Chao Chung, Peng is an assistant professor of Department of Aeronautics and Astronautics and International Bachelor Degree Program on Energy. He used to work in China steel corp. so he gave us a chance to contact CSC.

we met up with prof. Chung to get some suggestions which more proving our direction of our projects. Prof. Chung is proficient in energy field, different from giving us experimental technical advice, he gave us more about suggestion for action on device and idea.

He thinks that when we look for our advantages, we can take apart each of our research processes and experimental steps, and find every possible point that is more inefficient. The benefit of the whole project is very great. For example, what kind of way we can use to let every E.coli to eat most CO2, the way we replace our cultured E.coli. It is true that there are every existing way we can think more about how we can improve. Also, professor tell us we must be very understandable about our project, it includes our advantages, disadvantages, potential threat, our whole project process, and the most optimum environment we should provide for our E.coli. The more we realize our project, we can find more possibility of development. After talking with professor, we inspired more ideas and found direction about our project more clearly.

He gave us some advices in engineering pointview. Therefore, we tried to rewirte a big scope of our project and defined the points that we have to fulfill.

We had an opportunity to speak with Dr. Huang Chieh-Chen from National Chung Hsing University (NCHU) in the Asia Pacific Conference, whose work includes the reverse TCA cycle of engineered carbon capturing E. coli. Upon our discussion, Dr. Huang raised the issue of the low efficiency of one of our enzymes, Rubisco and suggested that we culture our engineered E.coli in an anaerobic condition. Since Rubisco, the ribulose-1,5-biphosphate carboxylase/oxygenase is an enzyme catalyzing the reaction of either carbon dioxide or oxygen with ribulose-1,5-biphosphate (RuBP) as the name itself suggests, putting our system in an aerobic condition would significantly lower the efficiency of capturing carbon dioxide due to the similarities between gaseous carbon dioxide and oxygen.

Prof. Jo-Shu Chang was under a lot of specialty of Biochemical Engineering, Microalgae Biotechnology, CO2 reutilization, Circular Economy; Biomass Energy, Environmental Biotechnology, Applied Microbiology. He complete a microalge carbon utilization system in ANAN Campus of NCKU which help the largest energy company in Taiwan (CPC, Taiwan) and Taiwan's No.2 CO2emission company (China Steel Co.) to develop the research platform of microalgae-based flue gas CO2 biofixation technology.Besides, Since2009, Prof. Chang served as the PI of National Energy Program (NEP) projects and focused on microalgae-based CO2 capture and utilization for biofuels production and biorefinery.

Our project is to reuse the carbon dioxide emitted form the industrial with a biological way. Although the pathway of carbon flow in E. coli and in microalge are quite different, it is really important for us to understand the current method of biological carbon utilization before we begin our research. We want to deeply understand the whole microalgae culture pool system including the position of CO2 pipeline, downstream process, pool cleaning and how is the progress of an research in lab turning into real application.What's more, we He subsequently proposed a 'zero emission' biohydrogen system integrating unit operation of cellulose hydrolysis, darkfermentation, photofermentation, microalgae CO2 fixation, microbial fuel cell and hydrogen fuel cell.

Prof. Chang let us visit how his project work in ANAN campus(link). Prof. Chang's team has been working with China Steel Co. and CPC, Taiwan to develop innovative microalgae-based flue gas CO2 reduction and biofuels production technologies. Therefore, they shared the progress of the whole technologies coming out. From the beginning, Prof. Chang was focus on the conversion of biomass feedstock and waste materials to energy. Large scale microalgae cultivation plants were constructed in China Steel Co. and on An-Nan campus of NCKU.

We visited Prof. Jo-Shu Chang discussing about our carbon capture system. We had a great time talk and he gave us three advises. One is to define the velocity of CO2 input in which scale of CO2 we want to solve with. Another one is to confirm a commercial product like proteins which can bring us some income to balance the cost. The last one is to calculate the whole carbon balance from the source of xylose to the process of waste. We were really appreciated that we can have a short meeting with prof. Chang.

Our secondary PI, Professor Hashimoto Masayuki is a project assistant professor from Institute of Molecular Medicine.

The host organism in our project is E. coli. Professor Hashimoto is familiar with the genetic manipulation of E. coli, therefore we introduced our idea to him. One of his research is about the effect of carbonic anhydrase (CA) to the growth of E. coli. Since we wanted to introduce heterologous CA into the E. coli, he suggested us to consider whether it will compete with the native CA of E. coli, which might affect the function of the heterologous CA. Also, Professor Hashimoto introduced the concept of homologous recombination to us as we had the idea to insert our construction into E. coli chromosome. However, we were suggested not to do the homologous recombination due to the limited time we had.

After the discussion, we started to review journals and found out the feasibility of expressing both native and non-native CA in E. coli. We found out that there were research which expressed heterologous CA successfully. Therefore we decided to clone the CA gene into the E .coli.