Competition Deliverables
We have or will meet all deliverables on the Competition Deliverables page.
Attributions
We have created a separated page on our wiki showing the attribution of each aspect clearly. You can click here to see more details.
Project Description
We have documented how we were inspired and finally selected our project on the “Project Description” page, and we also introduced how we originally achieved our goals. You can click here for more details.
Contribution
For the Contribution, we completed the experimental characterization of the previous parts (BBa_K2935068、BBa_K925000、BBa_K346085 and BBa_K2117000) about efficient production or accumulation in Yarrowia lipolytica. These investigations included the effect of IDH2 knockdown on lipid accumulation, verification of delta 12 desaturase function, introduction of the strong promoter PT7 and measurement of the expression intensity of the TEF promoter under different conditions, and these data were added to the corresponding BioBricks. All of these may be helpful to other teams and we hope it will make some contribution to the iGEM community.You can click here for more details.
Engineering Success
This year NNU-China 2022 has tackled and solved one or more of our project's problems and use synthetic biology tools to generate expected results. We introduced an entire EPA biosynthesis pathway into Yarrowia lipolytica Po1f, which is capable of guiding the conversion from the C18:2 to EPA step by step. In addition, many strategies were applied to enhance the EPA content, including inhibiting β-oxidation pathway, screening for different sources of elongases/desaturases, increasing the copy numbers of target genes, and replacement of promoter. All BioBricks were registered and tested, and most of them work well to perform their functions as expected.You can click here to see more details.
Collarboration
The NNU-China2022 team has participated in the joint organization of various universities many times. iGEMers from each university communicated and helped each other by sharing their project progress and activity experience. In addition, our team also participated in the world's largest regional iGEMer conference CCiC. We communicated with the University of Macao and Taiwan National Qsinghua University and offered assistance for Copenhagen University. Sharing the progress of the project with other teams and drawing lessons from the experience of various team activities at home and abroad has played a great role in promoting the improvement of our project. In addition, we co-hosted a meetup and established cooperation with several teams, including Hainan University, Worldshaper-Shanghai, University of Science and Technology of China and China Pharmaceutical University. We have exchanged and shared experience on the project theme, experiment scheme optimization, wiki design with other teams, providing a new perspective for the project.You can click here to see more details.
Human Practices
DHA and EPA are essential omega-3 polyunsaturated fatty acids, and have an irreplaceable role in human health. Inspired by related news and field trips to markets, we have learned about the huge market occupation of the omega-3 polyunsaturated fatty acids and the limitations of existing extraction methods. Through the survey by questionnaire, we learned about the public's understanding and acceptance of omega-3 unsaturated fatty acids and related products. We design our experiments by consulting experts in the field of fatty acid metabolism and synthetic biology. We also maintain close contact with other teams and share our experiences in experimental operation. In addition, we conducted online questionnaires and public education on synthetic biology via social media, visited several communities for education propaganda.You can click here to see more details.
Proposed Implementation
In order to ensure that the production process of EPA/DHA is environmentally friendly and stable in yield and quality, our team constructed an engineered Yeasts strain for the production of EPA/DHA through synthetic biology. On this basis, we improved the ability of the engineered strain to produce EPA/DHA by knocking down the PEX10 gene, screening different sources of fat elongase and desaturase, and increasing gene copy number without affecting the natural environment microorganism and human body. The engineered strain we constructed was grown and fermented in the experimental environment to obtain the required EPA/DHA. Under the condition of satisfying the growth of yeast, they can be used as raw materials of polyunsaturated fatty acids to be mass-produced in factories. At the same time, we will also make them as one of the raw materials to participate in the production of chicken feed. (We will comply with iGEM's safety and responsibility rules and will not conduct any live experiments within the scope of iGEM competition).You can click here to see more details.
Integrated human practice
This year, our project is focused on improving the low intake of polyunsaturated fatty acids in the population. Considering the actual situation and the brainstorming of the team, we decided to increase the DHA and EPA content in the daily nutrition of eggs, so that people can have DHA and EPA in their daily diet.
First, we were inspired by television reports about the serious pollution of the oceans and the decline in the quality of fish oil. Then through attending lectures, field trips, issuing questionnaires, visiting relevant departments and companies, we have a more comprehensive understanding of the social cognition of DHA/EPA.
Then, in order to determine the research direction of the project and the specific scheme, we consulted some professors. At the same time, we also found other consultants to clear the obstacles for our project and solve the problems in the progress of the project comprehensively.
Next, in order to make the project more socially acceptable, we organized offline public education activities on the topic of "synthetic biology" and distributed online questionnaires on polyunsaturated fatty acids and omega-3 eggs.
In the whole HP activity, each step complemented each other to form an organic whole.You can click here to see more details.
Improvement of an Existing Part
We improved the ability of BBa_K3645011 from iGEM20_Peking Team,to work more efficiently in Yarrowia lipolytica by using optimised related components. We first supposed whether replacing dCas9 with nCas9 will increase the targeting and editing efficiency of CBE. We also hypothesized that the expression level of CBE could be increased through using the stronger promoters thus increasing editing efficiency. For this,the improving of BBa_K3645011 are divided into three sections as follows: (i) the replacement of dCas9 by nick Cas9 (nCas9), (ii) promoter optimization and (iii) Cas9 without protospacer adjacent motif (PAM) limitation (SpRY)[5] to expand the editing range. You can click here to see more details.
Project Modeling
The intracellular metabolic activity of Yarrowia lipolytica is complex, and the synthesis path of intracellular polyunsaturated fatty acids is long, which makes it difficult to accumulate EPA directionally. Traditional metabolic engineering strategies are mostly based on a certain metabolic pathway or specific genes, without considering the dynamic impact of genetic disturbance on cell growth and product synthesis. As a mathematical model, genome scale metabolic network model is widely used in analyzing network characteristics, predicting cell phenotypes, guiding metabolic engineering, driving model discovery, studying evolutionary processes and analyzing interactions. We tried to use the genome scale metabolic network model of Y.lipolytica to systematically analyze the distribution of its intracellular metabolic flow, and combined with different algorithms to predict the transformation target of efficient EPA synthesis, providing guidance for the subsequent metabolic engineering transformation.And in the fermentation production, extracellular conditions (such as stirring and ventilation) will have a great impact on the yield. Therefore, it is necessary to obtain the optimal operating conditions through repeated screening of single factor experiments. This will be a time-consuming and laborious work, and the relationship between different parameters aggravate the difficulty of the experiment. In order to solve this problem, the computational fluid dynamics (CFD) technology was used to quantitatively characterize the flow field characteristics of the bioreactor. Furthermore, different fermentation conditions were compared in advance, and the best fermentation conditions were determined quickly.You can click here to see more details.
Proof of Concept
The overall goal of the project was to construct Yarrowia lipolytica as an omega-3 fatty acids production cell factory through the synthetic biology. In order to further investigate the performance of the engineered omega-3 producer, fed-batch fermentation was carried out using a 5-liter bioreactor. Moreover, in order to investigate the possibility of making the engineered strain into feed, we developed a set of technological process to make it into fungi powder.You can click here to see more details.
Partnership
Based on the similarity of team projects, NNU-China team established a partnership with NJTech_China team and NJXDF-CHN team. NJTech_China team has rich experience in competition, and has many ideas and breakthroughs. In the pv production part, NJtech_China team put forward specific suggestions to our team, and suggested that we can make full use of the advantages of normal University to carry out activities. The NJXDF-CHN team has been closely cooperating with our team, continuously following up the progress of the experimental part, and jointly designing and carrying out a series of synthetic biology education and publicity work.
We both hope to carry out our project in an innovative way. We have conducted in-depth cooperation in experimental design and implementation, human practice, education, web page production and other aspects, and provided a lot of suggestions to each other. The cooperation between the two teams is of great help to the promotion of the project of both sides. At the same time, the two teams also jointly held offline communication meetings and synthetic biology information sessions, and we will promote the progress of the project toward the common goal.You can click here to see more details.
Education and Communication
NNU-China understands that education and communication are effective ways to connect with the public and build bridges between science and society. Therefore, our team carried out a variety of educational publicity activities. We spread the knowledge about synthetic biology to the broad masses of the people. By effectively combining the advantages and professional characteristics of normal university, we have formulated various forms of popular science education activities with professional characteristics according to the age differences and different cultural and educational levels of the educatees. All our education and communication activities have received good social feedback. We have carried out the undergraduate curriculum reform activities of Nanjing Normal University, laboratory open day activity, several times of online and offline education popular science activities, as well as the operation of social media official account and the publication of popular science articles. All our activities were carried out after being approved by the relevant agencies in advance. During the education&communication activities, we strictly abide by relevant laws and regulations, epidemic prevention requirements and protect the personal rights and interests of the educated.You can click here to see more details.