Team:NPU-China/Human Practices

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Human Practices overview

Our project this year is to simplify the sequence of the Saccharomyces cerevisiae mitochondrial genome and achieve de novo synthesis. This topic is completely removed from our previous entries, and it is a totally blank area for us to explore. Therefore, there are four “Bigger Pictures” that we are enormously curious about and yearn to know:
Safety is always the first. Does our subject comply with the relevant laws and regulations? Is our subject within the security framework of the iGEM Organizing Committee? How do experts in this field view the safety of “artificial life”?
Objectives and significance count. Is this an innovative yet meaningful exploration? Can the project provide a reference or research basis for the further study in related fields, such as artificial life research, mitochondrial research, gene synthesis, etc.?
We do not need to understand the background knowledge of mitochondria, including genetic background, recent research progress, etc.
It is imperative for us to understand the technology of gene synthesis, including technological evolution, the latest research progress, mature commercial applications and so forth.
As follows the Human Practice activities we have carried out or partaken in this year are listed:
[1]. Took part in the 4th Synthetic Biology Young Scholar Forum;
[2]. HP with Dr.Jiang’s Lab;
[3]. HP with TSINGKE BioTech;
[4]. Participated in the 5th CCiC in Shanghai;
[5]. HP with Prof. Gu ;
[6]. HP with GENERAL BIOSYSTEMS;
[7]. HP with TACG BioTech;
[8]. Held the first iGEM Meetup in Shaanxi Province;
[9]. Attending the iGEM Summit of Nine Universities;

The following is a brief summary of our achievements of these four aspects that we have learned from the above exchanges and activities:

1.Bigger Picture of Safety:
We learned about China's genetically modified regulatory system, evaluation system and the laws and regulations pertaining to the research or commercial promotion of genetically modified gene from the speech by Mr. Zhang Xianfa, Deputy Secretary General of the National Agricultural Genetically Modified Organisms Safety Management Standardization Technical Committee and Deputy Director of the Department of Genetically Modified Biosafety and Intellectual Property of the Department of Science and Education of the Ministry of Agriculture. [1]
We confirmed that our researches are highly in compliance with relevant national laws and regulations. Also, we have strictly reported the safety-related project information in accordance with the requirements of the iGEM Organizing Committee and participated in the safety training of the laboratories. No safety incidents have occurred since the project was launched. (see Safety for details)
We resorted to Jiang Huifeng, a researcher specializing in biocomponent design and chassis modification, for his views on the safety issues of “artificial life”. [2]
Additionally, we were delighted that we happened to learn the GMP standard for medical Oligos synthesis from TSINKE BIOTECH and learned about their work on the strict control of medical primer safety. [3]

2.Bigger Picture of Project Importance：
From the speech by Academician Zhao Guoping, we learned that the “convergence technology” that combines bioinformatics, big data and synthetic biology is the future direction of development of life sciences. Being an innovative exploration, our project is intricately entwined with bioinformatics and synthetic biology. [4]
Our project has been unanimously affirmed and encouraged by Researcher Jiang Huifeng [2], Professor Gu Zhenglong [5], Researcher Dai Junyi [4], Dr. Du [6], etc. They maintain that our work is a brave scientific exploration that can provide empirical reference and research basis for artificial life research, mitochondrial research, and gene synthesis. Professor Gu Zhenglong [5] even directly proposed his willingness to cooperate with us for a longer time, hoping to further expand the research based on our work.
Dr. Xue Xiaoli [1] has set up a positive example for us to create a single-chromosome genome of Saccharomyces cerevisiae, inspiring us to challenge the impossible of life and strive to create new life.

3.Bigger Picture of Mitochondrial Research：
We contacted Professor Gu Zhenglong, who has long been working on mitochondrial research. Prof. Gu systematically introduced the biological and genetic background of mitochondria, through which we have learned about the important effects of mitochondria in cellular aerobic respiration, cancer and multi-gene regulation. Today, mitochondrial gene editing still remains a difficult task, and choosing to re-deliver through the entire genome may be a possible way that leads to the discovery of something new. [5]

4.Bigger Picture of Gene (Genome) Synthesis Technology：
Through the communication with Dr. Jiang [2] and the speech by Dr. Dai [4], we learned a lot about the artificial life, which was preliminarily a work that only God can achieve. Plus, we also accessed the classic cases where small-scale viral genes were synthesized to large-scale Saccharomyces cerevisiae genes.
We learned about OGAB gene synthesis technology from the speech by Professor Akihiko Kondo [1], the gene synthesis technology from TACG BioTech system [7], the answer to the "source of artificial life" from TSINGKE BioTech. [3], and the commercial application mode of gene synthesis technology from GENERAL BIOSYSTEMS [6]
Furthermore, we have gained a new perspective on DNA and life in the course of DNA Time Capsule activity. [10]

These exchanges and activities portrayed for us the complete “Bigger Pictures” that are directly or indirectly related to our project, which has greatly expanded our vision of the project and imparted us with substantially important knowledge and left valuable spiritual wealth to us.
Absolutely, what we have harvested from these HP activities is not merely confined to the exploration of “Bigger Picture”, benefitting us even more in the following four aspects:

Objective:
Recommendations from Dr. Jiang on the preferential selection of relatively small mitochondrial genomes [2] and Prof. Gu's introduction of the importance of mitochondria in life activities in eukaryotes and the difficulty of genetic editing [5], as well as other professionals' assessment of our project made us clear about the objective of our project this year: to creatively simplify the design of the Saccharomyces cerevisiae mitochondrial genome and achieve de novo synthesis to explore new areas of artificial life for the research on artificial life, mitochondrial and gene synthesis, offering our own experience and laying a foundation for deeper research.

Design:
Dr. Jiang suggested that we use a combination of multi-genome comparisons and homologous evolution analysis to figure out the strategy for the simplification of mitochondrial genomes based on existing gene annotation files. Dr. Cheng, a member in the research group of Dr. Jiang, specifically guided us through the design of the entire genome. [2]
Prof. Gu suggested that we add a GFP reporting module to this minimal genome for visual detection of new mitochondrial genome functions. [5]
During the design of experimental schemes, TACG BioTech suggested that we divide the whole genome so that the interface sequence between each fragment can be free of these complex sequence regions, which would facilitate subsequent splicing.
We referred to the work report of Researcher Wang Manli when the genome splicing encountered difficulties [1] and timely adjusted the experimental design, adding the plasmid replication stability module and the plasmid copy number control module, through which we ultimately realized the complete genome synthesis.

Execution:
TACG BioTech accorded us with a laboratory to carry out the genome synthesis work and complimentary support for the completion of the work. [7] TACG Biotech also helped us finish the synthesis of most of the kb-level primary fragments and specifically guided the subsequent synthesis of the entire genome. [7]
Dr. Jiang gave us the access to his laboratory and provided us with some reagent supplies. [2]
Dr. Jiang answered our questions in the mid-term progress of the experiment and gave feedback as well as guidance on the progress of the experiment and the achievements of the mid-term experiment. There were some repetitive and polluting events occurred in our experiments at that time. Dr. Jiang proposed that we might as well “return to zero” in light of the laboratory condition. Moreover, we were also kindly told to lavish due attention on the experimental operation and conduct a negative and positive control. [2]
Dr. Guo's experimental advice on extracting plasmids directly from a relatively larger amount of Saccharomyces cerevisiae showed no effect, but the accumulated valuable experience is conducive to the further development of our experiments. [4]
Ruan Jiangxing from the research group of Dr. Jiang guided our experiments of mitochondrial genome extraction and Saccharomyces cerevisiae rho0 cell preparation. [2] We obtained the important chassis for the subsequent S. cerevisiae genome transformation.
Prof. Gu was willing to help us with the following genome delivery work and assisted us in functional verification and longer-term functional extension studies based on the minimal genome. [5]

Presentation:
We introduced our project to other teams at CCiC[4], the first iGEM Meetup in Shaanxi [8] and iGEM Summit for Nine Universities [9], not merely boosting our oral expression and logical thinking, but also accumulating valuable materials in the question and answer session.
We received specific suggestions on how to improve the display of our poster at CCiC. [4]
We obtained suggestions and feedback with particular reference to our project display from the CCiC Organizing Committee. [4]

Intergrated HP with Prof. Gu

The background research on mitochondrial biology is the focus of our HP. For our project, which falls into basic researches and exploration, consulting senior scholars in related fields is probably the most effective approach to "crossing the threshold". With the help of Dr. Jiang, we contacted Professor Gu Zhenglong of Cornell University. Prof. Gu has long been committed to the function, evolution and correlation of mitochondria. Also, he devotes himself to the study of evolution of aerobic fermentation of yeast and the effects of this process on the physiology and multi-gene regulation of complex diseases such as cancer.
We have had an in-depth interaction with Prof. Gu in several substantial aspects, which are respectively project objectives and background research, genome design, project experimentation and subsequent genome delivery. And Prof. Gu indeed provided us with exceedingly valuable and specific advice and support.
Objectives and background:
We consulted Prof. Gu on the academic significance and possible specific applications of simplifying the mitochondrial genome of Saccharomyces cerevisiae and its de novo synthesis, and he was quite pleased to learn that we were designing and synthesizing the minimal mitochondrial genome. He positively confirmed the remarkable significance of our project, maintaining that the simplified design of the mitochondrial genome and its de novo synthesis are, till now, a field that entails further exploration. Chances are great that the research like this will provide a new engineered idea for the study of mitochondrial genes and their functions, as well as the treatment of diseases related to mitochondria. There is a high possibility to create a minimal model chassis, providing a new yet reliable reference for the researches in relevant fields.
Prof. Gu further said that with regard to mitochondrial research, the current method is only to knock out or add some certain genes. However, even such seemingly simple requirements have high operational difficulty in practical experiments. Even with the Crispr-Cas9 method, rapid and simple gene editing will not be that easy. On the one hand, it is difficult to express Cas9 protein in mitochondria, yet the use of a guide peptide to deliver Cas9 protein in cytoplasm is also not satisfactory. On the other hand, the difficulty of screening caused by multiple copies of mitochondria results in a low success rate at the same time.
It is a very convenient way to use the characteristics of Saccharomyces cerevisiae that it can survive in a reducing carbon source medium in the absence of a mitochondrial genome to deliver the entire de novo synthesized genome directly into the mitochondrial membrane. Of course, this also requires high-quality genomic DNA and efficient delivery methods.
Prof. Gu professed that he had long had plans to do this work. He has unique experience and skills in mitochondrial genome delivery technology, and we were more than glad that he expressed his willingness to offer us technical support in the late genome delivery.
Genomic design:
In designing the minimal S. cerevisiae mitochondrial genome, Prof. Gu suggested that we add a GFP reporter module to this minimal genome, which should make it easier to know if the genome's transcriptional translation system is working properly and also more vivid to display the results, rendering the project fancier.
Progress of experiments:
In the splicing process of genomic fragments, we encountered a serious problem, which was the spliced plasmids showed irregular base mutation or fragment loss. We thus consulted Prof. Gu for possible causes and solutions.
Prof. Gu shared with us his previous experimental experience, where he constructed the human mitochondrial genome into E. coli vector and transferred it into E. coli, and similar random mutations and fragment loss also occurred. He speculated that the reason might be the content of A and T bases in the mitochondrial genome is too high (about 85%), leading to the failure of balance of four nucleotides and the death of cells. Based on this experience, we adjusted the experimental protocol and used a low-copy vector to lay the foundation for the splicing of the genome that finally succeeded. The interaction with Prof. Gu allows us to have new solutions to the experimental problems we encountered.
Genome delivery and functional verification in the future work:
Prof. Gu expressed his willingness to help us with the following genome delivery and to assist us in functional verification and long-term functional extensions based on minimal genomes. The interaction and cooperation with Prof. Gu have laid a vitally crucial foundation for us to continue to improve our entire project.

Integrated HP with Dr. Jiang and his group

This year our project is to design and synthesize a minimal S. cerevisiae mitochondrial genome. As a basic study, we believe that it is very important to seek advice from professionals. Therefore, we have conducted detailed and extensive exchanges with Prof. Jiang Huifeng and his research group from Tianjin Institute of Industrial Biotechnology. We have obtained the full advice and support from Dr. Jiang and his research group for our project research, design and experimental development.
Dr. Jiang Huifeng, Deputy Director of the Key Laboratory of Systematic Microbial Engineering, Chinese Academy of Sciences and a researcher in Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Dr. Jiang focuses on the identification and design of novel biological parts. By combining omics technology and synthetic biology, he has developed new platforms to dig new genes for producing plant natural compounds and to design novel enzymes for one-carbon utilization.
Project research in the early period:
During the investigation of the previous project, we consulted Dr. Jiang about the most important research topic of synthetic biology in “Simplified Design and Synthesis of Life”. Dr. Jiang first briefed for us on the Syn1.0-Syn3.0 project that top scientists such as Craig Venter and Gibson have been devoted into, which centers around simplifying the design and de novo synthesis of the mycoplasma genome and achieving the breakthrough of artificially creating the simplest living body. We also learned the rapid development of genome synthesis technology in recent years, including the synthesis of artificial genome synthesis of Saccharomyces cerevisiae and E. coli. Dr. Jiang suggested we study the mitochondrial genome starting with a smaller genome size. On the one hand, there is no precedent for simplifying the design of mitochondrial genome and synthesizing it from the beginning. Such research bears a positive scientific exploration significance and can also provide some reference for the research of the simplest life design, mitochondrial research and etc. On the other hand, the mitochondrial genome scale is relatively small, with only several Ks. Compared with other living organism genomes, the synthesis cost and cycle are more advantageous, making the project more tangible. Dr. Jiang expressed his willingness to help us in genome design and synthesis. Additionally, Dr. Jiang also assisted us in contacting Professor Gu Zhenglong of Cornell University, who has long been committed to the study of the relationship between mitochondrial gene and function and basic researches on mitochondrial diseases.
After the exchange, we also had some exchanges with Nankai University, where we discussed the projects with each other this year. Our advisers gave them some suggestions for the competition.
What is more, we also consulted Dr. Jiang about the safety of artificially designed life. The "Pandora Box" effect that synthetic biotechnology may bring is a constant concern of the scientific community as well as the society. Dr. Jiang first affirmed our concern about this issue. He believes that this is a problem that all researchers need to consider. After all, our understanding of nature is still not comprehensive. There are always such defects in the researches based on experimental science, requiring researchers to seriously abide by the existing laws and regulations and conduct scientific research and exploration in the existing framework. Dr. Jiang also pointed out that blind panic was not necessary and the further study should not be therefore foregone. Safety should be judged according to the content of different research projects, rather than denied regardless.
Project design:
In the course of designing the minimal mitochondrial genome, Dr. Jiang suggested that we use the existing gene annotation files combined with multi-genomic comparison and homologous evolution analysis to determine the importance of the genes in the wild-type S. cerevisiae mitochondrial genome. He assigned Dr. Cheng Jian, who is responsible for the calculation of biological information in his research group, to guide us through the design of the entire genome. (see Design & Model for specific design).
Experiments:
During the process of genome synthesis, Dr. Jiang assigned his master student, Qi Jiangxing, to guide us on the extraction of Saccharomyces cerevisiae mitochondrial genome and the preparation of Saccharomyces cerevisiae rho0 cells (Saccharomyces cerevisiae that lost the mitochondrial genome). He gave us the access to their lab and provided us with some experimental reagent supplies.
Project progress and advice:
In the middle of the summer break, Dr. Jiang made a mid-term progress consultation for NKU-China and our team, and gave feedback and guidance on the progress and results of the mid-term experiment. There were some repetitive and polluting problems in our experiments at that time, thus Dr. Jiang suggested that we might as well “return to zero” for the lab, pay attention to the experimental operation and carry out a negative and positive control.

Trace journey of artificial life

——visit of TSINGKE BIOTECH
Easy for Judge:
Visited the primer synthesis workshop in person and learned the principle of primer synthesis and the complete synthesis process;
Found the “source of artificial life” --- deoxyoligonucleotide monomer;
For the first time we visually witnessed the apparent traits of a large number of primers.
Visited the GMP primer synthesis workshop and learned the specifications of safe production of medical primers. Discussed the future safety of medical primers with Ir. Chen.
Contents
Gene is the basic unit of life. The essence of artificial life is to artificially design and synthesize the genome of a living body. Then, as for the synthesis of genes, what do we use as raw materials? And how to synthesize? These are a technical background that is closely related to our project. In other words, where is the source of artificial life? With this kind of philosophy question, which is also the core issue of life sciences, we paid a visit to TSINGKE BIOTECH in Beijing to conduct a project survey and expand our “Bigger Picture” layout with the introduction of TACG BioTech.
Mr. Chen Yuntao, Director of the Primer Synthesis Workshop of TSINGKE BIOTECH, and Mr. Hao Yun, Marketing Manager, warmly received us.
Ir. Chen first introduced the synthesis of primers, which every student who has done molecular cloning has been exposed to. On the alkali-treated silicon-based surface, the protecting group-modified deoxyribonucleotides underwent a controlled addition of chemical "protection and deprotection" cycles, ultimately forming Oligos with a length of 15 bp to 250 bp. Ir. Chen then led us to visit their standard primer synthesis workshop, where more than 20 different models of the Oligos synthesizer in the gigantic workshop kept writing the book of life like a printer --- Oligos. He took us to one of their independently developed Oligos synthesizer with 192 flux, picking up a bottle containing oligonucleotide monomers and proudly said to us, "This is the answer to your research." Before our departure, we took a photo with Ir. Chen with the four monomers A, T, C, and G that can be used to write the book of life. It is conceivable that in the following 24 hours, these raw materials will be synthesized into primers and sent to researchers all over the country to write a grand blueprint for workers devoted into life sciences. Whenever we think of this scene, it is difficult for us to hide our excitement and ecstasy.
the “source of artificial life” --- deoxyoligonucleotide monomer
Ir. Chen continued to introduce us to the whole process of primer synthesis - ammonia hydrolysis - initial inspection - purification - quality inspection - lyophilization. He took a tube of yellow material from a primer lyophilizer, saying “this is the primer that you usually use". It was the first time for us to see the primers in person, which was an absolutely unexpected startling surprise. Ir. Chen explained to us that in this small test tube, there were Oligos with a number of tens of thousands of OD, which were tailored to our big customers, which indeed impressed us a lot.
After visiting the standard primer synthesis workshop, Ir. Chen also showed us around their GMP standard primer synthesis workshop, which is mainly used for the synthesis of Oligo probes for synthetic disease diagnosis. The GMP standard is a quality management standard that all medical drug synthesis processes in China need to strictly abide by. We didn't expect that even simple primers also had medical safety requirements, which once again expanded our "Bigger Picture" layout. We hold the firm belief that with the advancement of technology, genetic editing technologies like Crispr in the future will have substantive applications. So how does TSINGKE BIOTECH ensure the safety of the primers used in this process? Ir. Chen told us that they have been also paying attention to this issue and TSINGKE BIOTECH has now achieved the entire industry chain covering from oligonucleotide monomer preparation to primer synthesis and gene synthesis, and will strictly abide by relevant national laws and regulations as well as business ethics, ensuring the quality and safety of each primer from raw materials to delivery process.
The fruitful research process soon came to an end. Ir. Chen and Mr. Hao arranged the vehicles considerately to send us back and welcomed us to continue to carry out Human Practice at TSINGKE BIOTECH.

Exploration of artificially synthesized genome

Easy for Judge:
Gained a deeper and more comprehensive understanding of the commercial application model of gene synthesis technology;
Obtained the affirmation and encouragement from Dr. Du on our project;
Acquired the suggestion from Dr. Du that we should employ an E. coli chassis that knocks out homologous recombination systems to reduce erroneous rearrangement of genomic fragments.
Text:
Our trace journey of artificial life in Beijing TSINGKE Biotech allowed us to grasp the standard process of primer synthesis. Previously, TACG BioTech had explained the process and principles of conventional gene synthesis. We are equally curious as to how commercial gene synthesis works. In order to better understand the field of gene synthesis in a more integrated way, we came to GENERAL BIOSYSTEM (Anhui) Co., Ltd.
Dr. Du Pan, Minister of Department of Gene Synthesis, led us to visit their molecular probe production workshop, conventional primer synthesis workshop and gene synthesis workshop. During the visit, we learned that the process of industrial gene synthesis is to first design and synthesize primers with complementary sequences, and then use PCR to combine multiple primers to amplify a double-stranded DNA fragment, and finally sequence the correctness of the synthesized DNA. Usually this cycle takes around a week. Plus, he also compared the current gap between China and the world in the field of commercial gene synthesis: gene synthesis companies represented by TWIST have implemented chip-based high-throughput primer and gene synthesis, reducing the cost of primers to 1 cents/bp, while currently Chinese companies can decrease the cost to an extreme of around 3 cents/bp. At present, China's gene synthesis industry is overly dependent on human labor, nonetheless, there are already automated gene synthesis workshops in foreign countries. Dr. Du told us that although they are now building an automated gene synthesis platform, there is still a long way to go before the realization of actual production.
At the end of our visit, we introduced them to our project --- design and synthesis of the minimal mitochondrial genome of Saccharomyces cerevisiae. Dr. Du praised us for our courage to take the challenge of splicing complex mitochondrial genes. He thought that if we should succeed, it would be a positive demonstration.
Moreover, we also consulted Dr. Du about the difficulty of splicing fragments with low GC content. Dr. Du professed that the high AT regions of the genome are often the ones where gene segments are rearranged. Hence, He advised that we use the E. coli chassis, such as E.coli GXL-10 strain, which knocks out the homologous recombination system, to reduce the probability of mistaken recombination of the genome segments.

Integrated HP with TACG Biotech

Gene synthesis technology research:
We know that synthesizing a genome is not as simple as typing a code, so where does its technical difficulty lie? What are the existing gene synthesis technologies? With these questions we came to TACG Biotech.
Peng Kai, CTO of TACG Biotech, told us that the current process of a complete synthesis from bases to genomes is actually more like building blocks, rather than inputting word by word like writing as what many people may think. Gene synthesis is like building blocks, and Oligo is the smallest unit, which is chemically synthesized and bears the length of up to 250 bp (usually 60 bp to 90 bp). Usually it can be spliced into gene fragments of about 1 Kbp in one time in vitro, then the next round proceeds. The traditional fusion PCR, TA cloning, and the newly-minted emergence of Gibson Assembly, CPEC, in vitro homologous recombination and DATEL in the past 10 years have provided a variety of options for this process, greatly reducing the synthetic difficulty of the conventional kb-level genes. However, large-scale gene synthesis over 10K usually relies on the recombination and cloning system of the model chassis in order to obtain a better splicing effect. Therefore, how to obtain longer Oligos with lower cost in the preliminary stage and achieve large-scale gene fragment assembly with high efficiency in the future is a core technical obstacle that restricts the large-scale application of gene synthesis technology. In regard to Oligos synthesis, the high-throughput gene synthesis chip represented by TWIST reduced the synthesis cost of Oligos by ten times, yet the initial synthesis length did not improve. CTO Peng further introduced that in fact, organism itself is the most experienced in gene synthesis, so the bio-enzymatic Oligos gene synthesis technology is expected to achieve cheaper and greener Oligos synthesis with content length and even large-scale gene synthesis. TACG Biotech helped us contact TSINGKE Biotech to explore the source of synthetic genes and GENERAL SYSTEMS to learn more about commercial gene synthesis technology .
On top of the above, CTO Peng also introduced us to the most promising applications of current gene synthesis technology, employing DNA as a data storage medium to achieve high-density long-term preservation of data. In theory, only about 1 kilogram of DNA powder is needed to store all the data in the world today, yes, all of it! It is indeed startling! However, due to the high cost of synthesis and the hysteresis of sequencing reads, the technology has not been widely promoted. At present, many companies and research groups in the world are doing related research in this field. The description of CTO Peng inspired us to take the advantage of the utility of DNA storage to make DNA time capsules that store the kids’ dreams. And this was implemented later. (DNA time capsule link)
Genome mosaic scheme design:
There exists a low GC content and a very uneven distribution in the wild-type mitochondrial genome, presenting many GC-rich regions and AT-rich regions, as well as multiple repeat sequences. This is really a disaster for the splicing of the entire genome. Based on their experience, TACG Biotech suggested that we divide the entire gene combination so that the interface sequences between the fragments can be free of these complex sequence regions, thus facilitating subsequent splicing. For the synthesis of these individual fragments, TACG Biotech used the Oligo design software they developed to help us design these Oligos. The technique is to synthesize the longer Oligo containing the complex sequence areas. Of course, there will be some increase in the cost of synthesis, but it is worthwhile.
Genome synthesis：
Genome synthesis is a study that entails plenty of funds. After learning our financial predicament this year, TACG Biotech has generously opened the lab to us, providing us with most of the financial support needed to complete the synthesis. TACG Biotech aided us in synthesizing most of the kb-level fragments, and specifically guided the subsequent integration of the entire genome, which is essential for our whole project.
TACG Biotech conveyed their continuous support on gene synthesis for our next year's competition.

Participation of the 4th Synthetic Biology Young Scholar Forum

Name: 4th Synthetic Biology Young Scholar Forum
Objectives:
1. Track the current research progress in the field of synthetic biology, and get familiar with the background knowledge and cases, such as “artificial life”, “gene synthesis technology” and “biosafety”, expanding the “Bigger Picture” layout;
2. Learn from the forum about experience and practices related to project design and implementation.
Harvest:
Background and significance:
1、 Professor Akihiko Kondo from Kobe University introduced his large-scale multi-segment gene assembly system based on OGAB (Ordered Gene Assembly in B. subtilis). Originally, we had also thought up the idea of employing the OGAB method for genomic splicing. However, since we did not have the experience of molecular cloning experiments of Bacillus subtilis, we finally chose to synthesize the genome thorugh conventional methods.
2、 Dr. Xue Xiaoli from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, shared her amazing research achievement. She had successfully assembled the 16 chromosomes of Saccharomyces cerevisiae into a complete circular chromosome, which marks the artificially created new form of life of Saccharomyces cerevisiae. Dr. Xue's research provides us with a positive example that inspires us to challenge the impossible of life and strive to create new life.
Project design and execution:
Researcher Wang Manli from Wuhan Institute of Virology, Chinese Academy of Sciences, shared her de novo synthesis using pGF of a plant virus genome of approximately 145 kbp in length (plasmid of Genome Fast) as well as the creation of a controllable plant virus with normal function for biocontrol of pests. R. Wang also sets up a positive example for us. Afterwards, we reviewed the literature and thoroughly studied the principle and operation of pGF for genome synthesis, which had laid a solid foundation for us to ultimately achieve complete genome synthesis. The final genetic synthesis protocol we utilized bears a remarkable resemblance to hers.
Biosafety:
The forum also invited Mr. Zhang Xianfa, Deputy Secretary-General of the National Agricultural Genetically Modified Organisms Safety Management Standardization Technical Committee, Deputy Director of the Department of Genetically Modified Biosafety and Intellectual Property of the Department of Science and Education of the Ministry of Agriculture, to introduce the regulatory system and evaluation system of genetically modified organisms in China, especially the market access for genetically modified microbial products. Thanks to the briefing of Mr. Zhang, we got to know that the current regulations on genetic modification in China is Safety Management of Agricultural Genetically Modified Organisms. We have carefully reviewed the specific articles on the safety of genetically modified organisms in the Regulation and confirmed that our project this year is in compliance with the Regulation.
Note: Numerous relevant cases and techniques in the two-day forum had indeed impressed us egregiously. Although most of them were not fully digested, yet they provided us with thinking wires that guide us to optimize our project design and implementation.
Briefing on the Forum: Young scholars are the backbone and new force in the field of synthetic biology. The Synthetic Biology Young Scholar Forum was launched in 2015 by a group of Chinese synthetic biology young scholars, aiming to focus on the most advanced synthetic biological science theories and technologies and link to the world's top level of synthetic biology research, providing a platform for the exchange and cooperation among like-minded young synthetic biology researchers at home and abroad.
Time： July 7th to July 8th, 2018
Organizers: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Shenzhen Synthetic Biology Association.

iGEM University Joint Conference

Time: July 6, 2018, before the experiment mainly conducted during summer break
Activities: Online participation of the iGEM University Joint Conference hosted by Nankai University [along with the Nankai University Wiki link to the event.
Scale: eight schools participating in iGEM, a total of nine teams, including Peking iGEM 2018; Fudan; NKU_CHINA; Jilin_China 2018; TJU-China; TUST_China; NPU-China; SYSU-China;
Objectives:
1. Understand the content and progress of other outstanding teams' projects;
2. Learn the experience of other esteemed teams;
3. Introduce our team's project this year to other teams, and listen to their suggestions for project design and experiment implementation;
4. Seek cooperation and help from other teams.
Process and harvest:
In this conference, nine teams took turns to showcase this year's project. Jilin_China's project this year is also related to gene synthesis. Their modeling this year is absolutely impressive, which interested us to inquir them about their modeling. We also reached an agreement with Jilin University to help them complete the mini-interlab project [along with the link to the mini-lab page of this year's Jilin University.
Under the recommendation of Jilin University, we, too, consulted the Peking iGEM 2018 students about the modeling, and Peking University students shared their materials pertaining to modeling.
We have reached an agreement with TUST_China to help them examine water quality.
The SYSU-China presentation has inspired us to investigate the public's understanding of the understanding and recognition of artificial life through questionnaires.
One of TJU-China's four options this year is about treating mitochondrial diseases. We resorted to them with regard to the transformation method of mitochondrial genes, and they also generously accorded us with useful information about the mitochondrial genome editing that they had previously investigated.

2018 iGEM Meetup in Shaanxi

Time: 2018.7.17
Activities: 1st iGEM Meetup in Shaanxi held in the School of Life Sciences, Northwestern Polytechnical University.
Scale: 3 participating teams in Shaanxi, NPU-China, NWU-China and XJTU-China.
Objectives:
1. Acquiring the information of the projects and progress of other teams in Shaanxi Province;
2. Seeking for opportunities of cooperation with other teams;
3. Introducing the contents and progress of our project, and looking for advice and suggestions from other teams;
4. Sharing experience of partaking in iGEM.
Process and harvest:
At the meetup, the three teams showed their projects this year for the competition and their planning in project implementation, human practice, collaboration and other aspects. We also conducted deeper and vaster exchanges and discussions on team management, recruiting, training, etc., sharing and solving the problems that we encountered during the project.
Details:
1．We have encountered difficulties in implementing DNA time capsule activity. And XJTU-China conveyed their willingness to take us together to Mawu Primary School so that we could proceed to carry out our DNA time capsule activity smoothly.
2．NWU-China needed patch clamp to test the function of the modified strain, but they did not have one. And XJTU-China aided them in providing the instrument.
3．We advised XJTU-China on visas and some considerations for going to the US
4．We helped XJTU-China build a plasmid of the hairpin structure and verify the 5 We assisted NWU-China in analyzing the core enzymes of their projects and predicting the site of mutation.

Intergrated HP of Joining 5th CCIC

Time: 2018.8.27 – 2018.8.31
Activities: 5th CCiC (Conference of China iGEMer Community)
Scale: 63 participating teams in China
Objectives:
1. Seeking for advice from other teams on the design, implementation and display of our projects, thus bolstering our presentation;
2. Simulating Giant Jamboree, thus preparing for the competition;
3. Acquiring information of the contents and progress of other teams’ projects;
4. Searching for cooperation with other participants.
Process
CCIC virtually simulates the process of Giant Jamboree, with judges scoring, questioning and poster presentations. We showcased our project this year - MitoCRAFT: Designing and synthesizing the minimal Saccharomyces cerevisiae mitochondrial genome and communicated with other teams during the poster presentations.
CCIC this year has invited experts and scholars in the field of synthetic biology, including academician Zhao Guoping and researcher Dai Junxi. We elevated the affirmation and suggestions of the significance and the background of our project from the exchanges their speeches.
Harvest:
Background extension and assessment:
1. In the speech of academician Zhao Guoping, he introduced the development history of life sciences systematically, telling us that life sciences have entered the era of multidisciplinary intersection. "Convergence technology", which integrates “bioinformatics”, “big data” and “synthetic biology”, has become the direction of development and an important growth point of life sciences. Academician Zhao Guoping’s speech made us more confident in our participation in this year’s competition, since we have applied "bioinformatics" and "synthetic biology" to design and construct mitochondrial genomes in our project.
2. Researcher Dai Junxi is also a special guest of CCIC. Dr. Dai is a leading scientist in the field of genomics in China, obtaining outstanding achievements in the design and synthesis of Saccharomyces cerevisiae genomes. Dr. Dai shared with us his frustrating experience in the research of Saccharomyces cerevisiae genomes, which were quite similar to those of ours in the past few months when we had to solve various unforeseeable problems every day. This sort of empathy gave us a deeper understanding that genomic design and synthesis is very tough, yet Dr. Dai's role model has prodigiously inspired us to continue our exploration.
3. Dr. Dai also comprehensively introduced us to the development of gene synthesis technology, showing us the great process of scientists’ synthesizing from thousands of bp of viral genes to the realization of millions of bp of yeast chromosome, which, to a larger extent, has equipped us with a more systematic grasp of the gene synthesis technology, bolstering the layout of our “Bigger Picture”.
4. After the speech of Dr. Dai, we exchanged and discussed with him. He held a very optimistic attitude towards the innovation and application prospects of our project. He also reminded us with his own experience of that the challenge of what we are doing remains enormous. The difficulty of the project might accrue as our experiments proceed. He suggested that we speed up the experiment process and finish the experiment before Giant Jamboree, indeed giving us a sense of urgency but inspiring us to strive for better solutions.
Project execution:
Before we partook in CCiC, we encountered a very serious problem in the experiment. The genome assembled in yeast was difficult to extract enough concentration of genes needed for sequencing verification due to the low copy number of the plasmids. In the Q&A session after CCIC's presentation, we resorted to Dr. Guo Yutian for his consultancy, one of the judges for the session. He suggested that we directly increase the volume of the yeast solution used to extract the plasmid, and then concentrate the extracted plasmids with low concentration to obtain sufficient plasmids entailed by sequencing.
Project display:
CCIC highly simulates the process of Giant Jamboree, providing us with a precious opportunity to exercise our presentation and poster demonstration.
2. During the poster display, adviser of PKU-China, Li Cheng, suggested that we add the necessary textual explanations in the poster to help the audience understand the contents. He also reminded us to pay attention to the academic display specifications of the icons, so as to improve the display of the contents.
3. In the course of communicating with other teams, we gathered some puzzlements about their projects. For instance, is it possible to improve the GC content and reduce the difficulty of splicing by codon optimization of the whole genome? What is the formation mechanism of mitochondrial organelle membrane? These questions have helped us further boost the integrity of the collected materials and accumulate knowledge for the Q&A during the GJ presentation.
4. The CCIC Organizing Committee also gave us feedback with particular reference to our project, suggesting that we elaborate the project background and plans for how to verify the genome function in the future.