This summer, we collaborated with nine 2018 iGEM teams for a better progress in project of each other. We received help from 2 teams, SCU-China and Jiangnan in equipment and chassis cells. We also help 2 teams, CDHSU-CHINA and CSU_CHINA set up their team and mentor their work. Besides, we developed cooperative relationship with 4 team, BNU-China, NEFU_China, BUCT-China and UESTC-Software to fulfill our project. Moreover, it was the most fantastic thing that we have a spark with NCKU_Tainan of combining our project together and making use of our side product as well as solving their problems of xylose resource.
  • Cooperate
    It was the most fantastic thing that we have a spark with NCKU-Tainan of making use of our side product as well as solving their problems of xylose resource. Straw was a bio waste in agriculture while it wasn't easy to decompose. Our work successfully solved this problem with synthetic biology. We degrading straw into useful raw materials, which are glucose, xylose, ferule acid, and lignin. In addition, we aimed to convert glucose into butanol and hydrogen. But it still remains some problems of making use of our side product. Fortunately, the team, NCKU-Tainan contact with us and searching for solutions together. We also find that they met some problem during cost evaluation and carbon footprint when designing the whole CO2 utilization system.
    So they came up with the idea of combining our project together and end up building a completely eco-friendly system with negative carbon footprint. Xylose is one of the side product produced of us during straw degradation, it can be extracted and utilized in their system. At the same time, concerning the synthesis of xylose may produce greenhouse gas and chemicals, they would like to obtain xylose produced via biological method. So they can integrate their system at the downstream of our system.
    Through our collaboration, we can not only produce bioenergy, but have another powerful application in CO2 emission problem. NCKU-Tainan also provided us a valuable application way and helped us design a draft of device. Meanwhile, the xylose source from straw was really important to their E. coli CO2 utilization system which changed the positive carbon footprint into negative carbon footprint. This collaboration solved both the energy problem and environment problem. Click here to see them:
    We have established a partnership with NEFU_China and reached an agreement of helping examine the effectiveness of carrier. First, they cloned EGFP cDNA from pEGFPN2 vectors using NheI and BamHI. Second, they used EcoRI and NheI to cut pesc-trp plasmid, and then they inserted the EGFP sequence and pFUS1 into pesc-trp plasmid to complete the construction of pFUS1-EGFP carrier.We used EcoRI and BamHI to cut the pFUS1-EGFP carrier, we helped them to verify the correctness of pFUS1-EGFP carrier.
    And what we constructed is a plasmid containing GroES gene and GroEL gene. GroES / GroEL belongs to the chaperonin family of molecular chaperones and they can enhance the correct folding efficiency of proteins. NEFU_China was digested with BamH I-Vsp I and Bcu I-Vsp I to verify the validity of the plasmid. Click here to see them:
    We collaborate with BNU-China to optimize the project each other. During our experiment we found that the adhE gene which leads to the production of ethanol is competitive to butanol production in their synthetic pathway. So BNU-China give us their assistance in knocking out the adhE gene with their project. This year they are making effort in applying synthetic biology methods, constructing a novel pathway to screen mutants by giving target strains growth advantages. Therefore, they provides us their technical guidance for crisper cas9 and plasmids we need, as well as helping us design primers. At the same time we help them with their repeating experiment in detecting optimal concentration and optimal induction time of salicylic acid-induced expression. Click here to see them:
    One of the keys to experiment of BUCT-China was the construction of plasmid. Linking the operon they construct to the vector plasmid is the key to this step. The ends of the operon are BglII and XhoI digested ends, respectively, which are complementary to the vector plasmid. Thereafter, they performed an enzymatic ligation of the operon with the vector plasmid fragment under the conditions of a 16 ℃ reaction for 5 to 6 hours. Then, they transformed the enzyme-ligated plasmid into E. coli top10 strain and observed its growth (the AMP resistance gene was carried on the vector plasmid to confer selectivity). The expected result is 1, 3, 4 groups of bacteria grow, but 2 groups do not. However, they conducted a number of tests and all ended in failure. So, they contacted us to discuss the reasons for the failure and the solution. Finally, we proposed to change the enzyme ligation conditions, and changed the enzyme ligation conditions to a 4 degree reaction for 22 hours (overnight reaction) for enzyme ligation, and proceeded to verify the work. They finally conducted the experiment using the solution provided by us. Sure enough, the experimental results are very good. After that, they were able to carry out the subsequent operations.
    BUCT-China also helped us confirm the function of GroES & GroEL. After transforming the plasmid containing GroES & GroEL into DH5α strain, it was verified that the growth of the recombinant strain was better than that of the original strain at 1% butanol concentration. We invited BUCT-China to conduct repeated experiments to ensure the repeatability of the experiment. Through their data we found that we can come to the same conclusion. Under their verification, we confirmed that our GroESL in DH5α was correct. Click here for more:
    We offered many helps in the construction of their project. At their ‘Preliminary investigation’ stage, UESTC-Software communicated with us and we told them our needs for a new biobrick database. When their project has formed into the internal test version, we gave them valuable feedback. Our feedback has become one of the basis for their further adjustment.
    We also helped UESTC-Software validate the effects of their predictor. We provided them with the FRE sequence, then they predicted this sequence, and performed promoter optimization to remove unnecessary part. We constructed plasmids with predicted promoter, then transferred it to the host DH5α. The verification proves that the predicted promoter is very likely correct. And they helped us by using their database BioMaster. We needed some statistics aiding our experiments, so they recommended their database and we found what we need Click here to see them:
  • Acknowledge
    We have problems of lack of equipment on InterLab, so we turn to SCU-China for related equipment. They give us warmly help and instruct us not only in the way of using their equipment but also in analyses of our experimental data. With the help of SCU-China, we finally succeeded in our experiment of InterLab and received such a good result. Click here to see them:
    This year, we aims at using a bifunctional enzyme, xyn10D-fae1A, to directly break down the lignin-polysaccharide crosslink between straws and convert them into useful chemical materials. We introduced a group of cellulases (cex, cenA, xyl3A) to convert the cellulose produced by straw degradation into glucose, and then designed two pathways to ferment glucose to produce butanol and hydrogen respectively.
    However, there is a problem that when the conventional E. coli BW25113 is used as a chassis organism for butanol fermentation, the yield is very low, so we hope to replace the chassis cells to reach an increase in the yield of butanol.
    Through the discussions between our two teams, Jiangnan offered our team E. coli B0016-050 from their laboratory as a new chassis cell to help us make further increase in the yield of butanol. Click here to see them:
  • Support
    We mentored a new team, CSU_CHINA. We contact with members of CSU_CHINA through a meeting after we came back from Boston last year. We took them to an in-depth understanding of the iGEM competition, mentoring them on establishing a team and registration, as well as telling them a series of precautions. With our help, they join in the iGEM and set up their own team this year. Click here to see them:
    We held a lecture at Chengdu No.4 High School and give them a total introduction on iGEM. We help them know about iGEM and explained how to set up teams and how to register the competition in detail. This year, the school established their 2018 iGEM team, CDHSU-CHINA. Since then, we have been guiding them how to fulfill the criteria and giving instruction to them when deciding on their project. Click here to see them:
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