Team:FAFU-CHINA/Partnership
Partnership——BNU-CHINA
Through the igem community, we got in touch with BNU-China. As the projects of us had great similarities and we had already established good paternship during the past few competitions, we decided to help each other and build a more closer relationship.
From March to June：Brainstorming and communication
We got to know 2020 iGEM BNU-CHINA through the iGEM community. Based on the experience of many exchanges and cooperation in the past years, we established a partnership this year. Since the brainstorming in March and April, we have maintained close contact. In the process of project communication, our team invited BNU team to share the project plan and put forward their own suggestions for finalizing the final project.
Our team had a detailed discussion with BNU on the production of bioethanol from cellulose. At that time, we didn't think about the way to degrade cellulose. BNU suggested that we could transform Saccharomyces cerevisiae so that it could degrade lignocellulose and produce bioethanol at the same time. We adopted BNU's suggestion and designed our fermentation system from the perspective of eukaryotes.
July: Co-education
At the invitation of BNU, our team participated in a large-scale Popular Science Lecture led by Tsinghua University and the teaching and research center of Beijing Municipal Education Commission - synthetic biology from scratch. Both of our two teams are the first contact with online live broadcast activities. We have conducted many consultations and discussions in the preparation stage and live broadcast stage for the lack of relevant experience, so as to make our exhibition go smoothly.In this activity, we mainly introduced the application prospect of synthetic biology in the field of bioenergy. We hope to use the advanced technology of synthetic biology to improve the problems of low efficiency and high cost of biological fermentation in industrial production.
BNU China mainly introduces the new technologies of synthetic biology and diagnosis and treatment. In this live broadcast activity, we learned from BNU's live broadcast mode, successfully solved the problem of online handover of speakers, and made the popular science activities smoothly carried out and obtained good feedback.
August: Combine our projects and Further cooperation
After confirming the raw material of cellulose, we had an exchange with BNU on how to produce bioethanol from JUNCAO. The pretreatment, saccharification and fermentation of JUNCAO involve a large number of enzymes, so we and BNU agree that it is necessary to use co-culture system to achieve the above purpose.
With regard to the co-culture system, our initial idea was to produce minifibrosomes by co-culturing yeast. The microfibrosome consists of two parts, the different parts are produced by different yeast strains. One part is made up of cellulose exonuclease and dockerin,The other part is made up of cellulose endocellulase,linker,CBM,cohenson.
Fig 1. The composition of picrofiber corpuscles
The two part is secreted out of the cell environment by yeast, and then bound together through the specific interaction between cohenson and dockerin, and then forms a mini fibrosome. The principle of the action of the minifibrosomes is similar to that of the fibrosomes. The CBMs are guided to approach the cellulose substrate by CBM, and then the proximity effect of the enzymes is enhanced by the interaction between cohenson and dockerin, thus achieving higher degradation efficiency than free enzymes. However, the effect of the corresponding mini cellulosome is not as stable as that of the artificial cellulosome.
In the communication, BNU believes that our design lacks internal control means. Combined with the advantages of prokaryotic and eukaryotic co-culture, we carefully considered this problem and designed an integrated fermentation system for co culture of Escherichia coli and Saccharomyces cerevisiae.
Considering that the heterologous expression plasmids are easy to be lost due to continuous passage in the continuous fermentation process, which will seriously affect the fermentation efficiency of the system, so we want to integrate the lignocellulose gene into the genome of yeast. By using the integrated plasmid pKLac2 of yeast genome, we can insert the target gene into LAC4 promoter region.
We hope to use the ABC (automatic barcode creator) system designed by BNU to add barcode to the downstream of the target gene to analyze the stability of the heterologous expression of lignocellulose in the continuous passage, and then determine the optimal utilization time of the engineering bacteria in the continuous fermentation tank. BNU agreed with our idea and was willing to establish a cooperative relationship with us in terms of experiments. Therefore, in the following period, we will keep in touch with bnu-china, share the experience of experiments, HP and other aspects, and jointly promote the smooth progress of the project.
On the eve of CCIC, we discussed the design rationality of the integrated fermentation system with BNU online. First of all, we discussed with BNU whether the integrated fermentation system should be regulated by light or chemical induction. The cost of photoregulation is obviously lower than that of chemical induction, but chemical induction is more stable in terms of effect. In order to separate the degradation of lignin from the degradation of cellulose and hemicellulose, we finally chose chemical induction method. We expect that the effect of chemical induction is to induce the expression of lignin enzyme and inhibit the expression of cellulose and hemicellulase in the presence of lactose; when lactose is depleted, the expression of lignin enzyme stops and the inhibition of cellulase and hemicellulase expression is relieved.
we jointly participated in CCIC meeting, and after the meeting, we put forward suggestions for improving the video and poster displayed by the other party at the meeting. BNU suggested that we should supplement HP and experimental design in the video, and pointed out the problem that the sound and picture were not synchronized in the presentation process. We documented these issues and suggestions, hoping to avoid them in the final presentation video.
September: Graphic communication
At this time, the deadline for promotion video was very close. Our team members conducted online communication and discussed how to make promotion video and the style of promotion video.BNU suggested that we animate the fermentation process of bioethanol, which would help us to show our integrated fermentation system to the audience in a more intuitive way.
October:stories will go on
BNU-China contacted us again and exchanged the progress of the project.BNU designed two promoters, placing the GAPDH promoter upstream of the U6 promoter.The U6 promoter is used to express functional hgRNA.The GAPDH promoter is used to drive the barcode and the added polyA tail transcription.So that they can know the cell relationship and also know the transcriptional state of the cell.
This is similar to the purpose of our dual-plasmid system, and we applaud BNU's idea and suggest that they conduct further experiments to verify it.We have been in regular contact with BNU-China throughout the tournament.Long-term cooperation has promoted the common development of our project.So far, BNU has built an ABC (automatic barcode creator) system and a basic example in yeast: we built a dual-plasmid system in E. coli and successfully observed a green fluorescent signal in DH5α.
We both felt that the communication between the two teams was helpful, and the combination of our projects would bring even greater surprises.