1.Formed experimental group.
2.Conducted literature research.
3.Entered the lab for relevant skills training.
1.Recruited team members and determine the grouping of our practice sections(HP group, art group ,model group, wiki group).
2.Conducted project research organised project presentations.
3.Had a brainstorm and finalized our project idea.
1.Developed an experimental programme and confirmed the feasibility of the experiment.
2.The Escherichia coli NT1003 which could produce high lysine was used as the chassis strain, and designed and constructed a portion of the metabolic pathway for 1,5-pentanediol(1,5-PDO).
3.The Escherichia coli NT1003 which could produce high lysine was used as the chassis strain, and designed and constructed a portion of the metabolic pathway for 1,5-pentanediol(1,5-PDO).
4.Confirmed the selection of the carboxylic acid reductase pathway as the synthetic pathway for the subsequent study.
1.Determined the human practice.
2.Interviewed with Professor Bingzhi Li of Tianjin University and he approved our modular design of metabolic pathway in synthetic biology .
1.The plasmids pTrc99a-davB-davA-GabT and pACYCDuet-sfp-MmCAR-YahK were constructed.
2.These plasmids were then transferred into NT1003 to construct strain NT1003-P1. Through fermentation,the production pathway of glucose to 1,5-PDO was successfully verified.
1.Interviewed Professor Yingxiu Cao of Tianjin University and she suggested us to assess whether engineering of branched pathways could enhance the product titers. Furthermore, She emphasized the importance of creating a high-quality wiki and HP sections.
2.Professor Liya Liang was interviewed and she suggested replacing plasmids with higher copy numbers to enhance the expression of the 1,5-PDO synthesis module.
3.Interviewed with expert from Sinopec Beijing Research Institure Of Chemical Industry and discussed prospects for 1,5-pentanediol synthesis and other related topics.
4.Talked with high school team Nanjing-BioXstem and shared team case to help them better understand igem.
5.Participated in Nanjing iGEM association.
By metabolic flux analysis, the maximum theoretical yield of 1, 5-PDO can reach 0.847 mol/mol Glu via the lysine pathway using glucose as substrate.
1.Constructed the plasmid pRSFDuet-sfp-MmCAR-YahK.
2.Constructed the strain NT1003-P2: pTrc99a-davB-davA-GabT + pRSFDuet-sfp-MmCAR-YahK.
Through fermentation,it showed that the yield of 1,5-PDO was nearly 4-fold higher in NT1003-P2 than in NT1003-P1.
1.Interviewed with Professor Xinkun Ren of Nanjing University, and suggested that We can mutate the rate-limiting enzyme MmCAR to enhance the production.
2.Organized a science popularization event with the theme of iGEM for students majoring in Bioengineering at Nanjing Tech University.
3.Provided a demonstration on the usage of software such as SnapGene and GraphPad to the high school team Nanjing-BioXstem.
4.Conducted a preaching activity for middle school students.
Based on the model analysis, it had been predicted that the knockout of genes ackA and pta can enhance the production of 1,5-PDO.
1.By utilizing CRISPR-Cas9 technology, the genes ackA and pta were knocked out.
2.Using CRISPR-Cas9 technology, we had knocked out the genes ackA and pta and successfully constructed the strain NT1003-P2-ΔackA-pta.
3.Based on previous research in the laboratory, knocking out the gene YcjQ was identified and the strain NT1003-P2-ΔYcjQ.
Based on fermentation result, we decided to continue the modification using NT1003-P2-ΔYcjQ in subsequent experiments.
1.Interviewed Professor Jiaxin Xu, who suggested enzyme assembly as a strategy to enhance the yield of 1,5-PDO.
2.Participated in the 10th Conference of the China iGEMer Community (CCiC).
3.Participated in the Environmental Protection and Sustainability Summit (EPSS) hosted by Nanjing Normal University.
4.Took part in the 15th Training Course of Coating Formulation Design and the 10th Training Course of Waterborne Technology.
5.Participated in the National Chemical Engineering and Biochemical Annual Conference.
6.Held an online networking meeting with the NNU-CHINA team.
Based on iteration results,several genetic targets that can be used to optimize the fluxes to produce the production of 1, 5-pentadiol are predicted.
1.To enhance the carboxylic acid reductive efficiency of MmCAR enzyme, we conducted molecular docking and identified the mutation sites.
2.Constructed the plasmid PRSFDuet-sfp-MmCARQ302E-Yahk and PRSFDuet-sfp-MmCART390E-Yahk
These plasmids were then transferred into E. coli BL21.Through the Whole-cell catalysis, it determined that the mutation at position 302 yielded the best effect.
1.Constructed strain NT1003-P3-ΔYcjQ:pTrc99a-davB-davA-GabT+pRSFDuet-sfp-MmCARQ302E-YahK.
As fermentation was performed, the mutated MmCAR moderately increased the yield of 1,5-PDO
2.Constructed the plasmid pRSFDuet-sfp-MmCARQ302E-SpyTag-YahK-EutM-SpyCatcher.
3.Constructed strain NT1003-P4-ΔYcjQ: pTrc99a-davB-davA-GabT +pRSFDuet-sfp-MmCARQ302E-SpyTag-YahK-EutM-SpyCatcher.
Fermentation was carried out and the final pentylene glycol yield was boosted to 23 mM (2.4 g/L).
1.Came to Qiliqiao Community of Dingshan Street for a science popularization propaganda activity.
2.NJTech-China-B and College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University successfully held an activity called “Synthetic Biology Carnival”.
To further identify the performance of the E. coli NT1003-P4-ΔYcjQ, we performed a fed-batch fermentation using a 5-liter bioreactor. After fermentation of 80 h, the 1,5-PDO production was reached to 48 mM (5.0 g/L).
In order to promote the project, we visited Nanjing Shenghu New Material Technology Co. Ltd. Jing Wang, Yang Liao and Yue Yang provided suggestions on technology implementation and project promotion.