1. This year we initiated the first iGEM team meetup in Shaanxi Province, strengthening the contact and
collaboration of iGEM teams in this region.
2. We have conducted profound yet detailed cooperation of great significance with
Guided XJTU-China on their team construction and preparation for the
competition，XJTU-China also assisted us in
the construction of some standardized parts and contacted the primary school where we carried out our DNA Time
Capsule activity. Additionally, we aided NWU-China rationally analyze the core enzyme they try to transform and
offered our plan for them.
Aided Jilin-China in determining the induction temperature of 5 RNA
thermosensors, and they helped us characterize 2
Submitted the Newsletter to XMU-China.
1.Initiated the first iGEM meet up in Shaanxi Province.
At present, there are three official iGEM teams in Shaanxi Province: NPU-China (our team), XJTU-China, NWU-China.
Having been communicating with XJTU-China and NWU-China on WeChat in the past few months, we felt prodigiously more
and more eager to get together and hold a meetup as time went by. After a month of discussion, at the beginning of
the summer vacation, we three teams jointly held the first iGEM meet up in Shaanxi Province. During the meeting,
the three teams took turns to showcase their projects this year and had further exchange and discussion on team
building. We shared the problems encountered during the project and collectively strived to solve them. The results
of this meetup are as follows:
label We have encountered difficulties in implementing DNA time capsule activities. XJTU-China expressed their willingness to help us contact Mawu Primary School in Xi’an to enable our DNA time capsule activity to be carried out smoothly.
label NWU-China needed patch clamp to test the function of the modified strain they did not have one, and it was XJTU-China that helped get them one.
label We advised XJTU-China on visas and some considerations for going to the US.
label We intend to help XJTU-China build a plasmid for the hairpin structure and verify the function.
label We intend to help NWU-China analyze core enzymes they try to transform and predict mutation points.
2. Collaboration with XJTU-China
2.1 Guiding XJTU-China
XJTU-China is participating in iGEM for the first time this year. As the first team to participate in iGEM in Shaanxi Province, we were invited to Xi'an Jiaotong University to accord them with guidance and experience sharing.
As follows what we have shared during the exchange are listed:
label Our understanding and analysis of iGEM.
label 2016 and 2017 projects of our team and the competition planning for 2018.
label Entry experience and precautions.
label Team operation mode.
The participation of iGEM for two years has rendered it clear for us to understand what iGEM is about and what iGEM entails us to do. By sharing our comprehension of iGEM, we believe that XJTU-China will be able to suit the competition much faster. We gave them a reference for this year's competition by sharing our previous two-year entries and this year's plan. We summed up the lessons learned from the past two years’ preparations and some easy mistakes for them, hoping that they will take fewer detours. Funding has always been a headache for most teams participating in iGEM. We shared with them how our team works to save as much money as possible and get more funding.
In the later discussion, we:
label Answered their questions about participation and registration.
label Talked over the idea of their project in the preliminary stage and put forward our suggestions.
label Gave advice on scheduling and personnel management.
XJTU-China also made some suggestions for our project and planning this year.
Afterwards, our two teams also invited NWU-China to co-establish a WeChat group hence more close exchanges and cooperation in the future.
Figure 2. Guiding XJTU-China
2.2 XJTU-China’s aid for us in conducting DNA Time Capsule.
XJTU-China helped us contact Mawu Primary School in Xi'an, enabling our DNA Time Capsule activity to be implemented smoothly. (Click here for more information)
2.3 XJTU-China’s assistance for us in building five standardized plasmids
We later encountered several problems during the construction of the standardized plasmid. E. coli turned into red after transforming pSB1C3 plasmid. Plus, atp6 (BBa_K2707006), atp8 (BBa_K2707005), atp9 (BBa_K2707000), cob (BBa_K2707004) and var1 (BBa_K2707008) , these five fragments could not be constructed on the pSB1C3 plasmid after many attempts. XJTU-China helped us construct these five plasmids as soon as they knew about our obstacle.
Figure 3. XJTU-China assistance for us in constructing five parts. From left to right:
3．Collaboration with NWU-China
3.1 Support for NWU-China in analyzing KcsA protein
We used our laboratory's self-developed Automation Enzyme Mutation Design (AEMD) platform to help NWU-China analyze the KcsA protein, the core enzyme in their project, and work out the plan for the enzyme modification from both functional and stability aspects for NWU-China to carry out the next step of enzyme transformation.
Click here for more information!
4. Collaboration with Jilin-China
4.1 Aid for Jilin_China in verifying the accuracy of the five thermosensors’ measurements
We got to know Jilin-China at the iGEM summit initiated by NKU-China. This year both of our projects pertain to gene synthesis, so we have been in close contact since. Jilin_China constructed some heat-inducible RNA-based thermosensors onto the pSB1C3 vector, and installed the Anderson promoter J23104 upstream, superfolder GFP (sfGFP) downstream. The melting temperature of the thermosensors can be determined by measuring the expression level of sfGPF at different temperatures. They have measured the fluorescence expression intensity of five heat-inducible RNA-based therosensors (RT1-4, RT1-33, K2541003, K2541004, K2541010). However, the measurement results may vary under different laboratory and experimental conditions. To ensure the accuracy of their results, we assisted them in measuring the changes in fluorescence expression intensity of these 5 thermosensors.
Figure 4. Fluorescence expression intensity of RT1-4,RT1-33,K2541003, K2541004, K2541010
1. 100 μL of glycerol bacteria (plasmid containing different thermosensors) was cultured for 4-5 hours and Abs was measured after dilution, guaranteeing the initial Abs was 0.02 and the volume was made up to 8 mL;
2. 100 μL was added to a black 96-well plate, conducting 3 parallel experiments at different temperatures, measuring Abs and fluorescence used as the data for 0 h;
3. Incubated at 31 ° C, 35 ° C, 39 ° C respectively;
4. Measured Abs and fluorescence after incubation of 4h, 6h, 8h.
4.2 Jilin_China’s assistance for us in characterizing 2 parts (BBa_K2707004，BBa_K2707008)
In the later stage, due to the heavy burden of our experimental and academic tasks, there were not adequate staff to characterize all the parts. At the CCIC, Jilin_China came to our aid and proposed to characterize the parts after learning about our predicament. There existed low GC content in the mitochondrial genomes and a large amount of replication in E. coli, which jeopardized the balance of four nucleotides in the cell, resulting in strong cytotoxicity. Jilin_China helped us measure the growth curves of the two submitted parts: cob (BBa_K2707004) and var1 (BBa_K2707008), the sequences of which were examined to determine if they were toxic to cells.
These two parts are two of the eight proteins expressed by the mitochondrial genome of Saccharomyces cerevisiae. One is Cob encoding cytochrome oxidase b, which is a component of the electron transport chain, and another is Var1 that encodes ribosomal protein. Note that cob has been removed all introns.
Figure 5. The growth curves of cob and var1
Conclusion: These two genes were constructed on pB1C3, the amplification of which after being transferred into DH5α has little effect on bacterial growth, qualitatively proving that these two sequences are not toxic to cells.
1. 5 μL of glycerol bacteria were transferred to 5 ml of liquid LB, adding appropriate amount of chloramphenicol and culturing overnight;
2. Measured the OD value of the three samples and add a certain amount of each sample to 50ml LB (250ml triangle bottle), with the final OD value being 0.05;
3. Measured the OD value of 0 h with a spectrophotometer;
4. OD value was measured respectively at 0.9, 2.15, 3.1, 4.2, 5.2, 6.1, 7.2, 8.3, 9.1, 12 h. When the OD value exceeded 0.8, diluted before measuring.
6. Recorded the data, analyzed the charts and drew the conclusions.
5. Collaboration with XMU-China
5.1 Submitting Newsletter to XMU-China
We offered XMU-China a description including our team introduction and project description, which are recorded on Newsletter 2018 edited by XMU-China. Through this platform, more people are enabled to learn about our project and we have obtained more opportunities to communicate and cooperate with the external world.
Click here for more information!